chapter
stringlengths 1.97k
1.53M
| path
stringlengths 47
241
|
|---|---|
Electronegativity
The electronegativity of an atom is a measure of its affinity for electrons. The atoms of the various elements differ in their affinity for electrons.
This image distorts the conventional periodic table of the elements so that the greater the electronegativity of an atom, the higher its position in the table. Although fluorine (F) is the most electronegative element, it is the electronegativity of runner-up oxygen (O) that is exploited by life. The shuttling of electrons between carbon (C) and oxygen (O) atoms powers life.
1. Moving electrons against the gradient (O to C) — as occurs in photosynthesis — requires energy (and stores it).
2. Moving electrons down the gradient (C to O) — as occurs in cellular respiration — releases energy.
The relative electronegativity of two interacting atoms also plays a major part in determining what kind of chemical bond forms between them.
Chemical Bonds
Three main types of chemical bonds:Ionic Bond, Covalent Bond, Polar Covalent Bond.
Ionic Bond
Example of an ionic bond is : Sodium (Na) and Chlorine (Cl) = Ionic Bond. There is a large difference in electronegativity between Na and Cl atoms, so
• the chlorine atom takes an electron from the sodium atom
• converting the atoms into ions (Na+) and (Cl)
• These are held together by their opposite electrical charge forming ionic bonds
• Each sodium ion is held by 6 chloride ions while each chloride ion is, in turn, held by 6 sodium ions
• Result: a crystal lattice (not molecules) of common table salt (NaCl)
Covalent Bond
Example of a covalent bond is: Carbon (C) and Hydrogen (H) = Covalent Bond. There is only a small difference in electronegativity between the C and H atoms, so
• the two atoms share the electrons
• Result: a covalent bond (depicted as C:H or C-H)
• The atoms are held together by their mutual affinity for their shared electrons
• An array of atoms held together by covalent bonds forms a true molecule
Polar Covalent Bond
Example of a polar covalent bond is: Hydrogen (H) and Oxygen (O) = Polar Covalent Bond. There is a moderate difference in electronegativity, causing the oxygen atom to pull the electron of the hydrogen atom closer to itself. This results in a polar covalent bond. Oxygen does this with 2 hydrogen atoms to form a molecule of water
Molecules, like water, with polar covalent bonds are themselves polar; that is, have partial electrical charges across the molecule and may be attracted to each other (as occurs with water molecules). These species are good solvents for polar and/or hydrophilic compounds may form hydrogen bonds.
1.04: Noncovalent Bonding
Noncovalent Bonding
Noncovalent bonding does not involve sharing of electrons. Instead it:
• holds the two strands of the DNA double helix together (hydrogen bonds)
• folds polypeptides into such secondary structures as the alpha helix and the beta conformation
• enables enzymes to bind to their substrate
• enables antibodies to bind to their antigen
• enables transcription factors to bind to each other
• enables transcription factors to bind to DNA
• enables proteins (e.g. some hormones) to bind to their receptor
• permits the assembly of such macromolecular machinery as
• ribosomes
• actin filaments
• microtubules
• and many more
There are three principle kinds of noncovalent forces:
• ionic interactions
• hydrophobic interactions
• hydrogen bonds
Ionic Interactions
At any given pH, proteins have charged groups that may participate in binding them to each other or to other types of molecules. For example, as the figure shows, negatively-charged carboxyl groups on aspartic acid (Asp) and glutamic acid (Glu) residues may be attracted by the positively-charged free amino groups on lysine (Lys) and arginine (Arg) residues.
Ionic interactions are highly sensitive to
• changes in pH.
As the pH drops,
• H+ bind to the carboxyl groups (COO-) of aspartic acid (Asp) and glutamic acid (Glu), neutralizing their negative charge, and
• H+ bind to the unoccupied pair of electrons on the N atom of the amino (NH2) groups of lysine (Lys) and arginine (Arg) giving them a positive charge
The result: Not only does the net charge on the molecule change (it becomes more positive) but many of the opportunities that its R groups have for ionic (electrostatic) interactions with other molecules and ions are altered.
As the pH rises,
• H+ are removed from the COOH groups of Asp and Glu, giving them a negative charge (COO), and
• H+ are removed from the NH3+ groups of Lys and Arg removing their positive charge
The result: Again the net charge on the molecule changes (it becomes more negative) and, again, many of the opportunities its R groups have for electrostatic interactions with other molecules or ions are altered.
• salt concentration
Increasing salt concentration reduces the strength of ionic binding by providing competing ions for the charged residues.
Hydrophobic Interactions
The side chains (R groups) of such amino acids as phenylalanine and leucine are nonpolar and hence interact poorly with polar molecules like water. For this reason, most of the nonpolar residues in globular proteins are directed toward the interior of the molecule whereas such polar groups as aspartic acid and lysine are on the surface exposed to the solvent. When nonpolar residues are exposed at the surface of two different molecules, it is energetically more favorable for their two "oily" nonpolar surfaces to approach each other closely displacing the polar water molecules from between them.
The strength of hydrophobic interactions is not appreciably affected by changes in pH or in salt concentration.
Hydrogen Bonds
Hydrogen bonds can form whenever
• a strongly electronegative atom (e.g., oxygen, nitrogen) approaches
• a hydrogen atom which is covalently attached to a second strongly-electronegative atom
Some common examples:
• between the −C=O group and the H-N− group of nearby peptide bonds in proteins giving rise to the alpha helix and beta configuration
• Between −C=O groups and hydroxyl (H-O−) groups in
• serine and threonine residues of proteins and
• sugars
Noncovalent interactions are individually weak but collectively strong.
All three forms of noncovalent interactions are individually weak (on the order of 5 kcal/mole) as compared with a covalent bond (with its 90–100 kcal/mole of bond energy). And what strength these interactions do have requires that the interacting groups can approach each other closely (an angstrom or less). So we can conclude that all the examples given at the top of the page require:
• a substantial number of noncovalent interactions working together to hold the structures together
• a surface topography that enables substantial areas of two interacting surfaces to approach each other closely; that is, they must fit each other | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/01%3A_The_Chemical_Basis_of_Life/1.03%3A_Electronegativity_and_types_of_Chemical_Bonds.txt |
Hydrogen Bonds
Polar molecules, such as water molecules, have a weak, partial negative charge at one region of the molecule (the oxygen atom in water) and a partial positive charge elsewhere (the hydrogen atoms in water). Thus when water molecules are close together, their positive and negative regions are attracted to the oppositely-charged regions of nearby molecules. The force of attraction, shown here as a dotted line, is called a hydrogen bond. Each water molecule is hydrogen bonded to four others.
The hydrogen bonds that form between water molecules account for some of the essential — and unique — properties of water.
• The attraction created by hydrogen bonds keeps water liquid over a wider range of temperature than is found for any other molecule its size.
• The energy required to break multiple hydrogen bonds causes water to have a high heat of vaporization; that is, a large amount of energy is needed to convert liquid water, where the molecules are attracted through their hydrogen bonds, to water vapor, where they are not.
Two outcomes of this:
• The evaporation of sweat, used by many mammals to cool themselves, cools by the large amount of heat needed to break the hydrogen bonds between water molecules.
• Reduction of temperature extremes near large bodies of water like the ocean.
The hydrogen bond has only 5% or so of the strength of a covalent bond. However, when many hydrogen bonds can form between two molecules (or parts of the same molecule), the resulting union can be sufficiently strong as to be quite stable.
Multiple hydrogen bonds
• hold the two strands of the DNA double helix together
• hold polypeptides together in such secondary structures as the alpha helix and the beta conformation
• help enzymes bind to their substrate
• help antibodies bind to their antigen
• help transcription factors bind to each other
• help transcription factors bind to DNA
1.06: Acids and Bases
Acids
Acids are substances that donate protons (hydrogen ions, H+) to bases.
Bases
Bases are substances that accept protons from acids.
Example of Acid and Base formation
Hydrogen chloride (HCl) is a gas. Its two atoms are held together by a shared pair of electrons. However, the chlorine atom is so much more electronegative than hydrogen, that the bond between them is polar covalent.
When hydrogen chloride is bubbled through water, the nucleus of the hydrogen atom leaves and takes up residence at one of the unshared pairs of electrons in the water molecule. However, its electron remains behind still attached to the chlorine atom. "1" This ionization produces:
• a chloride ion (Cl)
• a hydronium ion (H3O+). "2"
The resulting mixture is called hydrochloric acid.
Now let us bubble ammonia gas (NH3) through the hydrochloric acid. Ammonia molecules have one pair of unshared electrons and these have a greater affinity for a proton than do the unshared electrons in the water molecule. Consequently, the proton shifts again ("3") to form a new ion, the ammonium ion (NH4+) and water ("4").
Because both the HCl molecule and the hydronium ion are proton donors, they meet the definition of an acid.
The water molecule in the first example and the ammonia in the second example accept protons; therefore each is a base.
While HCl is found in living systems (e.g., the gastric juice secreted by the stomach), the most common acids in biology are those containing the carboxyl group ("5").
The proton of the carboxyl group is easily removed forming the carboxyl ion ("6").
Acetic acid (CH3COOH) is a common example of a carboxylic acid. When mixed with water, some of the protons on its -COOH group are attracted to the unshared electron pairs of water molecules. Hydronium ions (H3O+) and acetate ions (CH3COO) result. Vinegar is a dilute solution of acetic acid.
Ammonia is also found (in low concentrations) in living matter. But the most common bases are those molecules that contain an amino group ("7"). The unshared pair of electrons serves as a proton acceptor, as it does in the ammonia molecule.
Bicarbonate ions ("8") also serve as an important base in living tissue. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/01%3A_The_Chemical_Basis_of_Life/1.05%3A_Hydrogen_Bonds.txt |
Molecular Weight
The weight of a molecule is the sum of the weights of the atoms of which it is made. The unit of weight is the dalton, one-twelfth the weight of an atom of 12C. Thus the molecular weight (MW) of water is 18 daltons. (We shall ignore the tiny error introduced by the presence of traces of other isotopes - 17O, 18O, and 2H among the predominant 1H and 16O atoms.)
Why is it important to know the molecular weight of a compound?
For an example, let us assume that you want to study the response of honeybees to solutions of various kinds of sugars. One way to do this would be to make up several different solutions and see which one the bees prefer to harvest.
You might offer the bees the choice between, say, a 35% solution of sucrose (common table sugar) and a 35% solution of glucose (a natural component of honey). This would involve, in each case, dissolving 350 parts by weight (e.g., grams) of sugar in 650 parts (g) of water, thus producing 1000 g of each solution. But there is a problem with this approach. The willingness of the honeybee to respond to the presence of sugar dissolved in water is dependent on the number of sugar molecules in a given volume of the solution.
The sucrose molecule (MW = 342) is almost twice as heavy as the glucose molecule (MW = 180). So a 35% solution of glucose would contain almost twice as many molecules as a 35% solution of sucrose. To correct the problem, you should make the solution with the weights of sucrose and glucose in a ratio of 342:180. Then you would have the same concentration of molecules in each; that is, drop for drop, each solution would contain the same number of molecules.
Mole
A mole is the quantity of a substance whose weight in grams is equal to the molecular weight of the substance. If you weight out exactly 342 grams (g) of sucrose, you will have weighed out 1 mole of it. Thus 1 mole of glucose weighs 180 g. Furthermore, if you dissolve 1 mole of a substance in enough water to make 1 liter (L) of solution, you have made a 1-molar (1 M) solution.
A 1 M solution of these sugars would probably be too strong for the experiment with the bees. It might be better to make up a liter of each solution containing 34.2 g and 18.0 g respectively. Such solutions would be designated one-tenth molar (0.1 M) solutions. Drop for drop, these two solutions would still contain exactly the same number of molecules because they are of the same molarity.
Avogadro's Number
How many molecules are there in a mole?
Solution
The number is approximately 6 x 1023. This number is called Avogadro's number after the chemist who first attempted to determine it.
Avogadro's number applies to a mole of any substance: molecule or ion. Thus we can properly refer to a mole of hydrogen ions (1 g).
1.08: pH
pH is a measure of the concentration of hydrogens ions (= H+) (= protons) in a solution. Numerically it is the negative logarithm of that concentration expressed in moles per liter (M).
Pure water spontaneously dissociates into ions, forming a 10-7 M solution of H+ (and OH-). The negative of this logarithm is 7, so the pH of pure water is 7.
Solutions with a higher concentration of H+ than occurs in pure water have pH values below 7 and are acidic. Solutions containing molecules or ions that reduce the concentration of H+ below that of pure water have pH values above 7 and are basic or alkaline.
Is pH important? Yes!
The properties of most proteins, enzymes for example, are sensitive to pH.
As the pH drops,
• H+ bind to the carboxyl groups (COO-) of aspartic acid (Asp) and glutamic acid (Glu), neutralizing their negative charge, and
• H+ bind to the unoccupied pair of electrons on the N atom of the amino (NH2 ) groups of lysine (Lys) and arginine (Arg) giving them a positive charge.
The result: Not only does the net charge on the molecule change (it becomes more positive) but many of the opportunities that its R groups have for ionic interactions with other molecules and ions are altered.
As the pH rises,
• H+ are removed from the COOH groups of Asp and Glu, giving them a negative charge (COO-), and
• H+ are removed from the NH3+ groups of Lys and Arg removing their positive charge.
The result: Again the net charge on the molecule changes (it becomes more negative) and, again, many of the opportunities its R groups have for ionic interactions with other molecules or ions are altered.
The pH of the cytosol within a human cell is about 7.4. BUT, this value masks the pH differences that are found in various compartments within the cell. For example,
• The interior of lysosomes is much more acidic (as low as pH 4) than the cytosol, and the enzymes within work best at these low pH values.
• The pH differential created within chloroplasts by the energy of the sun is harnessed to synthesize ATP which, in turn, powers the synthesis of food.
• The pH differential created within mitochondria during the respiration of food is harnessed to the synthesis of ATP which, in turn, powers most of the energy-consuming activities of the cell such as locomotion and biosynthesis of cell components. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/01%3A_The_Chemical_Basis_of_Life/1.07%3A_Molecular_Weight_and_the_Mole.txt |
• 2.1: Organic Molecules
• 2.2: Hydrocarbons
Hydrocarbons are organic molecules that consist exclusively, or primarily, of carbon and hydrogen atoms. They come in two flavors: (1) aliphatic hydrocarbons that consist of linear chains of carbon atoms and (2) aromatic hydrocarbons that which consist of closed rings of carbon atoms.
• 2.3: Fats
Fat molecules are made up of four parts: a molecule of glycerol (on the right) and three molecules of fatty acids.
• 2.4: Phospholipids
Phospholipids are fat derivatives in which one fatty acid has been replaced by a phosphate group and one of several nitrogen-containing molecules.
• 2.5: Cholesterol
The cholesterol molecule is a steroid that is essential to life. It has also been responsible for 17 Nobel Prizes, countless pages of reports in scientific journals and the popular press, and mounting anxiety on the part of health-conscious people. The human body contains about 100 g of cholesterol. Most of this is incorporated in the membranes from which cells are constructed and is an indispensable component of them. The insulating layers of myelin wound around neurons are rich in cholesterol.
• 2.6: Carbohydrates
Carbohydrates were once thought to represent "hydrated carbon". However, the arrangement of atoms in carbohydrates has little to do with water molecules. Starch and cellulose are two common carbohydrates. Both are macromolecules with molecular weights in the hundreds of thousands. Both are polymers built from repeating units, monomers, much as a chain is built from its links. The monomers of both starch and cellulose are the same: units of the sugar glucose.
• 2.7: Amino Acids
Amino acids are the building blocks (monomers) of proteins. 20 different amino acids are used to synthesize proteins. The shape and other properties of each protein is dictated by the precise sequence of amino acids in it.
• 2.8: Enantiomers
In three-dimensional (3D) space, the four covalent bonds of carbon atoms point toward the corners of a regular tetrahedron.
• 2.9: Polypeptides
Polypeptides are chains of amino acids. Proteins are made up of one or more polypeptide molecules. The amino acids are linked covalently by peptide bonds. The picture below shows how three amino acids are linked by peptide bonds into a tripeptide.
• 2.10: Proteins
Proteins are macromolecules. They are constructed from one or more unbranched chains of amino acids; that is, they are polymers. An average eukaryotic protein contains around 500 amino acids but some are much smaller (the smallest are often called peptides) and some much larger (the largest to date is titin a protein found in skeletal and cardiac muscle; one version contains 34,350 amino acids in a single chain!).
• 2.11: Rules of Protein Structure
The function of a protein is determined by its shape. The shape of a protein is determined by its primary structure (sequence of amino acids). The sequence of amino acids in a protein is determined by the sequence of nucleotides in the gene (DNA) encoding it. The function of a protein (except when it is serving as food) is absolutely dependent on its three-dimensional structure.
• 2.12: Glycoproteins
Glycoproteins have carbohydrate attached to them — a process called glycosylation.
• 2.13: Nucleotides
• 2.14: Proteomics
While we humans probably have only some 21 thousand genes, we probably make at least 10 times that number of different proteins. The great majority of our genes produce pre-mRNAs that are alternatively-spliced. The study of proteomics is important because proteins are responsible for both the structure and the functions of all living things. Genes are simply the instructions for making proteins. It is proteins that make life.
Contributors and Attributions
• John W. Kimball. This content is distributed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license and made possible by funding from The Saylor Foundation.
• Thumbnail: Cellulose molecular structure (CC BY-SA 3.0 Unported; Pintor4257 via Wikipedia)
02: The Molecules of Life
Functional groups
The various functional groups include:
• hydroxyl group (-OH)
• carboxyl group [-COOH]
• carbonyl group (-C=O)
• amino group -NH2
Organic Molecules
Various organic molecules formed by these groups are as follows:
Alcohols
Organic molecules with a hydroxyl group (-OH).
Methanol [CH3OH] and ethanol (beverage alcohol)[CH3CH2OH] are common examples.
Sugars are also alcohols.
Carboxylic Acids
Contain one or more carboxyl groups [-COOH].
Many of the intermediates in the breakdown of foodstuffs by cellular respiration are carboxylic acids.
Aldehydes
Contain a carbon atom to which is attached one hydrogen atom and — by a double bond — one oxygen atom.
Formaldehyde [HCHO] is a powerful disinfectant and preservative (it denatures proteins).
Acetaldehyde is produced during the conversion of pyruvic acid to ethanol when yeast ferment sugars. The converse is also true — acetaldehyde is produced in the liver as it metabolizes ingested ethanol (and may be the prime culprit in a "hangover").
Phosphoglyceraldehyde is an intermediate in glycolysis and the "dark reaction" of photosynthesis
Ethers
Formed when two carbon atoms are linked by an oxygen atom.
Diethyl ether is a commonly-used anesthetic.
Esters
The removal of a molecule of water between the -OH group of an alcohol and the -OH group of a
• carboxylic acid (-COOH) [shown in the diagram] or
• phosphoric acid
produces an ester.
• Fats are triesters of three fatty acids and glycerol (the alcohol).
• Phospholipids are also esters.
• Nucleotides are esters of nucleosides and phosphoric acid.
• The nucleotides of DNA and RNA are linked by a double ester linkage called a phosphodiester bond.
Ketones
Organic molecules with a carbonyl group (-C=O) between two hydrocarbon portions.
Ketones are synthesized in the liver, usually from fatty acids.
When glucose metabolism is suppressed, during starvation or in diabetics, fatty acids are used as a source of energy. But instead of entering the citric acid cycle, the acetyl-CoA produced from them is converted into the ketone acetoacetate. Some of this is then converted into acetone (which can be smelled on the breath of patients whose diabetes is out of control).
Amines
Organic molecules with an amino group, -NH2. Some examples:
• all the amino acids (lysine has two of them).
• the thyroid hormones thyroxine (T4) and triiodothyronine (T3)
• Many neurotransmitters:
• adrenaline and noradrenaline
• dopamine
• serotonin (5-hydroxytryptamine)
• histamine
Amides
Amides are organic molecules containing a carbonyl group (-C=O) attached to a nitrogen atom. The peptide bond between the amino acids linked in a polypeptide is also called an amide bond.
Contributors and Attributions
John W. Kimball. This content is distributed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license and made possible by funding from The Saylor Foundation. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.01%3A_Organic_Molecules.txt |
Hydrocarbons are organic molecules that consist exclusively, or primarily, of carbon and hydrogen atoms. They come in two flavors: (1) aliphatic hydrocarbons that consist of linear chains of carbon atoms and (2) aromatic hydrocarbons that which consist of closed rings of carbon atoms.
Aliphatic Hydrocarbons
The simplest is methane, CH4. Next is ethane, C2H6.
The fatty acids in fats are aliphatic hydrocarbons. If a chain holds all the hydrogen atoms it can, the molecule is said to be saturated. The fatty acids in tristearin are all saturated.
If two adjacent carbon atoms each lose a hydrogen atom, a double bond forms between them. Such a molecule is said to be unsaturated.
Example : Ethylene H2C=CH2
The fatty acids in trilinolein and linolenic acid are examples of unsaturated fatty acids
Aromatic Hydrocarbons
The building block of aromatic hydrocarbons is the benzene ring. The arrangement of atoms is shown on the left. The version in the center is often used to simplify diagrams of molecular structures. The three double bonds are not restricted to the positions shown but are free to pass around the ring. This is sometimes indicated by drawing the benzene ring as it is on the far right.
Some examples of biological molecules that incorporate the benzene ring:
• the amino acids tyrosine and phenylalanine
• cholesterol and its various derivatives, such as the sex hormones: estrogens and testosterone
• the herbicide, 2,4-D
The carotenoid, beta-carotene, is a hydrocarbon that has both aliphatic and aromatic portions.
2.03: Fats
Fat molecules are made up of four parts: a molecule of glycerol (on the right) and three molecules of fatty acids. Each fatty acid consists of a hydrocarbon chain with a carboxyl group at one end. The glycerol molecule has three hydroxyl groups, each able to interact with the carboxyl group of a fatty acid. Removal of a water molecule at each of the three positions forms a triglyceride. The three fatty acids in a single fat molecule may be all alike (as shown here for tristearin) or they may be different. They may contain as few as 4 carbon atoms or as many as 24.
Because fatty acids are synthesized from fragments containing two carbon atoms, the number of carbon atoms in the chain is almost always an even number. In animal fats, 16-carbon (palmitic acid) and 18-carbon (stearic acid - shown here) fatty acids are the most common.
Unsaturated Fats
Some fatty acids have one or more double bonds between their carbon atoms. They are called unsaturated because they could hold more hydrogen atoms than they do. Monounsaturated fats have a single double bond in their fatty acids and polyunsaturated fats, such as trilinolein shown here, have two or more.
Double bonds are rigid and those in natural fats introduce a kink in the molecule. This prevents the fatty acids from packing close together and as a result, unsaturated fats have a lower melting point than do saturated fats. Because most of them are liquid at room temperature, we call them oils. Corn oil, canola oil, cottonseed oil, peanut oil, and olive oil are common examples. As this list suggests, plant fats tend to be unsaturated (therefore "oils"). Fats from such animals as cattle tend to be saturated.
Trans Fatty Acids
The most abundant (and least expensive) source of fat is from plant oils but many cooking applications, particularly baked products, need solid fats. The food industry uses hydrogenated oils for things like shortening and margarine. In hydrogenation, plant oils are exposed to hydrogen at a high temperature and in the presence of a catalyst.
Two things result:(1) some double bonds are converted into single bonds and (2) other double bonds are converted from cis to trans configuration. Both these effects straighten out the molecules so they can lie closer together and become solid rather than liquid.
Omega fatty acids
One system for naming unsaturated fatty acids is to indicate the position of the first double bond counting from the opposite end from the carboxyl group. That terminal carbon atom (shown here in blue) is called the omega carbon atom. Thus a monounsaturated fatty acid with its single double bond after carbon #3 (counting from and including the omega carbon) is called an omega-3 fatty acid. But so is a polyunsaturated fatty acid, such as linolenic acid (shown here), if its first double bond is in that position.
Some studies have suggested that omega-3 fatty acids help protect against cardiovascular disease. For this reason, a Dietary Reference Intake (DRI) of 1.1 grams/day for women (1.6 for men) was established in September 2002.
2.04: Phospholipids
Phospholipids are fat derivatives in which one fatty acid has been replaced by a phosphate group and one of several nitrogen-containing molecules.
Example 2.4.1: Phosphatidyl ethanolamine (also known as cephalin)
The hydrocarbon chains are hydrophobic (as in all fats). However, the charges on the phosphate and amino groups (in red) make that portion of the molecule hydrophilic. The result is an amphiphilic molecule.
Phospholipids like phosphatidyl ethanolamine are major constituents of cell membranes. These molecules form a phospholipid bilayer with their hydrophilic (polar) heads facing their aqueous surroundings (e.g., the cytosol) and their hydrophobic tails facing each other. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.02%3A_Hydrocarbons.txt |
The cholesterol molecule is a steroid that is essential to life. It has also been responsible for 17 Nobel Prizes, countless pages of reports in scientific journals and the popular press, and mounting anxiety on the part of health-conscious people. The human body contains about 100 g of cholesterol. Most of this is incorporated in the membranes from which cells are constructed and is an indispensable component of them. The insulating layers of myelin wound around neurons are especially rich in cholesterol.
The structure of Cholesterol
Uses of Cholesterol
Cholesterol is starting ingredient for the synthesis of the steroid hormones including progesterone, estrogens, androgens (e.g., testosterone), glucocorticoids (e.g., cortisol), and mineralocorticoids (e.g., aldosterone). Cholesterol is also the precursor from which the body synthesizes vitamin D.
Another major use of cholesterol is the synthesis of bile acids. These are synthesized in the liver from cholesterol and are secreted in the bile. They are essential for the absorption of fat from the contents of the intestine. A clue to the importance of cholesterol is that more than 90% of the bile acids are not lost in the feces but are reabsorbed from the lower intestine and recycled to the liver. There is some loss, however, and to compensate for this and to meet other needs, the liver synthesizes some 1500–2000 mg of new cholesterol each day. It synthesizes cholesterol from the products of fat metabolism.
There is also an unceasing transport of cholesterol in the blood between the liver and all the other tissues. Most of this cholesterol travels as low density lipoproteins (LDLs). Each LDL particle is a sphere filled with ~1,500 molecules of cholesterol complexed with fatty acids and coated with a layer of phospholipids and a single molecule of a protein called apolipoprotein B (apoB). Cells that need cholesterol trap and ingest LDLs by receptor-mediated endocytosis.
Problems caused by cholesterol
Cholesterol can also create problems.
• High levels of LDL cholesterol lead to the development of atherosclerosis: cholesterol-rich deposits (plaques) that form on the inside of blood vessels and predispose to heart attacks.
• Cholesterol in the bile can crystallize to form gall stones that may block the bile ducts.
Typical lipid values in humans
The level of cholesterol in the blood is measured in milligrams per deciliter (mg/dl), which is equivalent to parts per 100,000. The levels range from less than 50 in infants to an average of 215 in adults and to 1,200 or more in individuals suffering from a rare, inherited disorder called familial hypercholesterolemia. For those of us in the normal range, approximately two-thirds of our cholesterol is transported as LDLs. Most of the rest is carried by so-called high density lipoproteins (HDLs).
Because of their relationship to cardiovascular disease, the analysis of serum lipids has become an important health measure. The table shows the range of typical values as well as the values above (or below) which the subject may be at increased risk of developing atherosclerosis.
LIPID Typical values (mg/dl) Desirable (mg/dl)
Cholesterol (total) 170–210 <200
LDL cholesterol 60–140 <130
HDL cholesterol 35–85 >40
Triglycerides 40–150 <135
• Total cholesterol is the sum of
• HDL cholesterol
• LDL cholesterol and
• 20% of the triglyceride value
• Note that
• high LDL values are bad, but
• high HDL values are good (because HDL cholesterol transports cholesterol from the tissues back to the liver where it is secreted in the bile).
• Using the various values, one can calculate a
cardiac risk ratio = total cholesterol divided by HDL cholesterol
• A cardiac risk ratio greater than 7 is considered a warning.
In May of 2001, a panel of the National Institutes of Health recommended a more aggressive attack on reducing cholesterol levels in the U.S. population. In addition to a better diet and more exercise, they urged that many more people at risk of developing heart disease, such as
• smokers
• diabetics
• people with high blood pressure and/or
• obesity
be put on cholesterol-lowering drugs.
There are several types:
• drugs that interfere with the ability of the liver to synthesize cholesterol by blocking the action of the enzyme HMG-CoA reductase. These are the "statins", e.g., lovastatin (Mevacor®), pravastatin (Pravachol®), atorvastatin (Lipitor®).
• insoluble powders ("colestipol", "cholestyramine") that bind to bile acids in the intestine so that instead of being reabsorbed they are eliminated in the feces. In compensation, the liver increases its consumption of blood-borne cholesterol. The main drawback to these drugs is that they are gritty powders and must be consumed in rather large amounts.
• nicotinic acid (niacin);
• "fibric acids" such as gemfibrozil and clofibrate.
Careful attention to diet may by itself lead to a reduction in cholesterol levels. In one study, men with high (>265 mg/dl) levels were able to lower these an average of 3.5% (10 mg/dl) by diet alone. Their diets were low in fat as well as low in cholesterol, and it was not — and still is not — clear as to what aspect of the diet contributed to the modest reduction. Cholesterol is made from fat and lowering the proportion of fat in the diet will probably help. Favoring unsaturated fats over saturated fats appears to be beneficial. There is little evidence that lowering one's intake of cholesterol is, by itself, useful. An average intake of cholesterol of 300–500 mg per day is joined in the intestine by several times that amount that has been synthesized by the liver and appears to have little or no effect on blood levels of cholesterol. So when choosing between the pat of butter and the pat of margarine, it is not the 30-odd mg of cholesterol in the butter (vs. 0 in the margarine) but its high content of saturated fat (over 3 times that in the margarine) that is probably significant. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.05%3A_Cholesterol.txt |
Carbohydrates have the general molecular formula CH2O, and thus were once thought to represent "hydrated carbon". However, the arrangement of atoms in carbohydrates has little to do with water molecules. Starch and cellulose are two common carbohydrates. Both are macromolecules with molecular weights in the hundreds of thousands. Both are polymers (hence "polysaccharides"); that is, each is built from repeating units, monomers, much as a chain is built from its links. The monomers of both starch and cellulose are the same: units of the sugar glucose.
Monosaccharides
Three common sugars share the same molecular formula: C6H12O6. Because of their six carbon atoms, each is a hexose.
They are:
• glucose, "blood sugar", the immediate source of energy for cellular respiration
• galactose, a sugar in milk (and yogurt)
• fructose, a sugar found in honey
Although all three share the same molecular formula (C6H12O6), the arrangement of atoms differs in each case. Substances such as these three, which have identical molecular formulas but different structural formulas, are known as structural isomers. Glucose, galactose, and fructose are "single" sugars or monosaccharides. Two monosaccharides can be linked together to form a "double" sugar or disaccharide.
Disaccharides
Three common disaccharides:
• sucrose — common table sugar = glucose + fructose
• lactose — major sugar in milk = glucose + galactose
• maltose — product of starch digestion = glucose + glucose
Although the process of linking the two monomers is rather complex, the end result in each case is the loss of a hydrogen atom (H) from one of the monosaccharides and a hydroxyl group (OH) from the other. The resulting linkage between the sugars is called a glycosidic bond. The molecular formula of each of these disaccharides is
\[C_{12}H_{22}O_{11} = 2 C_6H_{12}O_6 − H_2O\]
All sugars are very soluble in water because of their many hydroxyl groups. Although not as concentrated a fuel as fats, sugars are the most important source of energy for many cells. Carbohydrates provide the bulk of the calories (4 kcal/gram) in most diets, and starches provide the bulk of that. Starches are polysaccharides.
Polysaccharides
There are three primary polysaccharide polymer systems of interest: Starches, Glycogen and Cellulose.
Starches
Starches are polymers of glucose. Two types are found:
• amylose consists of linear, unbranched chains of several hundred glucose residues (units). The glucose residues are linked by a glycosidic bond between their #1 and #4 carbon atoms.
• amylopectin differs from amylose in being highly branched. At approximately every thirtieth residue along the chain, a short side chain is attached by a glycosidic bond to the #6 carbon atom (the carbon above the ring). The total number of glucose residues in a molecule of amylopectin is several thousand.
2.07: Amino Acids
Amino acids are the building blocks (monomers) of proteins. 20 different amino acids are used to synthesize proteins. The shape and other properties of each protein is dictated by the precise sequence of amino acids in it.
Each amino acid consists of an alpha carbon atom to which is attached
• a hydrogen atom
• an amino group (hence "amino" acid)
• a carboxyl group (-COOH). This gives up a proton and is thus an acid (hence amino "acid")
• one of 20 different "R" groups. It is the structure of the R group that determines which of the 20 it is and its special properties. The amino acid shown here is Alanine
Table 2.7.1: Types of Amino Acids. For each amino acid both three-letter and single letter codes are given
Alanine Ala A hydrophobic
Arginine Arg R free amino group makes it basic and hydrophilic
Asparagine Asn N carbohydrate can be covalently linked ("N-linked) to its -NH
Aspartic acid Asp D free carboxyl group makes it acidic and hydrophilic
Cysteine Cys C oxidation of their sulfhydryl (-SH) groups link 2 Cys (S-S)
Glutamic acid Glu E free carboxyl group makes it acidic and hydrophilic
Glutamine Gln Q moderately hydrophilic
Glycine Gly G so small it is amphiphilic (can exist in any surroundings)
Histidine His H basic and hydrophilic
Isoleucine Ile I hydrophobic
Leucine Leu L hydrophobic
Lysine Lys K strongly basic and hydrophilic
Methionine Met M hydrophobic
Phenylalanine Phe F very hydrophobic
Proline Pro P causes kinks in the chain
Serine Ser S carbohydrate can be covalently linked ("O-linked") to its -OH
Threonine Thr T carbohydrate can be covalently linked ("O-linked") to its -OH
Tryptophan Trp W scarce in most plant proteins
Tyrosine Tyr Y a phosphate or sulfate group can be covalently attached to its -OH
Valine Val V hydrophobic
The Essential Amino Acids
Humans must include adequate amounts of 9 amino acids in their diet.
• Histidine
• Isoleucine
• Leucine
• Lysine
• Methionine (and/or cysteine)
• Phenylalanine (and/or tyrosine)
• Threonine
• Tryptophan
• Valine
These "essential" amino acids cannot be synthesized from other precursors. However, cysteine can partially meet the need for methionine (they both contain sulfur), and tyrosine can partially substitute for phenylalanine. Two of the essential amino acids, lysine and tryptophan, are poorly represented in most plant proteins. Thus strict vegetarians should ensure that their diet contains sufficient amounts of these two amino acids. 19 of the 20 amino acids listed above can exist in two forms in three dimensions.
2.08: Enantiomers
In three-dimensional (3D) space, the four covalent bonds of carbon atoms point toward the corners of a regular tetrahedron. The molecule represented to the below is methane (\(CH_4\)).
Whenever a carbon atom has four different structures bonded to it, two different molecules can be formed.
Example 2.8.1: Alanine
The amino acid alanine.
Bonded to its alpha carbon atom are four different groups:
• a carboxyl group (COO)
• an amino group (NH3+)
• a methyl group (CH3)(its R group)
• a hydrogen atom
If you orient the molecule so that you look along it from the COO group to the NH3+ group, the methyl (R) group can extend out to the left, forming L-alanine (shown on the left) or to the right, forming D-alanine (on the right). Although they share the same chemical formula, they are not interchangeable any more than a left-hand glove is interchangeable with right-hand glove.
19 of the 20 amino acids used to synthesize proteins can exist as L- or D- enantiomorphs. The exception is glycine, which has two (indistinguishable) hydrogen atoms attached to its alpha carbon. L amino acids are used exclusively for protein synthesis by all life on our planet. (Some D amino acids are used for other purposes).
Does chirality really matter? Yes. The function of a protein is determined by its shape. A protein with a D amino acid instead of L will have its R group sticking out in the wrong direction (Figure 2.8.3).
Many other kinds of organic molecules exist as enantiomers. Usually only one form is active in biological systems. For example, if one form binds to a receptor protein on the surface of a cell, the other probably cannot. With their protein catalysts (enzymes), cells usually synthesize only one form. However, chemical synthesis in the laboratory or pharmaceutical factory usually produces equal amounts of the two enantiomers — called a racemic mixture.
Example 2.8.2: Albuterol
The drug albuterol (e.g., Proventil®) contains equal amounts of two enantiomers. Only one of them is effective, and the other may be responsible for the occasional unpleasant side-effects associated with the drug (which is used to dilate the bronchi, e.g, during an attack of asthma). The active form can now be synthesized pure, and — called levalbuterol (Xopenex®) — is available by prescription.
Two enantiomers of albuterol: (R)-(−)-salbutamol (top) and (S)-(+)-salbutamol (bottom)
Enantiomers are also called optical isomers because their solutions rotate the plane of polarized light passing through them. If one enantiomer rotates light in the clockwise direction, a solution of the other enantiomer will rotate it in the opposite direction.
2.09: Polypeptides
The sequence of amino acids in a polypeptide is dictated by the codons in the messenger RNA (mRNA) molecules from which the polypeptide was translated. The sequence of codons in the mRNA was, in turn, dictated by the sequence of codons in the DNA from which the mRNA was transcribed. Proteins are made up of one or more polypeptide molecules. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.06%3A_Carbohydrates.txt |
Proteins are macromolecules. They are constructed from one or more unbranched chains of amino acids; that is, they are polymers. An average eukaryotic protein contains around 500 amino acids but some are much smaller (the smallest are often called peptides) and some much larger (the largest to date is titin a protein found in skeletal and cardiac muscle; one version contains 34,350 amino acids in a single chain!).
Every function in the living cell depends on proteins.
• Motion and locomotion of cells and organisms depends on proteins. [Examples: Muscles, Cilia and Flagella]
• The catalysis of all biochemical reactions is done by enzymes, which contain protein.
• The structure of cells, and the extracellular matrix in which they are embedded, is largely made of protein. [Examples: Collagens] (Plants and many microbes depend more on carbohydrates, e.g., cellulose, for support, but these are synthesized by enzymes.)
• The transport of materials in body fluids depends of proteins.
• The receptors for hormones and other signaling molecules are proteins.
• Proteins are an essential nutrient for heterotrophs.
• The transcription factors that turn genes on and off to guide the differentiation of the cell and its later responsiveness to signals reaching it are proteins.
• and many more — proteins are truly the physical basis of life.
The protein represented here displays many of the features of proteins. Let's examine some of them as you scroll down the image. The protein consists of two polypeptide chains, a long one on the left of 346 amino acids — it is called the heavy chain — and a short one on the right of 99 amino acids. The heavy chain is shown as consisting of 5 main regions or domains:
• three extracellular domains, designated here as N (includes the N-terminal), C1, and C2;
• a transmembrane domain where the polypeptide chain passes through the plasma membrane of the cell;
• a cytoplasmic domain (with the C terminal) within the cytoplasm of the cell.
Inteins
Another, very rare, post-translational modification is the later removal of a section of the polypeptide and the splicing together (with a peptide bond) of the remaining N-terminal and C-terminal segments. The portion removed is called an intein (a "protein intron"), and the ligated segments are called exteins ("protein exons"). Genes encoding inteins have been discovered in a variety of organisms, including
• some "true" bacteria such as
• Bacillus subtilis
• several mycobacteria
• several blue-green algae (cyanobacteria)
• some Archaea such as
• Methanococcus jannaschii
• Aeropyrum pernix
• and a few unicellular eukaryotes, e.g., budding yeast (Saccharomyces cerevisiae).
• None has been found in the genomes of multicellular eukaryotes like Drosophila, C. elegans, or the green plant Arabidopsis.
How proteins get their shape
The function of a protein is determined by its shape. The shape of a protein is determined by its primary structure(sequence of amino acids). The sequence of amino acids in a protein is determined by the sequence of nucleotides in the gene (DNA) encoding it. The function of a protein (except when it is serving as food) is absolutely dependent on its three-dimensional structure. A number of agents can disrupt this structure thus denaturing the protein.
• changes in pH (alters electrostatic interactions between charged amino acids)
• changes in salt concentration (does the same)
• changes in temperature (higher temperatures reduce the strength of hydrogen bonds)
• presence of reducing agents (break S-S bonds between cysteines)
None of these agents breaks peptide bonds, so the primary structure of a protein remains intact when it is denatured. When a protein is denatured, it loses its function.
Example 2.10.1
• A denatured enzyme ceases to function.
• A denatured antibody no longer can bind its antigen.
Often when a protein has been gently denatured and then is returned to normal physiological conditions of temperature, pH, salt concentration, etc., it spontaneously regains its function (e.g. enzymatic activity or ability to bind its antigen). This tells us
• The protein has spontaneously resumed its native three-dimensional shape.
• Its ability to do so is intrinsic; no outside agent was needed to get it to refold properly.
However, there are:
• enzymes that add sugars to certain amino acids, and these may be essential for proper folding;
• proteins, called molecular chaperones, that may enable a newly-synthesized protein to acquire its final shape faster and more reliably than it otherwise would.
Chaperones
Although the three-dimensional (tertiary) structure of a protein is determined by its primary structure, it may need assistance in achieving its final shape.
• As a polypeptide is being synthesized, it emerges (N-terminal first) from the ribosome and the folding process begins.
• However, the emerging polypeptide finds itself surrounded by the watery cytosol and many other proteins.
• As hydrophobic amino acids appear, they must find other hydrophobic amino acids to associate with. Ideally, these should be their own, but there is the danger that they could associate with nearby proteins instead — leading to aggregation and a failure to form the proper tertiary structure.
To avoid this problem, the cells of all organisms contain molecular chaperones that stabilize newly-formed polypeptides while they fold into their proper structure. The chaperones use the energy of ATP to do this work.
Chaperonins
Some proteins are so complex that a subset of molecular chaperones — called chaperonins — is needed. Chaperonins are hollow cylinders into which the newly-synthesized protein fits while it folds. The inner wall of the cylinder is lined with hydrophobic amino acids which stabilize the hydrophobic regions of the polypeptide chain while it folds safely away from the
• watery cytosol and
• other proteins outside.
Chaperonins also use ATP as the energy source to drive the folding process.
As mentioned above, high temperatures can denature proteins, and when a cell is exposed to high temperatures, several types of molecular chaperones swing into action. For this reason, these chaperones are also called heat-shock proteins (HSPs). Not only do molecular chaperones assist in the folding of newly-synthesized proteins, but some of them can also unfold aggregated proteins and then refold the protein properly. Protein aggregation is the cause of disorders such as Alzheimer's disease, Huntington's disease, and prion diseases (e.g., "mad-cow" disease). Perhaps some day ways will be found to treat these diseases by increasing the efficiency of disaggregating chaperones.
Despite the importance of chaperones, the rule still holds: the final shape of a protein is determined by only one thing: the precise sequence of amino acids in the protein. And the sequence of amino acids in every protein is dictated by the sequence of nucleotides in the gene encoding that protein. So the function of each of the thousands of proteins in an organism is specified by one or more genes.
Primary Structure
The primary structure of a protein is its linear sequence of amino acids and the location of any disulfide (-S-S-) bridges. Note the amino terminal or "N-terminal" (NH3+) at one end; carboxyl terminal ("C-terminal") (COO-) at the other.
Secondary Structure
Most proteins contain one or more stretches of amino acids that take on a characteristic structure in 3-D space. The most common of these are the alpha helix and the beta conformation.
Alpha Helix
The R groups of the amino acids all extend to the outside.
• The helix makes a complete turn every 3.6 amino acids.
• The helix is right-handed; it twists in a clockwise direction.
• The carbonyl group (-C=O) of each peptide bond extends parallel to the axis of the helix and points directly at the -N-H group of the peptide bond 4 amino acids below it in the helix. A hydrogen bond forms between them [-N-H·····O=C-]
Beta Conformation
• consists of pairs of chains lying side-by-side and
• stabilized by hydrogen bonds between the carbonyl oxygen atom on one chain and the -NH group on the adjacent chain.
• The chains are often "anti-parallel"; the N-terminal to C-terminal direction of one being the reverse of the other.
Tertiary Structure
Tertiary structure refers to the three-dimensional structure of the entire polypeptide chain.
The images (courtesy of Dr. D. R. Davies) represent the tertiary structure of the antigen-binding portion of an antibody molecule. Each circle represents an alpha carbon in one of the two polypeptide chains that make up this protein. (The filled circles at the top are amino acids that bind to the antigen.) Most of the secondary structure of this protein consists of beta conformation, which is particularly easy to see on the right side of the image.
Do try to fuse these two images into a stereoscopic (3D) view. I find that it works best when my eyes are about 18" from the screen and I try to relax so that my eyes are directed at a point behind the screen.
Where the entire protein or parts of a protein are exposed to water (e.g., in blood or the cytosol), hydrophilic R groups — including R groups with sugars attached , are found at the surface; hydrophobic R groups are buried in the interior.
Importance of Tertiary structure
The function of a protein (except as food) depends on its tertiary structure. If this is disrupted, the protein is said to be denatured, and it loses its activity. Examples:
• denatured enzymes lose their catalytic power
• denatured antibodies can no longer bind antigen
A mutation in the gene encoding a protein is a frequent cause of altered tertiary structure.
• Curiously, tiny amounts of the mutant version can trigger the alpha-to-beta conversion in the normal protein. Thus the mutant version can be infectious. There have been several cases in Europe of people ill with Creutzfeldt-Jakob disease that may have acquired it from ingesting tiny amounts of the mutant protein in their beef.
• A number of other proteins altered by a point mutation in the gene encoding them, e.g.,
• fibrinogen
• lysozyme
• transthyretin (a serum protein that transports thyroxin and retinol (vitamin A) in the blood)
can form insoluble amyloid deposits in humans.
The many hydrogen bonds that can form between the polypeptide backbones in the beta conformation suggests that this is a stable secondary structure potentially available to many proteins and so a tendency to form insoluble aggregates is as well. Avoidance of amyloid formation may account for the large investment in the cell in
• chaperones
• proteasomes
as well as the crucial importance of particular amino acid side chains in maintaining a globular, and hence soluble, tertiary structure.
Protein Domains
The tertiary structure of many proteins is built from several domains. Often each domain has a separate function to perform for the protein, such as:
• binding a small ligand (e.g., a peptide in the molecule shown here)
• spanning the plasma membrane (transmembrane proteins)
• containing the catalytic site (enzymes)
• DNA-binding (in transcription factors)
• providing a surface to bind specifically to another protein
In some (but not all) cases, each domain in a protein is encoded by a separate exon in the gene encoding that protein. In the histocompatibility molecule shown here ,
• three domains α1, α2, and α3 are each encoded by its own exon.
• Two additional domains a transmembrane domain and a cytoplasmic domainare also encoded by separate exons.
• 2-microglobulin, "β2m", is NOT a domain of this molecule. It is a separate molecule that binds to the three alpha domains (red line and circle) by noncovalent forces only. The complex of these two proteins is an example of quaternary structure.)
This image (courtesy of P. J. Bjorkman from Nature 329:506, 1987) is a schematic representation of the extracellular portion of HLA-A2, a human class I histocompatibility molecule. It also illustrates two common examples of secondary structure: the stretches of beta conformation are represented by the broad green arrows (pointing N -> C terminal); regions of alpha helix are shown as helical ribbons. The pairs of purple spheres represent the disulfide bridges. A correspondence between exons and domains is more likely to be seen in recently-evolved proteins. Presumably, "exon shuffling" during evolution has enabled organisms to manufacture new proteins, with new functions, by adding exons from other parts of the genome to encode new domains (rather like Lego® pieces).
Quaternary Structure
Complexes of 2 or more polypeptide chains held together by noncovalent forces (usually) but in precise ratios and with a precise 3-D configuration. The noncovalent association of a molecule of beta-2 microglobulin with the heavy chain of each class I histocompatibility molecule is an example.
Protein Kinesis
All proteins are synthesized by ribosomes using the information encoded in molecules of messenger RNA (mRNA). The various destinations for proteins occur in two major sets:
• one set for those proteins synthesized by ribosomes that remain suspended in the cytosol, and
• a second set for proteins synthesized by ribosomes that are attached to the membranes of the endoplasmic reticulum (ER) forming "rough endoplasmic reticulum" (RER).
Some of the important destinations for proteins are:
• the cytosol
• the nucleus
• mitochondria
• chloroplasts
• peroxisomes | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.10%3A_Proteins.txt |
The function of a protein is determined by its shape. The shape of a protein is determined by its primary structure (sequence of amino acids). The sequence of amino acids in a protein is determined by the sequence of nucleotides in the gene (DNA) encoding it. The function of a protein (except when it is serving as food) is absolutely dependent on its three-dimensional structure. A number of agents can disrupt this structure thus denaturing the protein.
• changes in pH (alters electrostatic interactions between charged amino acids)
• changes in salt concentration (does the same)
• changes in temperature (higher temperatures reduce the strength of hydrogen bonds)
• presence of reducing agents (break S-S bonds between cysteines)
Often when a protein has been gently denatured and then is returned to normal physiological conditions of temperature, pH, salt concentration, etc., it spontaneously regains its function (e.g. enzymatic activity or ability to bind its antigen). This tells us that the protein has spontaneously resumed its native three-dimensional shape. Moreover, this ability is intrinsic; no outside agent was needed to get it to refold properly.
However, there are enzymes that add sugars to certain amino acids, and these may be essential for proper folding. These proteins, called molecular chaperones, enable a newly-synthesized protein to acquire its final shape faster and more reliably than it otherwise would.
Chaperones
Although the three-dimensional (tertiary) structure of a protein is determined by its primary structure, it may need assistance in achieving its final shape.
• As a polypeptide is being synthesized, it emerges (N-terminal first) from the ribosome and the folding process begins.
• However, the emerging polypeptide finds itself surrounded by the watery cytosol and many other proteins.
• As hydrophobic amino acids appear, they must find other hydrophobic amino acids to associate with. Ideally, these should be their own, but there is the danger that they could associate with nearby proteins instead — leading to aggregation and a failure to form the proper tertiary structure.
Despite the importance of chaperones, the rule still holds: the final shape of a protein is determined by only one thing: the precise sequence of amino acids in the protein. And the sequence of amino acids in every protein is dictated by the sequence of nucleotides in the gene encoding that protein. So the function of each of the thousands of proteins in an organism is specified by one or more genes.
2.12: Glycoproteins
Glycoproteins have carbohydrate attached to them — a process called glycosylation. The attachment is a covalent linkage to:
• the hydroxyl (-OH) group of the R group of serine or threonine - called "O-linked" in both cases or to
• the amino group (-NH2) in the R group of asparagine - called "N-linked"
The carbohydrate consists of short, usually branched, chains of
• plain sugars (e.g., glucose, galactose)
• amino sugars (sugars with an amino group, e.g., N-acetylglucosamine), and
• acidic sugars (sugars with a carboxyl group, e.g., sialic acid)
Sugars are very hydrophilic thanks to their many -OH groups. Their presence
• makes glycoproteins far more hydrophilic than they would be otherwise and
• are often essential for the proper folding of the protein into its tertiary structure
Most of the proteins exposed to the watery surroundings at the surface of cells are glycoproteins.
This image shows the primary structure of glycophorin A, a glycoprotein that spans the plasma membrane ("Lipid bilayer") of human red blood cells. Each RBC has some 500,000 copies of the molecule embedded in its plasma membrane.
• Fifteen carbohydrate chains are "O-linked" to serine (Ser) and threonine (Thr) residues
• One carbohydrate chain is "N-linked" to the asparagine (Asn) at position 26
Two polymorphic versions of glycophorin A, which differ only at residues 1 and 5, occur in humans. These give rise to the MN blood groups
• The M allele encodes Ser at position 1 (Ser-1) and Gly at position 5 (Gly-5)
• The N allele encodes Leu-1 and Glu-5
Genotype to Phenotype
Individuals who inherit two N alleles (are homozygous) have blood group N.
• Individuals who are homozygous for the M allele have blood group M.
• Heterozygous individuals produce both proteins and have blood group MN.
Glycophorin A is the most important attachment site by which the parasite Plasmodium falciparum invades human red blood cells. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.11%3A_Rules_of_Protein_Structure.txt |
Nucleic acids are linear, unbranched polymers of nucleotides. Nucleotides consist of three parts.
A five-carbon sugar (hence a pentose). Two kinds are found:
• deoxyribose, which has a hydrogen atom attached to its #2 carbon atom (designated 2'), and
• ribose, which has a hydroxyl group there.
Deoxyribose-containing nucleotides, the deoxyribonucleotides, are the monomers of deoxyribonucleic acids (DNA). Ribose-containing nucleotides, the ribonucleotides, are the monomers of ribonucleic acids (RNA).
The Purines The Pyrimidines
A nitrogen-containing ring structure called a nucleobase (or simply a base). The nucleobase is attached to the 1' carbon atom of the pentose. In DNA, four different nucleobases are found:
• two purines, called adenine (A) and guanine (G)
• two pyrimidines, called thymine (T) and cytosine (C)
RNA contains:
• The same purines, adenine (A) and guanine (G).
• RNA also uses the pyrimidine cytosine (C), but instead of thymine, it uses the pyrimidine uracil (U).
Nucleoside
The combination of a nucleobase and a pentose is called a nucleoside.
One (as shown in the first figure), two, or three phosphate groups. These are attached to the 5' carbon atom of the pentose. The product in each case is called a nucleotide. Both DNA and RNA are assembled from nucleoside triphosphates.
• For DNA, these are dATP, dGTP, dCTP, and dTTP.
• For RNA, these are ATP, GTP, CTP, and UTP.
In both cases, as each nucleotide is attached, the second and third phosphates are removed.
Table 2.13.1: The nucleosides and their mono-, di-, and triphosphates
Nucleobase Nucleoside Nucleotides
DNA Adenine (A) Deoxyadenosine dAMP dADP dATP
Guanine (G) Deoxyguanosine dGMP dGDP dGTP
Cytosine (C) Deoxycytidine dCMP dCDP dCTP
Thymine (T) Deoxythymidine dTMP dTDP dTTP
RNA Adenine (A) Adenosine AMP ADP ATP
Guanine (G) Guanosine GMP GDP GTP
Cytosine (C) Cytidine CMP CDP CTP
Uracil (U) Uridine UMP UDP UTP
2.14: Proteomics
Genome
One complete set of genes in an organism (a haploid set).
Except for occasional unrepaired damage to its DNA (= mutations), the genome is fixed.
Transcriptome
The most common definition: All the messenger RNA (mRNA) molecules transcribed from the genome.
Varies with the differentiated state of the cell and the activity of the transcription factors that turn gene transcription on (and off).
Speaking strictly, one would define the transcriptome as all the RNA molecules — which includes a wide variety of untranslated, nonprotein-encoding RNA — transcribed from the DNA of the genome. It is now thought that ~75% of our DNA is transcribed into RNA although only 1.5% of this is messenger RNA for protein synthesis.
Metabolome
All the metabolic machinery, e.g.,
• enzymes
• coenzymes
• small metabolites, like
• the intermediates in glycolysis and cellular respiration
• nucleotides
present in a cell at a given time.
Varies with the differentiated state of the cell and its current activities.
Proteome
The proteome is the protein complement of the genome. It is quite a bit more complicated than the genome because a single gene can give rise to a number of different proteins through
• alternative splicing of the pre-messenger RNAs (pre-mRNAs)
• RNA editing of the pre-messenger RNAs
• attachment of carbohydrate residues to form glycoproteins
• addition of phosphate groups to some of the amino acids in the protein
While we humans probably have only some 21 thousand genes, we probably make at least 10 times that number of different proteins. The great majority of our genes produce pre-mRNAs that are alternatively-spliced.
The study of proteomics is important because proteins are responsible for both the structure and the functions of all living things. Genes are simply the instructions for making proteins. It is proteins that make life.
The set of proteins within a cell varies
• from one differentiated cell type to another (e.g. red blood cell vs lymphocyte) and
• from moment to moment, depending on the activities of the cell, e.g.,
• getting ready to duplicate its genome;
• repairing damage to its DNA;
• responding to a newly-available nutrient or cytokine;
• responding to the arrival of a hormone | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/02%3A_The_Molecules_of_Life/2.13%3A_Nucleotides.txt |
• 3.1: Animal Cells
The idealized animal cell contains many structures.
• 3.2: Cell Membranes
One universal feature of all cells is an outer limiting membrane called the plasma membrane. In addition, all eukaryotic cells contain elaborate systems of internal membranes which set up various membrane-enclosed compartments within the cell. Cell membranes are built from lipids and proteins.
• 3.3: The Nucleus
The nucleus is the hallmark of eukaryotic cells; the very term eukaryotic means having a "true nucleus".
• 3.4: Ribosomes
Ribosomes are the protein-synthesizing machines of the cell. They translate the information encoded in messenger RNA (mRNA) into a polypeptide.
• 3.5: Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a system of membrane-enclosed sacs and tubules in the cell. Their lumens are probably all interconnected, and their membranes are continuous with the outer membrane of the nuclear envelope. All the materials within the system are separated from the cytosol by a membrane.
• 3.6: Golgi Apparatus
The Golgi apparatus is a cell structure mainly devoted to processing the proteins synthesized in the endoplasmic reticulum (ER). Some of these will eventually end up as integral membrane proteins embedded in the plasma membrane. Other proteins moving through the Golgi will end up in lysosomes or be secreted by exocytosis (e.g., digestive enzymes).
• 3.7: Centrosomes and Centrioles
Centrioles are built from a cylindrical array of 9 microtubules, each of which has attached to it 2 partial microtubules. Centrioles are a key feature of eukaryotic cells and presumably arose with the first eukaryotes.
• 3.8: Lysosomes and Peroxisomes
Lysosomes are roughly spherical bodies enclosed by a single membrane. They are manufactured by the Golgi apparatus and contain over 50 different kinds of hydrolytic enzymes including proteases, lipases, nucleases, and polysaccharidases. The pH within the lysosome is about pH 5, substantially less than that of the cytosol (~pH 7.2). All the enzymes in the lysosome work best at an acid pH.
• 3.9: Protein Kinesis
All proteins are synthesized by ribosomes using the information encoded in molecules of messenger RNA (mRNA). This process is called translation and is described in Gene Translation: RNA -> Protein. Our task here is to explore the ways that these proteins are delivered to their proper destinations.
• 3.10: The Proteasome
Lysosomes and proteasomes are two major intracellular devices in which damaged or unneeded proteins are broken down. Protein degradation is as essential to the cell as protein synthesis. For example, to supply amino acids for fresh protein synthesis, to remove excess enzymes, and to remove transcription factors that are no longer needed.
• 3.11: The Cytoskeleton
The cytoskeleton is made up of three kinds of protein filaments: Actin filaments (also called microfilaments), Intermediate filaments, and Microtubules. Cells contain elaborate arrays of protein fibers that serve such functions as establishing cell shape, providing mechanical strength, and locomotion. These fibers participate in chromosome separation in mitosis and meiosis and intracellular transport of organelles.
• 3.12: Cilia
These whiplike appendages extend from the surface of many types of eukaryotic cells. If there are many of them, they are called cilia. If only one, or a few, they are flagella. Flagella also tend to be longer than cilia but are otherwise similar in construction.
• 3.13: Animal Tissues
Cells contain elaborate arrays of protein fibers that serve such functions as establishing cell shape, providing mechanical strength, and locomotion. These fibers participate in chromosome separation in mitosis and meiosis and intracellular transport of organelles.
• 3.14: Adipose Tissue
Two kinds of adipose tissue are found in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT). White adipose tissue is the most common and is the fat that so many of us complain of acquiring. Brown adipose tissue is present in small mammals (e.g., mice) and in newborn humans. Most of it disappears in adult humans.The cells in both types of fat are called adipocytes although they differ in origin, structure, and function in the two types of tissue.
• 3.15: Junctions between Cells
In many animal tissues (e.g., connective tissue), each cell is separated from the next by an extracellular coating or matrix. However, in some tissues (e.g., epithelia), the plasma membranes of adjacent cells are pressed together. Four kinds of junctions occur in vertebrates: Tight junctions Adherens junctions Gap junctions Desmosomes In many plant tissues, it turns out that the plasma membrane of each cell is continuous with that of the adjacent cells.
• 3.16: Plant Cells
Plant cells are eukaryotic and have many of the structures found in animal cells. Plant cells differ from animal cells as they lack centioles and intermediate filament; they also do not have plastids and a cell wall and large vacuoles.
• 3.17: Chloroplasts
The chloroplast is made up of 3 types of membrane: A smooth outer membrane which is freely permeable to molecules. A smooth inner membrane which contains many transporters: integral membrane proteins that regulate the passage in an out of the chloroplast of small molecules like sugars proteins synthesized in the cytoplasm of the cell but used within the chloroplast A system of thylakoid membranes
• 3.18: Chlorophylls and Carotenoids
Two types of chlorophyll are found in plants and the green algae: chlorophyll a and chlorophyll b.
• 3.19: Plant Tissues
A mature vascular plant (any plant other than mosses and liverworts), contains several types of differentiated cells. These are grouped together in tissues. Some tissues contain only one type of cell. Others consist of several cells.
• 3.20: Apoptosis
Apoptosis is a process of programmed cell death that occurs in multicellular organisms. There are two ways in which cells die: They are killed by injurious agents or they are induced to commit suicide.
• 3.21: Collagens
Collagens are insoluble, extracellular glycoproteins found in all animals. They are the most abundant proteins in the human body and are essential structural components of all connective tissues such as cartilage, bone, tendons, ligaments, fascia, skin. Gelatin is solubilized collagen. 29 types of collagens have been found in humans.
• 3.22: Chromatophores
Chromatophores are irregularly shaped, pigment-containing cells. If the pigment is melanin, they are called melanophores. Chromatophores are common in crustaceans, cephalopod mollusks, lizards and amphibians, and some fishes.
• 3.23: Diffusion, Active Transport and Membrane Channels
All cells acquire the molecules and ions they need from their surrounding extracellular fluid (ECF). There is an unceasing traffic of molecules and ions in and out of the cell through its plasma membrane (Examples: glucose, sodium, and calcium ions). In eukaryotic cells, there is also transport in and out of membrane-bounded intracellular compartments such as the nucleus, endoplasmic reticulum, and mitochondria.
• 3.24: Endocytosis
In endocytosis, the cell engulfs some of its extracellular fluid (ECF) including material dissolved or suspended in it. A portion of the plasma membrane is invaginated, coated with molecules of the protein clathrin, and pinched off forming a membrane-bounded vesicle called an endosome.
• 3.25: Exocytosis
Exocytosis is the reverse of endocytosis and that is just as well. In 30 minutes an active cell like a macrophage can endocytose an amount of plasma membrane equal to its complete plasma membrane. So the cell must have a mechanism to restore the normal amount of plasma membrane. Exocytosis is that mechanism.
Thumbnail: A diagram of a typical prokaryotic cell. (Public Domain; LadyofHats).
03: The Cellular Basis of Life
This schematic represents an idealized animal cell, e.g., a liver cell. The columns to the left and right of the labels contain links to discussions of the particular structures.
Intermediate filaments
Plasma membrane
Peroxisome
Vacuole
Lysosome
Nucleolus
Centrioles
Nucleus
Nuclear envelope
Golgi apparatus
Pinocytotic vesicle
Actin filaments
Glycogen granules
Smooth endoplasmic reticulum
Microtubules
Ribosomes
Mitochondrion
Rough endoplasmic reticulum
Cytosol
3.02: Cell Membranes
One universal feature of all cells is an outer limiting membrane called the plasma membrane. In addition, all eukaryotic cells contain elaborate systems of internal membranes which set up various membrane-enclosed compartments within the cell. Cell membranes are built from lipids and proteins.
The Plasma Membrane
The plasma membrane serves as the interface between the machinery in the interior of the cell and the extracellular fluid (ECF) that bathes all cells. The lipids in the plasma membrane are chiefly phospholipids like phosphatidyl ethanolamine. Phospholipids are amphiphilic with the hydrocarbon tail of the molecule being hydrophobic; its polar head hydrophilic. As the plasma membrane faces watery solutions on both sides, its phospholipids accommodate this by forming a phospholipid bilayer with the hydrophobic tails facing each other. Substantial amounts of cholesterol are tucked within the hydrocarbon tails (not shown).
Integral Membrane Proteins
Many of the proteins associated with the plasma membrane are tightly bound to it. Some are attached to lipids in the bilayer. In others — the transmembrane proteins — the polypeptide chain actually traverses the lipid bilayer (Figure 3.2.2). The figure shows a transmembrane protein that passes just once through the bilayer and another that passes through it 7 times. All G-protein-coupled receptors (e.g., receptors of peptide hormones, and odors) each span the plasma membrane 7 times.
In all these cases, the portion within the lipid bilayer consists primarily of hydrophobic amino acids. These are usually arranged in an alpha helix so that the polar -C=O and -NH groups at the peptide bonds can interact with each other rather than with their hydrophobic surroundings. Those portions of the polypeptide that project out from the bilayer tend to have a high percentage of hydrophilic amino acids. Furthermore, those that project into the aqueous surroundings of the cell are usually glycoproteins, with many hydrophilic sugar residues attached to the part of the polypeptide exposed at the surface of the cell. Some transmembrane proteins that span the bilayer several times form a hydrophilic channel through which certain ions and molecules can enter (or leave) the cell.
Peripheral Membrane Proteins
These are more loosely associated with the membrane. They are usually attached noncovalently to the protruding portions of integral membrane proteins (Figure 3.2.3). Membrane proteins are often restricted in their movements. A lipid bilayer is really a film of oil. Thus we might expect that structures immersed in it would be relatively free to float about. For some membrane proteins, this is the case. For others, however, their mobility is limited:
• Some of the proteins exposed at the interior face of the plasma membrane are tethered to cytoskeletal elements like actin microfilaments.
• Some proteins are the exterior face of the plasma membrane are anchored to components of the extracellular matrix like collagen.
• Integral membrane proteins cannot pass through the tight junctions found between some kinds of cells (e.g., epithelial cells). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.01%3A_Animal_Cells.txt |
The nucleus is the hallmark of eukaryotic cells; the very term eukaryotic means having a "true nucleus".
The Nuclear Envelope
The nucleus is enveloped by a pair of membranes enclosing a lumen that is continuous with that of the endoplasmic reticulum. The inner membrane is stabilized by a meshwork of intermediate filament proteins called lamins. The nuclear envelope is perforated by thousands of nuclear pore complexes (NPCs) that control the passage of molecules in and out of the nucleus.
Chromatin
The nucleus contains the chromosomes of the cell. Each chromosome consists of a single molecule of DNA complexed with an equal mass of proteins. Collectively, the DNA of the nucleus with its associated proteins is called chromatin.
Most of the protein consists of multiple copies of 5 kinds of histones. These are basic proteins, bristling with positively charged arginine and lysine residues. (Both Arg and Lys have a free amino group on their R group, which attracts protons (H+) giving them a positive charge.) Just the choice of amino acids you would make to bind tightly to the negatively-charged phosphate groups of DNA.
Chromatin also contains small amounts of a wide variety of nonhistone proteins. Most of these are transcription factors (e.g., the steroid receptors) and their association with the DNA is more transient.
The image on the right shows the 5 histones separated by electrophoresis. These 5 proteins vary little from one cell type to another or even from one species to another. However, the many nonhistone proteins in chromatin (also shown on the right) do vary from one cell type to another and from one species to another. (Courtesy of Gary S. Stein and Janet Swinehart Stein, University of Florida.)
Two copies of each of four kinds of histones H2A, H2B, H3, H4 form a core of protein, the nucleosome core. Around this is wrapped about 147 base pairs of DNA. From 20–60 bp of DNA link one nucleosome to the next. Each linker region is occupied by a single molecule of histone 1 (H1). This region is longer (50–150 bp) adjacent to the promoters of genes which presumably makes more room for the binding of transcription factors.
Nucleosomes Nucleosome Schematics
The binding of histones to DNA does not depend on particular nucleotide sequences in the DNA but does depend critically on the amino acid sequence of the histone. Histones are some of the most conserved molecules during the course of evolution. Histone H4 in the calf differs from H4 in the pea plant at only 2 amino acids residues in the chain of 102. The above electron micrograph (courtesy of David E. Olins and Ada L. Olins) for nucleosomes shows chromatin from the nucleus of a chicken red blood cell (birds, unlike most mammals, retain the nucleus in their mature red blood cells). The arrows point to the nucleosomes. You can see why the arrangement of nucleosomes has been likened to "beads on a string".
The formation of nucleosomes helps somewhat, but not nearly enough, to make the DNA sufficiently compact to fit in the nucleus. In order to fit 46 DNA molecules (in humans), totaling over 2 meters in length, into a nucleus that may be only 10 µm across requires more extensive folding and compaction. Interactions between the exposed "tails" of the core histones causes nucleosomes to associate into a compact fiber 30 nm in diameter. These fibers are then folded into more complex structures whose precise configuration is uncertain and which probably changes with the level of activity of the genes in the region.
Histone Modifications
Although their amino acid sequence (primary structure) is unvarying, individual histone molecules do vary in structure as a result of chemical modifications that occur later to individual amino acids. These include adding:
• acetyl groups (CH3CO−) to lysines
• phosphate groups to serines and threonines
• methyl groups to lysines and arginines
Although 75–80% of the histone molecule is incorporated in the core, the remainder — at the N-terminal — dangles out from the core as a "tail" (not shown in the figure). Most of the chemical modifications occur on these tails, especially of H3 and H4. Most of theses changes are reversible. For example, acetyl groups are added by enzymes called histone acetyltransferases (HATs)(not to be confused with the "HAT" medium used to make monoclonal antibodies) and are also removed by histone deacetylases (HDACs). More often than not, acetylation of histones occurs in regions of chromatin that become active in gene transcription. This makes a kind of intuitive sense as adding acetyl groups neutralizes the positive charges on Lys thus reducing the strength of the association between the highly-negative DNA and the highly-positive histones.
However, there is surely more to the story. Acetylation of Lys-16 on H4 ("H4K16ac") prevents the interaction of their "tails" needed to form the compact 30-nm structure of inactive chromatin and thus is associated with active genes (note that this case involves interrupting protein-protein not protein-DNA interactions). Methylation, which also neutralizes the charge on lysines (and arginines), can either stimulate or inhibit gene transcription in that region.
• Adding 3 methyl groups to lysine-4 and/or lysine-36 in H3 (H3K4me3 and H3K36me3 respectively) is associated with active gene transcription while
• trimethylation of lysine-9 and/or lysine-27 in H3 (H3K9me3 and H3K27me3 respectively) is associated with inactive genes. (These include those imprinted genes that have been permanently inactivated in somatic cells.)
• And adding phosphates causes the chromosomes to become more — not less — compact as they get ready for mitosis and meiosis.
In any case, it is now clear that histones are a dynamic component of chromatin and not simply inert DNA-packing material.
Histone Variants
We have genes for 8 different varieties of histone 1 (H1). Which variety is found at a particular linker depends on such factors as the type of cell, where it is in the cell cycle, and its stage of differentiation. In some cases, at least, a particular variant of H1 associates with certain transcription factors to bind to the enhancer of specific genes turning off expression of those genes.
Some other examples of histone variants:
• H3 is replaced by CENP-A ("centromere protein A") at the nucleosomes near centromeres. Failure to substitute CENP-A for H3 in this regions blocks centromere structure and function.
• H2A is replaced by the variant H2A.Z at gene promoters and enhancers.
• All the "standard" histones are replaced by variants as sperm develop.
In general, the "standard" histones are incorporated into the nucleosomes as new DNA is synthesized during S phase of the cell cycle. Later, some are replaced by variant histones as conditions in the cell dictate.
Chromosome Territories
During interphase, little can be seen of chromatin structure (except for special cases like the polytene chromosomes of Drosophila and some other flies). Although each chromosome is greatly elongated, it tends to occupy a discrete region within the nucleus called its territory. This can be demonstrated by:
• directing a tiny laser beam at a small portion of the nucleus. If all the chromosomes were intertwined, one would expect that all would receive some damage. That does not occur — only one or two chromosomes are damaged.
• Fluorescent stains specific for a particular chromosome stain only two regions in the nucleus — revealing the territory of the two homologs.
"Kissing" Chromosomes
Portions of one chromosome can loop out of its territory and interact with part of a different chromosome looping out from its territory. These are "kissing" chromosomes. The examples that have been found so far indicate that these interactions are another way of coordinating the activity of genes residing on different chromosomes.
The human genome contains many genes — scattered along different chromosomes — that are turned on by the arrival of a single signal. Among the many genes activated by estrogen, are TIFF1 on chromosome 21 and GREB1 on chromosome 2. Using FISH analysis, researchers at the University of California in San Diego showed that within a little as 2 minutes after exposing cells to estrogen, the TIFF1 and GREB1 loci move from their respective chromosome territories and "kiss".
In the mouse, naive helper T cells — awaiting a signal to direct them to become either Th1 cells or Th2 cells have
• the part of chromosome 10 carrying the gene for interferon-gamma (a Th1 cytokine) kissing
• the part of chromosome 11 carrying the genes for IL-4 and IL-5 (Th2 cytokines).
When the cell receives the signals committing it to one path or the other, the two regions separate, the appropriate one going to a region of active transcription; the other to a region of heterochromatin.
And still another example (in this case, two loci far apart on the same chromosome "kiss"):
In the head region of the Drosophila larva, expression of the homeobox (HOX) genes Antp and Abd-B is shut down. FISH analysis shows that these two loci — 10,000,000 base pairs apart on chromosome III — are brought together in the nucleus bound by proteins that prevent their transcription.
Euchromatin versus Heterochromatin
The density of the chromatin that makes up each chromosome (that is, how tightly it is packed) varies along the length of the chromosome. Dense regions are called heterochromatin and less dense regions are called euchromatin. Heterochromatin is found in parts of the chromosome where there are few or no genes, such as
• centromeres and
• telomeres. This heterochromatin is found in all types of cells in the organism.
• is also found in gene-rich regions of the chromosome but where the genes are inactive; that is, not transcribed. The location of this heterochromatin varies from one type of differentiated cell to another (as we would expect — a liver cell, for example, should shut down expression of genes that are not needed for its functions.
• is densely-packed.
• is greatly enriched with transposons and other types of DNA that does not contribute to the proteome.
• is replicated late in S phase of the cell cycle.
• has reduced crossing over in meiosis.
• is localized near the inner surface of the nuclear envelope, in most animal cells.
• The histones in the nucleosomes of heterochromatin show characteristic modifications:
• decreased acetylation;
• increased methylation of lysine-9 in histone H3 (H3K9), which now provides a binding site for heterochromatin protein 1 (HP1), which blocks access by the transcription factors needed for gene transcription
• increased methylation of lysine-27 in histone H3 (H3K27)
Euchromatin
• is found in parts of the chromosome that contain many active genes.
• is loosely-packed in loops of 30-nm fibers.
• separated from adjacent heterochromatin by insulators.
• In animal cells, euchromatin and thus active gene transcription occurs near the center of the nucleus.
• The genes in euchromatin show
• decreased methylation of the cytosines in CpG sites of the gene's promoter(s)
• increased acetylation of nearby histones
• decreased methylation of lysine-9 and lysine-27 in histone H3
Nucleosomes and Transcription
Transcription factors cannot bind to their promoter if the promoter is blocked by a nucleosome. One of the first functions of the assembling transcription factors is to either expel the nucleosome from the site where transcription begins or at least to slide the nucleosomes along the DNA molecule. Either action exposes the gene's promoter so that the transcription factors can then bind to it.
The actual transcription of protein-coding genes is done by RNA polymerase II (Pol II or RNAP II). In order for it to travel along the DNA to be transcribed, a complex of proteins removes the nucleosomes in front of it and then replaces them after Pol II has transcribed that portion of DNA and moved on.
Nucleosomes and DNA Replication
As is the case in transcription, the DNA helix must open to allow DNA replication to proceed. This, too, requires that the nucleosomes preceding the replication fork be removed and then quickly reassembled as the leading and lagging strands are synthesized.
The Nucleolus
During the period between cell divisions, when the chromosomes are in their extended state, one or more of them (10 in human cells) have loops extending into a spherical mass called the nucleolus. Here are synthesized three (of the four) kinds of RNA molecules (28S, 18S, 5.8S) used in the assembly of the large and small subunits of ribosomes.
28S, 18S, and 5.8S ribosomal RNA is transcribed (by RNA polymerase I) from hundreds to thousands of tandemly-arranged rDNA genes distributed (in humans) on 10 different chromosomes. The rDNA-containing regions of these 10 chromosomes cluster together in the nucleolus.
(In yeast, the 5S rRNA molecules — as well as transfer RNA molecules — are also synthesized (by RNA polymerase III) in the nucleolus.)
Once formed, rRNA molecules associate with the dozens of different ribosomal proteins used in the assembly of the large and small subunits of the ribosome.
But proteins are synthesized in the cytosol — and all the ribosomes are needed in the cytosol to do their work — so there must be a mechanism for the transport of these large structures in and out of the nucleus. This is one of the functions of the nuclear pore complexes.
Nuclear Pore Complexes (NPCs)
The nuclear envelope is perforated with thousands of pores. Each is constructed from multiple copies of several dozen different proteins called nucleoporins.
The entire assembly forms an aqueous channel connecting the cytosol with the interior of the nucleus ("nucleoplasm"). When materials are to be transported through the pore, it opens up to form a channel some 27–41 nm wide — large enough to get such large assemblies as ribosomal subunits through.
Transport through the nuclear pore complexes is active; that is, it requires
• energy
• many different carrier molecules each specialized to transport a particular cargo
• docking molecules in the NPC (represented here as colored rods and disks)
Import into the nucleus
Proteins are synthesized in the cytosol and those needed by the nucleus must be imported into it through the NPCs. They include:
• all the histones needed to make the nucleosomes
• all the ribosomal proteins needed for the assembly of ribosomes
• all the transcription factors (e.g., the steroid receptors) needed to turn genes on (and off)
• all the splicing factors needed to process pre-mRNA into mature mRNA molecules; that is, to cut out intron regions and splice the exon regions.
Probably all of these proteins has a characteristic sequence of amino acids — called a nuclear localization sequence (NLS) — that target them for entry.
Export from the nucleus
Molecules and macromolecular assemblies exported from the nucleus include:
• the ribosomal subunits containing both rRNA and proteins
• messenger RNA (mRNA) molecules (accompanied by proteins)
• transfer RNA (tRNA) molecules (also accompanied by proteins)
• transcription factors that are returned to the cytosol to await reuse
Both the RNA and protein molecules contain a characteristic nuclear export sequence (NES) needed to ensure their association with the right carrier molecules to take them out to the cytosol.
Contributors and Attributions
John W. Kimball. This content is distributed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license and made possible by funding from The Saylor Foundation. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.03%3A_The_Nucleus.txt |
Ribosomes are the protein-synthesizing machines of the cell. They translate the information encoded in messenger RNA (mRNA) into a polypeptide.
Shape, size and function
Ribosomes are roughly spherical with a diameter of ~20 nm, they can be seen only with the electron microscope. Figure \(1\) is an electron micrograph showing clusters of ribosomes. These clusters, called polysomes, are held together by messenger RNA (mRNA). They can make up 25% of the dry weight of cells (e.g., pancreas cells) and specialize in protein synthesis. A single pancreas cell can synthesize 5 million molecules of protein per minute.
In eukaryotes, ribosomes that synthesize proteins for use within the cytosol (e.g., enzymes of glycolysis) are suspended in the cytosol. The specific ribosomes that synthesize proteins destined for secretion (by exocytosis), the plasma membrane (e.g., cell surface receptors), and lysosomes. These ribosomes are attached to the cytosolic face of the membranes of the endoplasmic reticulum. As the polypeptide is synthesized, it is extruded into the interior (lumen) of the endoplasmic reticulum. Then, before these proteins reach their final destinations, they undergo a series of processing steps in the Golgi apparatus.
Ribosomes that synthesize 13 of the proteins destined for the inner membrane of mitochondria are found within the mitochondrion itself and are quite different in structure from the others. The ribosomes of bacteria, eukaryotes, and mitochondria differ in many details of their structure (Table \(1\)). However, despite these differences, the basic operations of bacterial, eukaryotic, and mitochondrial ribosomes are very similar.
Bacterial (70S) Eukaryotic (80S) Mitochondrial (55S)
Table \(1\): Comparison of Ribosome Structure in Bacteria, Eukaryotes, and Human Mitochondria
Large Subunit 50S 60S 39S
rRNAs
(1 of each)
23S (2904 nts) 28S (4700 nts) 16S (1560 nts)
5S (120 nts) 5S (120 nts)
5.8S (160 nts)
Proteins 35 47 50
Small Subunit 30S 40S 28S
rRNA 16S (1542 nts) 18S (1900 nts) 12S (950 nts)
Proteins 20 33 30
S values are the sedimentation coefficient: a measure of the rate at which the particles are spun down in the ultracentrifuge. S values are not additive. nts = nucleotides.
3.05: Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a system of membrane-enclosed sacs and tubules in the cell. Their lumens are probably all interconnected, and their membranes are continuous with the outer membrane of the nuclear envelope. All the materials within the system are separated from the cytosol by a membrane. The endoplasmic reticulum is the site where the cell manufactures most of the membranes of the cell (plasma membrane, Golgi apparatus, lysosomes, nuclear envelope), lipids (including lipids for membranes, e.g., of the mitochondria, that are not made by the ER), and transmembrane proteins and secreted proteins. The ER comes in two versions: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER).
Rough Endoplasmic Reticulum (RER)
The RER is typically arranged as interconnecting stacks of disc-like sacs. The cytosolic surface of the RER is studded with ribosomes engaged in protein synthesis. As the messenger RNA is translated by the ribosome, the growing polypeptide chain is inserted into the membrane of the RER. Proteins destined to be secreted by the cell or shipped into the lumen of certain other organelles like the Golgi apparatus and lysosomes pass all the way through into the lumen of the RER. Transmembrane proteins destined for the plasma membrane or the membrane of those organelles are retained within the membrane of the RER.
In either case, the portion of the protein within the lumen of the RER is subject to extensive glycosylation (primarily N-linked) in Figure \(1\). The RER takes up a large proportion of the cytoplasm of cells specialized for protein synthesis such as cells secreting digestive enzymes (e.g. the pancreas cell above) and antibody-secreting plasma cells
Smooth Endoplasmic Reticulum (SER)
The SER differs from the RER in lacking attached ribosomes and usually being tubular rather than disc-like. A major function of the SER is the synthesis of lipids from which various cell membranes are made and like steroids, they are secreted from the cell. The SER represents only a small portion of the ER is most cells, e.g. serving as transport vesicles for the transport of protein to the Golgi apparatus. However, it is a prominent constituent of some cells, especially the cells of the adrenal cortex (which secrete steroid hormones), the cells of the liver (hepatocytes) where it synthesis lipids for secretion of lipoproteins, and the sarcoplasmic reticulum of muscle cells is SER. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.04%3A_Ribosomes.txt |
The Golgi apparatus is a cell structure mainly devoted to processing the proteins synthesized in the endoplasmic reticulum (ER). Some of these will eventually end up as integral membrane proteins embedded in the plasma membrane. Other proteins moving through the Golgi will end up in lysosomes or be secreted by exocytosis (e.g., digestive enzymes). The major processing activity is glycosylation: the adding of sugar molecules to form glycoproteins. In some cells, e.g., mucus-secreting cells in epithelia, the amount of carbohydrate so far exceeds that of the protein that the product is called a mucopolysaccharide (also known as a proteoglycan). In plant cells, the Golgi secretes the cell plate and cell wall.
Small peptides, e.g., some hormones and neurotransmitters, are typically too small to be synthesized directly by ribosomes. Instead, the ribosomes on the ER synthesize a large precursor protein that is later cut up into small peptide fragments as it traverses the Golgi. For example, proopiomelanocortin (POMC) is a polypeptide of 241 amino acids from which is cut ACTH, alpha and beta MSH, beta- endorphin, and others. POMC is cleaved to give rise to multiple peptide hormones.
• α-MSH produced by neurons in the arcuate nucleus has important roles in the regulation of appetite and sexual behavior, while α-MSH secreted from the intermediate lobe of the pituitary regulates the production of melanin.
• ACTH is a peptide hormone that regulates the secretion of glucocorticoids from the adrenal cortex.
• β-Endorphin and [Met]enkephalin are endogenous opioid peptides with widespread actions in the brain.
The Golgi consists of a stack of membrane-bounded cisternae located between the endoplasmic reticulum and the cell surface (Figure \(2\)). Many different enzymes (proteins) are present in the Golgi to perform its various synthetic activities. So there must be mechanisms to sort out the processed proteins and send them on to their destinations while reclaiming processing proteins (e.g., glycosylases) for reuse.
The Outbound Path (Membrane Fission)
Two mechanisms appear to participate in the migration of proteins from the endoplasmic reticulum through the Golgi apparatus.
• Mechanism 1: Transition vesicles pinch off from the surface of the endoplasmic reticulum carrying integral membrane proteins, soluble proteins awaiting processing, and processing enzymes. Pinching off requires that the vesicle be coated with COPII (Coat Protein II). The transition vesicles move toward the cis Golgi on microtubules. As they do so, their COPII coat is removed and they may fuse together forming larger vesicles; these fuse with the cis Golgi. Sugars are added to proteins in small packets so many glycoproteins have to undergo a large number of sequential steps of glycosylation, each requiring its own enzymes. These steps take place as shuttle vesicles carry the proteins from cis to medial to the trans Golgi compartments. At the outer face of the trans Golgi, vesicles pinch off and carry their completed products to their various destinations.
• Mechanism 2: In addition to the pinching off and fusing of shuttle vesicles, the cisternae of the Golgi actually migrate themselves, that is, the cis Golgi gradually migrates up the stack becoming a medial and finally a trans Golgi (Figure \(3\)).
The Inbound Path (Membrane Fusion)
The movement of cisternal contents through the stack means that essential processing enzymes are also moving away from their proper site of action. Using a variety of signals, the Golgi separates the products from the processing enzymes that made them and returns the enzymes back to the endoplasmic reticulum. This transport is also done by pinching off vesicles, but the inbound vesicles are coated with COPI (coat protein I). A vesicle recognize its correct target by involving pairs of complementary integral membrane proteins:
• v-SNAREs = "vesicle SNAREs" — on the vesicle surface
• t-SNAREs = "target SNAREs" — on the surface of the target membrane
v-SNAREs and t-SNAREs bind specifically to each other thanks to the complementary structure of their surface domains. Binding is followed by fusion of the two membranes (Figure \(4\)).
The Golgi is Not a Static Organelle
The Golgi breaks up and disappears at the onset of mitosis. By telophase of mitosis, the Golgi reappears. How it is recreated is still uncertain. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.06%3A_Golgi_Apparatus.txt |
Centrioles
Centrioles are built from a cylindrical array of 9 microtubules, each of which has attached to it 2 partial microtubules. Figure \(1\) is an electron micrograph showing a cross section of a centriole with its array of nine triplets of microtubules.
When a cell enters the cell cycle and passes through S phase, each centriole is duplicated. A "daughter" centriole grows out of the side of each parent ("mother") centriole. Thus centriole replication — like DNA replication (which is occurring at the same time) — is semiconservative.
• Functional microtubules grow out only from the "mother".
• When stem cells divide, one daughter cell remains a stem cell; the other goes on to differentiate. In two animal systems that have been examined (mouse glial cells and Drosophila male germline cells), the cell that receives the old ("mother") centriole remains a stem cell while the one that receives what had been the original "daughter" centriole goes on to differentiate. (You can read about these findings in Wang, X., et. al., Nature, 15 October 2009.)
Centrioles are a key feature of eukaryotic cells and presumably arose with the first eukaryotes. A few groups have since lost their centrioles including most fungi (but not the primitive chytrids), "higher" plants (but not the more primitive mosses, ferns, and cycads with their motile sperm) and animal eggs lose their centriole during meiosis and must have it restored by the sperm that fertilizes it
In nondividing cells, the mother centriole can attach to the inner side of the plasma membrane forming a basal body. In almost all types of cell, the basal body forms a nonmotile primary cilium. In cells with a flagellum, e.g. sperm, the flagellum develops from a single basal body. (While sperm cells have a basal body, eggs have none. So the sperm's basal body is absolutely essential for forming a centrosome which will form a spindle enabling the first division of the zygote to take place.)
In ciliated cells such as the columnar epithelial cells of the lungs and ciliated protozoans like the paramecium, many basal bodies form, each producing a beating cilium. Most of their centrioles are produced by repeated duplication of the daughter centriole of centrosome and are temporarily assembled in a special organelle called the deuterosome (not to be confused with deuterostome). Centrioles organize the centrosome in which they are embedded.
Centrosomes
The centrosome is located in the cytoplasm usually close to the nucleus. It consists of two centrioles — oriented at right angles to each other — embedded in a mass of amorphous material containing more than 100 different proteins.It is duplicated during S phase of the cell cycle. Just before mitosis, the two centrosomes move apart until they are on opposite sides of the nucleus. As mitosis proceeds, microtubules grow out from each centrosome with their plus ends growing toward the metaphase plate. These clusters of microtubules are called spindle fibers. Figure \(2\) shows microtubules growing in-vitro from an isolated centrosome. The centrosome was supplied with a mixture of alpha and beta tubulin monomers spontaneously assembled into microtubules only in the presence of centrosomes.
Spindle fibers have three destinations:
1. Some attach to one kinetochore of a dyad with those growing from the opposite centrosome binding to the other kinetochore of that dyad.
2. Some bind to the arms of the chromosomes.
3. Still others continue growing from the two centrosomes until they extend between each other in a region of overlap.
All three groups of spindle fibers participate in
• the assembly of the chromosomes at the metaphase plate at metaphase.
1. Microtubules attached to opposite sides of the dyad shrink or grow until they are of equal length.
2. Microtubules motors attached to the kinetochores move them
• toward the minus end of shrinking microtubules (a dynein);
• toward the plus end of lengthening microtubules (a kinesin).
3. The chromosome arms use a different kinesin to move to the metaphase plate.
• the separation of the chromosomes at anaphase.
1. The sister kinetochores separate and, carrying their attached chromatid,
2. move along the microtubules powered by minus-end motors, dyneins, while the microtubules themselves shorten (probably at both ends).
3. The overlapping spindle fibers move past each other (pushing the poles farther apart) powered by plus-end motors, the "bipolar" kinesins.
4. In this way the sister chromatids end up at opposite poles.
Functions of Centrosomes
In addition to their role in spindle formation, centrosomes play other important roles in animal cells:
• Formation of the network of microtubules that participate in making the cytoskeleton.
• Signaling that it is o.k. to proceed to cytokinesis. Destruction of both centrosomes with a laser beam prevents cytokinesis even if mitosis has been completed normally.
• Signaling that it is o.k. for the daughter cells to begin another round of the cell cycle; specifically to duplicate their chromosomes in the next S phase. Destruction of one centrosome with a laser beam still permits cytokinesis but the daughter cells fail to enter a new S phase.
• Segregating signaling molecules (e.g., mRNAs) so that they pass into only one of the two daughter cells produced by mitosis. In this way, the two daughter cells can enter different pathways of differentiation even though they contain identical genomes.
• In at least some developing neurons, the position of the centrosome establishes the point at which the axon will grow out.
Centrosomes and Cancer
Cancer cells often have more than the normal number of centrosomes. They also are aneuploid (have abnormal numbers of chromosomes), and considering the role of centrosomes in chromosome movement, it is tempting to think that the two phenomena are related. Mutations in the tumor suppressor gene p53 seem to predispose the cell to excess replication of the centrosomes. Chromosome movement in mitosis also involves polymerization and depolymerization of microtubules.
Because the hallmark of cancer cells is uncontrolled mitosis, both vincristine and Taxol are used as anticancer drugs. Vincristine, a drug found in the Madagascar periwinkle (a wildflower), binds to tubulin dimers preventing the assembly of microtubules. This halts cells in metaphase of mitosis and Taxol, a drug found in the bark of the Pacific yew, prevents depolymerization of the microtubules of the spindle fiber. This, in turn, stops chromosome movement, and thus prevents the completion of mitosis. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.07%3A_Centrosomes_and_Centrioles.txt |
A cell is composed of many different organelles and microbodies (or cytosomes) is a type of organelle that is found in the cells of plants, protozoa, and animals. Organelles in the microbody family include peroxisomes, glyoxysomes, glycosomes and hydrogenosomes.
Lysosomes
Lysosomes are roughly spherical bodies enclosed by a single membrane. They are manufactured by the Golgi apparatus (Figure \(1\)) and contain over 50 different kinds of hydrolytic enzymes including proteases, lipases, nucleases, and polysaccharidases. The pH within the lysosome is about pH 5, substantially less than that of the cytosol (~pH 7.2). All the enzymes in the lysosome work best at an acid pH, which reduces the risk of their digesting their own cell if they should escape from the lysosome.
Materials within the cell scheduled for digestion are first deposited within lysosomes. These may be:
• other organelles, such as mitochondria, that have ceased functioning properly and have been engulfed in autophagosomes
• food molecules or, in some cases, food particles taken into the cell by endocytosis
• foreign particles like bacteria that are engulfed by neutrophils
• antigens that are taken up by
• "professional" antigen-presenting cells like dendritic cells (by phagocytosis) and
• B cells (by binding to their antigen receptors (BCRs) followed by receptor-mediated endocytosis.
At one time, it was thought that lysosomes were responsible for killing cells scheduled to be removed from a tissue; for example, the resorption of its tail as the tadpole metamorphoses into a frog. This is incorrect. These examples of programmed cell death (PCD) or apoptosis take place by an entirely different mechanism.
In some cells, lysosomes have a secretory function — releasing their contents by exocytosis.
• Cytotoxic T cells (CTL) secrete perforin from lysosomes.
• Mast cells secrete some of their many mediators of inflammation from modified lysosomes.
• Melanocytes secrete melanin from modified lysosomes.
• The exocytosis of lysosomes provides the additional membrane needed to quickly seal wounds in the plasma membrane.
Lysosomal Storage Diseases
Lysosomal storage diseases are caused by the accumulation of macromolecules (proteins, polysaccharides, lipids) in the lysosomes because of a genetic failure to manufacture an enzyme needed for their breakdown. Neurons of the central nervous system are particularly susceptible to damage. Most of these diseases are caused by the inheritance of two defective alleles of the gene encoding one of the hydrolytic enzymes. Examples include:
• Tay-Sachs disease and Gaucher's disease — both caused by a failure to produce an enzyme needed to break down sphingolipids (fatty acid derivatives found in all cell membranes).
• Mucopolysaccharidosis I (MPS-I). Caused by a failure to synthesize an enzyme (α-L-iduronidase) needed to break down proteoglycans like heparan sulfate. In April 2003, the U.S. Food and Drug Administration approved a synthetic version of the enzyme, laronidase (Aldurazyme®), as a possible treatment. This enzyme (containing 628 amino acids) is manufactured by recombinant DNA technology.
However, one lysosomal storage disease, I-cell disease ("inclusion-cell disease"), is caused by a failure to "tag" (by phosphorylation) all the hydrolytic enzymes that are supposed to be transported from the Golgi apparatus to the lysosomes. Lacking the mannose 6-phosphate (M6P) tag, they are secreted from the cell instead. The result: all the macromolecules incorporated in lysosomes remain undegraded forming "inclusion bodies" in the cell.
Peroxisomes
Peroxisomes, also called microbodies, are about the size of lysosomes (0.5–1.5 µm) and like them are enclosed by a single membrane. They also resemble lysosomes in being filled with enzymes. However, peroxisomes bud off from the endoplasmic reticulum, not the Golgi apparatus (the source of lysosomes) and the enzymes and other proteins destined for peroxisomes are synthesized in the cytosol. Each contains a peroxisomal targeting signal (PTS) that binds to a receptor molecule that takes the protein into the peroxisome and then returns for another load. Two peroxisomal targeting signals have been identified: a 9-amino acid sequence at the N-terminal of the protein and a tripeptide at the C-terminal. Each has its own receptor to take it to the peroxisome.
Functions of the peroxisomes in the human liver inlcude:
• Breakdown (by oxidation) of excess fatty acids.
• Breakdown of hydrogen peroxide (H2O2), a potentially dangerous product of fatty-acid oxidation. It is catalyzed by the enzyme catalase.
• Participates in the synthesis of cholesterol. One of the enzymes involved, HMG-CoA reductase, is the target of the popular cholesterol-lowering "statins".
• Participates in the synthesis of bile acids.
• Participates in the synthesis of the lipids used to make myelin.
• Breakdown of excess purines (AMP, GMP) to uric acid.
Peroxisomes are also present in plant cells where they participate is such functions as symbiotic nitrogen fixation and photorespiration.
Peroxisome Disorders
A variety of rare inherited disorders of peroxisome function occur in humans. Most involve mutant versions of one or another of the enzymes found within peroxisomes. For example: X-linked adrenoleukodystrophy (X-ALD) results from a failure to metabolize fatty acids properly. One result is deterioration of the myelin sheaths of neurons. The disorder occurs in young boys because the gene is X-linked. An attempt to find an effective treatment was the subject of the 1992 film Lorenzo's Oil. A few diseases result from failure to produce functional peroxisomes. For example: Zellweger syndrome results from the inheritance of two mutant genes for one of the receptors (PXR1) needed to import proteins into the peroxisome. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.08%3A_Lysosomes_and_Peroxisomes.txt |
Proteins are the major building blocks of life. Eukaryotic cells synthesize proteins for thousands of different functions. Some examples:
• to build the components of the cytosol (e.g. microtubules, glycolytic enzymes);
• to build the receptors and other molecules exposed at the surface of the cell embedded in the plasma membrane;
• to supply some of the components of the mitochondria and (in plant cells) chloroplasts;
• proteins secreted from the cell to supply the needs of other cells and tissues (e.g. collagens to support cells, hormones to signal them).
All proteins are synthesized by ribosomes using the information encoded in molecules of messenger RNA (mRNA). This process is called translation and is described in Gene Translation: RNA -> Protein. Our task here is to explore the ways that these proteins are delivered to their proper destinations.
The various destinations for proteins occur in two major sets: (1) one set for those proteins synthesized by ribosomes that remain suspended in the cytosol, and (2) a second set for proteins synthesized by ribosomes that are attached to the membranes of the endoplasmic reticulum (ER) forming "rough endoplasmic reticulum" (RER). This electron micrograph (courtesy of Keith Porter) shows the RER in a bat pancreas cell. The clearer areas are the lumens. So the first decision that must be made as a ribosome begins to translate a mRNA into a polypeptide is whether to remain free in the cytosol or to bind to the ER.
Pathways Through the Endoplasmic Reticulum (ER)
The decision to enter the ER is dictated by the presence of a signal sequence on the growing polypeptide. The signal sequence consists of the first portion of the elongating polypeptide chain (so the signal sequence occurs at the amino terminal of the polypeptide). Typical signal sequences contain 15–30 amino acids. The precise amino acid sequence varies surprisingly from one protein to the next, but all signal sequences include many hydrophobic amino acids.
If a signal sequence is present,
• translation ceases after it has been synthesized.
• The signal sequence is recognized by and is bound by a signal recognition particle (SRP).
• The complex of ribosome with its nascent polypeptide and the SRP binds to a receptor on the surface (facing the cytosol) of the ER.
• The SRP leaves and translation recommences.
• The growing polypeptide chain is extruded through a pore in the ER membrane and into the lumen of the ER.
• The signal sequence is usually clipped off the polypeptide unless the polypeptide is to be retained as an integral membrane protein.
• Other proteins, called molecular chaperones, present in the lumen of the ER, bind the growing polypeptide chain and assist it to fold into its correct tertiary structure.
• Sugar residues may be added to the protein. The process is called glycosylation and often is essential for proper folding of the final product, a glycoprotein.
Note
The 1999 Nobel Prize in Physiology or Medicine was awarded to Dr. Günter Blobel for his discovery of the signal sequence and other intrinsic signals that enable proteins to reach their proper destinations.
Destinations of proteins synthesized within the ER
There are two options.
1. proteins glycosylated with residues of mannose-6-phosphate will leave the Golgi in transport vesicles that eventually fuse with lysosomes (path 2 in fig. 3.9.1).
2. proteins that do not receive this marker, leave in transport vesicles that eventually fuse with the plasma membrane (path 1 in fig. 3.9.1). These are integral membrane proteins that become exposed at the surface of the cell (forming receptors and the like) and proteins in solution within the transport vesicle. These are discharged from the cell. This secretory process is called exocytosis.
The Signal Recognition Particle (SRP)
The signal recognition particle in mammalian cells is made from:
• a single small (7S) molecule of RNA
• six different molecules of protein
It contains binding sites for the signal sequence, the ribosome, and an SRP receptor, also called the docking protein, on the cytosol face of the membranes of the ER.
Destinations of Proteins Synthesized By Free Ribosomes
Ribosomes synthesizing a protein without a signal sequence do not bind to the ER and continue synthesis until the polypeptide is completed. Chaperones are also present in the cytosol that help the protein assume its final three-dimensional configuration. Some of the important destinations for these proteins are:
• The cytosol itself. Such proteins as the enzymes of glycolysis, tubulins for making microtubules, and actin for making microfilaments are simply released from the ribosome and go to work.
• The nucleus. Many proteins — histones, transcription factors, and ribosomal proteins are notable examples — must move from the cytosol into the interior of the nucleus. They are targeted to the nucleus by their nuclear localization sequence, a sequence of 7–41 amino acids of which the basic amino acids lysine and arginine are characteristic members. These proteins are actively transported through pores in the nuclear envelope into the interior.
• Mitochondria. Although the mitochondrion has its own genome and protein-synthesizing machinery, most of the proteins used by mitochondria are Proteins destined for mitochondrion contain a characteristic signal sequence. This is recognized and bound by a chaperone called mitochondrial stimulation factor (MSF). MSF targets the protein to a receptor embedded in the outer membrane of the mitochondrion. Other factors and receptors shepherd proteins through the intermembrane space to the inner mitochondrial membrane (e.g. some proteins of the electron transport chain) and the matrix.
• encoded by genes in the nucleus of the cell
• synthesized in the cytosol
• must be imported into the mitochondrion.
• Chloroplasts. Chloroplasts, like mitochondria, have their own genome and their own protein-synthesizing machinery. But also like mitochondria, most of the proteins used in chloroplasts are encoded by genes in the nucleus of the cell, are synthesized by ribosomes in the cytosol, and must then be imported into the chloroplast. Proteins destined for chloroplasts are recognized by their characteristic transit sequence. Chaperones are also needed to get them to their final destination: stroma, thylakoid membrane, etc.
• Peroxisomes. Proteins destined for peroxisomes are synthesized with a peroxisomal targeting signal (PTS) that binds to a receptor molecule that takes the protein into the peroxisome and then returns for another load.
Two peroxisomal targeting signals have been identified:
Each has its own receptor to take it to the peroxisome.
• a 9-amino acid sequence at the N-terminal of the protein
• a tripeptide at the C-terminal. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.09%3A_Protein_Kinesis.txt |
Protein degradation is as essential to the cell as protein synthesis. For example, to supply amino acids for fresh protein synthesis, to remove excess enzymes, and to remove transcription factors that are no longer needed. There are two major intracellular devices in which damaged or unneeded proteins are broken down. They are lysosomes and proteasomes
Lysosomes
Lysosomes deal primarily with extracellular proteins, e.g., plasma proteins, that are taken into the cell, e.g., by endocytosis. They are cell-surface membrane proteins that are used in receptor-mediated endocytosis. The proteins (and other macromolecules) are engulfed by autophagosomes.
Proteasomes
Proteasomes deal primarily with endogenous proteins; that is, proteins that were synthesized within the cell such as transcription factors, cyclins (which must be destroyed to prepare for the next step in the cell cycle) and proteins encoded by viruses and other intracellular pathogens. Proteasomes also address proteins that are folded incorrectly because of translation errors, or they are encoded by faulty genes or they have been damaged by other molecules in the cytosol. Structure of the Proteasome in the Core Particle (CP) and the Regulatory Particle (RP) as shown in Figure 3.10.2.
The core particle is made of 2 copies of each of 14 different proteins that are assembled in groups of 7 forming a ring. The 4 rings are stacked on each other (like 4 doughnuts) along a common center (Figure 3.10.3).
There are two identical RPs, one at each end of the core particle. Each is made of 19 different proteins (none of them the same as those in the CP). 6 of these are ATPases and some of the subunits have sites that recognize the protein ubiquitin. Ubiquitin is a small protein (76 amino acids) that is conserved throughout all the kingdoms of life (Figure 3.10.4) and is virtually identical in sequence whether in bacteria, yeast, or mammals. Ubiquitin is used by all these creatures to target proteins for destruction (hence the name based off of the "ubiquitous" term).
The Process
Proteins destined for destruction are conjugated to a molecule of ubiquitin which binds to the terminal amino group of a lysine residue. Additional molecules of ubiquitin bind to the first forming a chain and this complex then binds to ubiquitin-recognizing site(s) on the regulatory particle. The protein is unfolded by the ATPases using the energy of ATP, which is translocated into the central cavity of the core particle. Several active sites on the inner surface of the two middle "doughnuts" break various specific peptide bonds of the chain, which produces a set of peptides averaging about 8 amino acids long. These leave the core particle by an unknown route where they may be further broken down into individual amino acids by peptidases in the cytosol. However, in mammals, they may be incorporated in a class I histocompatibility molecule to be presented to the immune system as a potential antigen. The regulatory particle releases the ubiquitins for reuse
Antigen Processing by Proteasomes
In mammals, activation of the immune system leads to the release of the cytokine interferon-gamma. This causes three of the subunits in the core particle to be replaced by substitute subunits; the peptides generated in this altered proteasome are picked up by TAP (= transporter associated with antigen processing) proteins and transported from the cytosol into the endoplasmic reticulum where each enters the groove at the surface of a class I histocompatibility molecule. This complex then moves through the Golgi apparatus and is inserted in the plasma membrane where it can be "recognized" by CD8+ T cells. It is probably no coincidence that the genes encoding the three substitute core particle subunits, TAP and all the MHC (major histocompatibility complex) molecules are clustered together on the same chromosome (#6 in humans). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.10%3A_The_Proteasome.txt |
Cells contain elaborate arrays of protein fibers that serve such functions as establishing cell shape, providing mechanical strength, and locomotion. These fibers participate in chromosome separation in mitosis and meiosis and intracellular transport of organelles. The cytoskeleton is made up of three kinds of protein filaments: Actin filaments (also called microfilaments), Intermediate filaments, and Microtubules.
Actin Filaments
Monomers of the protein actin polymerize to form long, thin fibers. These are about 8 nm in diameter and, being the thinnest of the cytoskeletal filaments, are also called microfilaments (in skeletal muscle fibers they are called "thin" filaments). Some functions of actin filaments are:
• form a band just beneath the plasma membrane that
• provides mechanical strength to the cell
• links transmembrane proteins (e.g., cell surface receptors) to cytoplasmic proteins
• pinches dividing animal cells apart during cytokinesis
• generate cytoplasmic streaming in some cells
• generate locomotion in cells such as white blood cells and the amoeba
• interact with myosin ("thick") filaments in skeletal muscle fibers to provide the force of muscular contraction
Intermediate Filaments
These cytoplasmic fibers average 10 nm in diameter (and thus are "intermediate" in size between actin filaments (8 nm) and microtubules (25 nm) (as well as of the thick filaments of skeletal muscle fibers). There are several types of intermediate filament, each constructed from one or more proteins characteristic of it.
• keratins are found in epithelial cells and also form hair and nails;
• nuclear lamins form a meshwork that stabilizes the inner membrane of the nuclear envelope;
• neurofilaments strengthen the long axons of neurons;
• vimentins provide mechanical strength to muscle (and other) cells.
Despite their chemical diversity, intermediate filaments play similar roles in the cell: providing a supporting framework within the cell. For example, the nucleus in epithelial cells is held within the cell by a basketlike network of intermediate filaments made of keratins.
Different kinds of epithelia use different keratins to build their intermediate filaments. Over 20 different kinds of keratins have been found, although each kind of epithelial cell may use no more than 2 of them. Up to 85% of the dry weight of squamous epithelial cells can consist of keratins.
Microtubules
Microtubules are straight, hollow cylinders whose wall is made up of a ring of 13 "protofilaments" and have a diameter of about 25 nm. They are variable in length but can grow 1000 times as long as they are wide. They are built by the assembly of dimers of alpha tubulin and beta tubulin. Microtubules are found in both animal and plant cells. In plant cells, microtubules are created at many sites scattered through the cell. In animal cells, the microtubules originate at the centrosome. The attached end is called the minus end; the other end is the plus end.
Microtubules grow at the plus end by the polymerization of tubulin dimers (powered by the hydrolysis of GTP), and shrink by the release of tubulin dimers (depolymerization) at the same end. They participate in a wide variety of cell activities. Most involve motion. The motion is provided by protein "motors" that use the energy of ATP to move along the microtubule.
Microtubule motors
There are two major groups of microtubule motors kinesins (most of these move toward the plus end of the microtubules) and dyneins (which move toward the minus end)
Examples
• The rapid transport of organelles, like vesicles and mitochondria, along the axons of neurons takes place along microtubules with their plus ends pointed toward the end of the axon. The motors are kinesins.
• The migration of chromosomes in mitosis and meiosis takes place on microtubules that make up the spindle fibers. Both kinesins and dyneins are used as motors
Cilia and Flagella
Cilia and flagella are built from arrays of microtubules. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.11%3A_The_Cytoskeleton.txt |
These whiplike appendages extend from the surface of many types of eukaryotic cells. If there are many of them, they are called cilia. If only one, or a few, they are flagella. Flagella also tend to be longer than cilia but are otherwise similar in construction.
Function of Cilia and Flagella
Cilia and flagella move liquid past the surface of the cell. For single cells, such as sperm, this enables them to swim. For cells anchored in a tissue, like the epithelial cells lining our air passages, this moves liquid over the surface of the cell (e.g., driving particle-laden mucus toward the throat). Both cilia and flagella consist of:
• a cylindrical array of 9 filaments consisting of:
• a complete microtubule (the A-microtubule) extending into the tip of the cilium. When a cilium is being disassembled, protein complexes move down from the tip of the cilium traveling along A-microtubules.
• a partial microtubule (the B-microtubule) that doesn't extend as far into the tip. When the cilium is growing, its protein components move up toward the tip of the cilium traveling along B-microtubules.
• cross-bridges of the motor protein dynein that extend from the complete microtubule of one filament to the partial microtubule of the adjacent filament.
• a pair of single microtubules running up through the center of the bundle, producing the "9+2" arrangement.
• The entire assembly is sheathed in a membrane that is an extension of the plasma membrane.
This electron micrograph (Figure \(1\)) shows a cilium in cross section. Each cilium (and flagellum) grows out from, and remains attached to, a basal body embedded in the cytoplasm. Basal bodies are identical to centrioles and are, in fact, produced by them. For example, one of the centrioles in developing sperm cells — after it has completed its role in the distribution of chromosomes during meiosis — becomes a basal body and produces the flagellum
The Sliding-Filament Model of Bending
Motion of cilia and flagella is created by the microtubules sliding past one another. This requires motor molecules of dynein, which link adjacent microtubules together, and the energy of ATP. Dynein powers the sliding of the microtubules against one another — first on one side, then on the other. The bending of cilia (and flagella) has many parallels to the contraction of skeletal muscle fibers.
Testing the Model
Remember, the partial microtubules do not extend as far into the tip as the complete microtubules. So if a slice is made a short distance back from the tip:
• A straight cilium should show the complete pattern (center of diagram).
• In a bent cilium, approximately half the filaments on the upper side should be retracted because of the greater arc on the convex side. So the partial microtubules would disappear being drawn below the plane of the slice. As seen here, bending to the left causes the partial microtubules 4, 5, 6, 7, and 8 to disappear.
• When the cilium bends the other way, the partial microtubules on the opposite side disappear while they reappear on what is now the lower or concave side.
• Electron micrographs (made by Peter Satir) have verified this model precisely.
Other Parallels
There are other parallels between the sliding filaments of skeletal muscle and the sliding microtubules of cilia. Both are powered by ATP. Both motors —dynein in cilia, myosin in skeletal muscle — are ATPases and both are regulated by calcium ions.
The Primary Cilium
Motile, "9+2", cilia are found only on certain cells in the vertebrate body, e.g., the epithelia lining the airways. But almost every cell in mammals has — or had — a single primary cilium. The primary cilium grows out of the older of the two centrioles that the cell inherited following mitosis. The primary cilium does not beat because it lacks the central pair of microtubules; that is, it is "9+0". Where functions have been identified, they all involve sensory reception. Some examples are as follows:
• Mechanoreceptors: A primary cilium extends from the apical surface of the epithelial cells lining the kidney tubules and monitors the flow of fluid through the tubules. Inherited defects in the formation of these cilia cause polycystic kidney disease.
• Chemoreceptors: We detect odors by receptors on the primary cilium of olfactory neurons. Many types of cells detect extracellular signaling molecules, e.g., nutrients, growth factors, hormones, with receptors localized on their primary cilium. These signals may be transduced into the nucleus where they alter gene expression.
• Photoreceptors: The outer segment of the rods in the vertebrate retina is also derived from a primary cilium. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.12%3A_Cilia.txt |
The development of a fertilized egg into a newborn child requires an average of 41 rounds of mitosis ($2^{41} = 2.2 \times 10^{12}$). During this period, the cells produced by mitosis enter different pathways of differentiation; some becoming blood cells, some muscle cells, and so on. There are more than 100 visibly-distinguishable kinds of differentiated cells in the vertebrate animal. These are organized into tissues; the tissues into organs. Groups of organs make up the various systems — digestive, excretory, etc. — of the body (Figure $1$ and Table $1$).
The actual number of differentiated cell types is surely much larger than 100. All lymphocytes, for example, look alike but actually represent a variety of different functional types, e.g., B cells, T cells of various subsets. The neurons of the central nervous system must exist in a thousand or more different functional types, each representing the result of a particular pathway of differentiation. This page will give a brief introduction to the major types of animal tissues.
Table $1$: Classification of Animal Tissues
Epithelial Tissues Linings and Coverings Simple Epithelia
Classifying or Naming Epithelia Stratified Epithelia
Glands Exocrine Glands
Endocrine Glands
Connective Tissues Fluid Connective Tissues Lymph
Blood
Connective Tissues Proper Loose Connective Tissues
Loose Connective Tissues and Inflammation
Dense Connective Tissues
Supportive Connective Tissues Osseous Tissue
Cartilage
Muscle Tissues Non-striated Smooth muscle
Striated Skeletal Muscle
Cardiac Muscle
Nervous Tissues Neurons Multipolar Neurons in CNS
Nerves Nerves of the PNS
Receptors Miessner's and Pacinian Corpuscles
Epithelial
Epithelial tissue is made of closely-packed cells arranged in flat sheets. Epithelia form the surface of the skin, line the various cavities and tubes of the body, and cover the internal organs. Epithelia that form the interface between the internal and external environments. Skin as well as the lining of the mouth and nasal cavity. These are derived from ectoderm. Inner lining of the GI tract, lungs, urinary bladder, exocrine glands, vagina and more. These are derived from endoderm.
The apical surface of these epithelial cells is exposed to the "external environment", the lumen of the organ or the air.
• Mesothelia. These are derived from mesoderm.
• pleura — the outer covering of the lungs and the inner lining of the thoracic (chest) cavity.
• peritoneum — the outer covering of all the abdominal organs and the inner lining of the abdominal cavity.
• pericardium — the outer lining of the heart.
• Endothelia. These are derived from mesoderm. The inner lining of the heart, all blood and lymphatic vessels.
The basolateral surface of all epithelia is exposed to the internal environment - extracellular fluid (ECF). The entire sheet of epithelial cells is attached to a layer of extracellular matrix that is called the basement membrane or, better (because it is not a membrane in the biological sense), the basal lamina.
The function of epithelia always reflects the fact that they are boundaries between masses of cells and a cavity or space. Some examples include:
• The epithelium of the skin protects the underlying tissues from mechanical damage, ultraviolet light, dehydration and invasion by bacteria
• The columnar epithelium of the intestine secretes digestive enzymes into the intestine and absorbs the products of digestion from it.
• An epithelium also lines our air passages and the alveoli of the lungs. It secretes mucus which keeps it from drying out and traps inhaled dust particles. Most of its cells have cilia on their apical surface that propel the mucus with its load of foreign matter back up to the throat.
Muscle
Three kinds of muscle are found in vertebrates. Skeletal muscle is made of long fibers whose contraction provides the force of locomotion and other voluntary body movements. Smooth muscle lines the walls of the hollow structures of the body, such as the intestine, urinary bladder, uterus, and blood vessels. Its contraction, which is involuntary, reduces the size of these hollow organs. The heart is made of cardiac muscle.
Connective
The cells of connective tissue are embedded in a great amount of extracellular material. This matrix is secreted by the cells. It consists of protein fibers embedded in an amorphous mixture of protein-polysaccharide ("proteoglycan") molecules. Supporting connective tissue gives strength, support, and protection to the soft parts of the body.
• cartilage. Example: the outer ear
• bone. The matrix of bone contains collagen fibers and mineral deposits. The most abundant mineral is calcium phosphate, although magnesium, carbonate, and fluoride ions are also present.
Dense connective tissue is often called fibrous connective tissue and include Tendons and Ligaments. Tendons connect muscle to bone with a The matrix is principally Type I collagen, and the fibers are all oriented parallel to each other. Tendons are strong but not elastic. Ligaments attach one bone to another and contain both collagen and also the protein elastin. Elastin permits ligaments to be stretched.
Loose connective tissue is distributed throughout the body. It serves as a packing and binding material for most of our organs. Sheets of loose connective tissue that bind muscles and other structures together are called fascia. Collagen, elastin, and other proteins are found in the matrix of loose connective tissue. Both dense and loose connective tissue are derived from cells called fibroblasts, which secrete the extracellular matrix.
Adipose Tissue
Adipose tissue is "fat". There are two kinds found in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT). The WAT in which the cells, called adipocytes, have become almost filled with oil, which is confined within a single membrane-enclosed droplet. Virtually all of the "fat" in adult humans is white adipose tissue. BAT in which the adipocytes contain many small droplets of oil as well as many mitochondria. White adipose tissue and brown adipose tissue differ in function as well as cellular structure. These differences are described elsehwhere.
New adipocytes in white adipose tissue are formed throughout life from a pool of precursor cells. These are needed to replace those that die (after an average life span of 10 years). Whether the total number of these adipocytes increases in humans becoming fatter as adults is still uncertain. If not, why do so many of us get fatter as we age? Because of the increased size of individual adipocytes as they become filled with oil. The adipocytes of white adipose tissue secrete several hormones, including leptin and adiponectin.
Nerve
Nerve tissue is composed of nerve cells called neurons and glial cells. Neurons are specialized for the conduction of nerve impulses; a typical neuron consists of a cell body which contains the nucleus; a number of short fibers — dendrites — extending from the cell body and a single long fiber, the axon. The nerve impulse is conducted along the axon. The tips of axons meet other neurons at junctions called synapses, muscles (called neuromuscular junctions) and glands.
Glia
Glial cells surround neurons. Once thought to be simply support for neurons (glia = glue), they turn out to serve several important functions. There are three types:
• Schwann cells. These produce the myelin sheath that surrounds many axons in the peripheral nervous system.
• Oligodendrocytes. These produce the myelin sheath that surrounds many axons in the central nervous system (brain and spinal cord).
• Astrocytes. These, often star-shaped cells are clustered around synapses and the nodes of Ranvier where they perform a variety of functions such as:
• modulating the activity of neurons
• supplying neurons with materials (e.g. glucose and lactate) as well as some signaling molecules
• regulating the flow of blood to their region of the brain. It is primarily the metabolic activity of astrocytes that is being measured in brain imaging by positron-emission tomography (PET) and functional magnetic resonance imaging (fMRI).
• pruning away (by phagocytosis) weak synapses
In addition, the central nervous system contains many microglia — mobile cells (macrophages) that respond to damage (e.g., from an infection) by engulfing cell debris and secreting inflammatory cytokines like tumor necrosis factor (TNF-α) and interleukin-1 (IL-1). Microglia are also active in the healthy brain, at least in young mice where, like astrocytes, they engulf synapses thus reducing the number of synapses in the developing brain.
Blood
The bone marrow is the source of all the cells of the blood. These include red blood cells (RBCs or erythrocytes), five kinds of white blood cells (WBCs or leukocytes), and platelets (or thrombocytes). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.13%3A_Animal_Tissues.txt |
Two kinds of adipose tissue are found in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT). White adipose tissue is the most common and is the fat that so many of us complain of acquiring. Brown adipose tissue is present in small mammals (e.g., mice) and in newborn humans. Most of it disappears in adult humans.The cells in both types of fat are called adipocytes although they differ in origin, structure, and function in the two types of tissue.
Table 3.14.1: Two classifications of Adipocytes
WAT Adipocytes BAT Adipocytes
a narrow rim of cytoplasm with its nucleus
pressed near the margin of the cell
surrounding
Cytoplasm throughout the cell with a central nucleus and
a single large membrane-enclosed lipid droplet many small lipid droplets
few mitochondria many mitochondria (providing the brown color)
modest blood supply rich blood supply
serves as a depot of stored energy function is to generate heat
New adipocytes in white adipose tissue are formed throughout life from a pool of precursor cells. These are needed to replace those that die (after an average life span of 10 years). Whether the total number of these adipocytes increases in humans becoming fatter as adults is still uncertain. If not, why do so many of us get fatter as we age? Because of the increased size of individual adipocytes as they become filled with oil.
The adipocytes of white adipose tissue secrete several hormones, including leptin, asprosin (which, during fasting, causes a rapid release of blood sugar (glucose) from the liver) and adiponectin. In addition to serving as a major source of energy reserves, white adipose tissue also provides some mechanical protection and insulation to the body. Obesity is the excessive accumulation of white adipose tissue.
Brown adipose tissue provides a vital source of heat to maintain body temperature in small mammals (with their high surface to volume ratio) and infants (who usually cannot shiver when they are cold).
Adipose tissue activation mechanism
Brown adipose tissue is activated by the following mechanism when the body temperature drops:
• Cold activates the sympathetic nervous system.
• Noradrenaline is released by the postganglionic neurons.
• The noradrenaline binds to G-protein-coupled receptors on the adipocytes surface.
• The second messenger cAMP is generated and moves into the nucleus where
• it binds to the promoter of the gene encoding an enzyme that converts thyroxine (T4) to triiodothyronine (T3).
• T3 enters the nucleus and bind to the promoter of the gene encoding uncoupling protein1 (UCP1).
• UCP1 inserts into the inner membrane of the mitochondria where
• it allows the protons that have been pumped out into the intermembrane space by the electron transport chain
• to return to the matrix without having to pass through ATP synthase.
So instead of cellular respiration (of fatty acids and glucose) generating ATP, it generates heat.
WAT can Acquire the Properties of BAT
In mice and perhaps in humans, skeletal muscles that have undergone a period of vigorous exercise secrete a protein hormone called irisin. Irisin acts on white adipose tissue to give it the properties of brown adipose tissue:
• an increase in the number of mitochondria and lipid droplets;
• a marked increase in the synthesis of UCP1;
• an increase in the rate of cellular respiration but with the energy released as heat rather than fueling the synthesis of ATP.
These brown-like fat cells derived from white fat cells have been called "beige" or "brite" cells. Lean adult humans have deposits of beige cells in the neck and upper chest regions. When they are exposed to cold, their beige cells are activated. Obese people have few or no beige cells. Probably their layers of white adipose tissue provide such good insulation that they are in less danger of heat loss. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.14%3A_Adipose_Tissue.txt |
In many animal tissues (e.g., connective tissue), each cell is separated from the next by an extracellular coating or matrix. However, in some tissues (e.g., epithelia), the plasma membranes of adjacent cells are pressed together. Four kinds of junctions occur in vertebrates:
1. Tight junctions
2. Adherens junctions
3. Gap junctions
4. Desmosomes
In many plant tissues, it turns out that the plasma membrane of each cell is continuous with that of the adjacent cells. The membranes contact each other through openings in the cell wall called Plasmodesmata.
Tight Junctions
Epithelia are sheets of cells that provide the interface between masses of cells and a cavity or space (a lumen). The portion of the cell exposed to the lumen is called its apical surface. The rest of the cell (i.e., its sides and base) make up the basolateral surface. Tight junctions seal adjacent epithelial cells in a narrow band just beneath their apical surface. They consist of a network of claudins and other proteins. Tight junctions perform two vital functions:
1. They limit the passage of molecules and ions through the space between cells. So most materials must actually enter the cells (by diffusion or active transport) in order to pass through the tissue. This pathway provides tighter control over what substances are allowed through.
2. They block the movement of integral membrane proteins (red and green ovals) between the apical and basolateral surfaces of the cell. Thus the special functions of each surface, for example receptor-mediated endocytosis at the apical surface and exocytosis at the basolateral surface can be preserved.
The Epithelia of the Human Lung
A report by Vermeer, et al., in the 20 March 2003 issue of Nature provides a striking example of the role of tight junctions. The epithelial cells of the human lung express
• a growth stimulant, called heregulin, on their apical surface and
• its receptors on the basolateral surface. (These receptors also respond to epidermal growth factor (EGF), and mutant versions have been implicated in cancer.
As long as the sheet of cells is intact, there is no stimulation of its receptors by heregulin thanks to the seal provided by tight junctions. However, if the sheet of cells becomes broken, heregulin can reach its receptors. The result is an autocrine stimulation of mitosis leading to healing of the wound. Several disorders of the lung the chronic bronchitis of cigarette smokers, asthma, cystic fibrosis increase the permeability of the airway epithelium. The resulting opportunity for autocrine stimulation may account for the proliferation (piling up) of the epithelial cells characteristic of these disorders.
Adherens Junctions
Adherens junctions provide strong mechanical attachments between adjacent cells. They hold cardiac muscle cells tightly together as the heart expands and contracts and hold epithelial cells together. Adherens junctions seem to be responsible for contact inhibition and some are present in narrow bands connecting adjacent cells. Others are present in discrete patches holding the cells together . Adherens junctions are built from cadherins that are transmembrane proteins (Figure 3.15.2; red labeled) whose extracellular segments bind to each other and whose intracellular segments bind to catenins (Figure 3.15.2; yellow labeled), which are connected to actin filaments
Human synthesize some 80 different types of cadherins. In most cases, a cell expressing one type of cadherin will only form adherens junctions with another cell expressing the same type. This is because molecules of cadherin tend to form homodimers, not heterodimers. Inherited mutations in a gene encoding a cadherin can cause stomach cancer. Mutations in a gene (APC), whose protein normally interacts with catenins, are a common cause of colon cancer. Loss of functioning adherens junctions may accelerate the edema associated with sepsis and tumor metastasis.
Gap Junctions
Gap junctions are intercellular channels some 1.5–2 nm in diameter. These permit the free passage between the cells of ions and small molecules (up to a molecular weight of about 1000 daltons). They are cylinders constructed from 6 copies of transmembrane proteins called connexins. Because ions can flow through them, gap junctions permit changes in membrane potential to pass from cell to cell.
Examples of gap junctions include:
• The action potential in heart (cardiac) muscle flows from cell to cell through the heart providing the rhythmic contraction of the heartbeat.
• At some so-called electrical synapses in the brain, gap junctions permit the arrival of an action potential at the synaptic terminals to be transmitted across to the postsynaptic cell without the delay needed for release of a neurotransmitter.
• As the time of birth approaches, gap junctions between the smooth muscle cells of the uterus enable coordinated, powerful contractions to begin.
Several inherited disorders of humans such as certain congenital heart defects and certain cases of congenital deafness have been found to be caused by mutant genes encoding connexins.
Desmosomes
Desmosomes are localized patches that hold two cells tightly together. They are common in epithelia (e.g., the skin). Desmosomes are attached to intermediate filaments of keratin in the cytoplasm. Pemphigus is an autoimmune disease in which the patient has developed antibodies against proteins (cadherins) in desmosomes. The loosening of the adhesion between adjacent epithelial cells causes blistering. Carcinomas are cancers of epithelia. However, the cells of carcinomas no longer have desmosomes. This may partially account for their ability to metastasize.
Hemidesmosomes
These are similar to desmosomes but attach epithelial cells to the basal lamina ("basement membrane") instead of to each other. Pemphigoid is an autoimmune disease in which the patient develops antibodies against proteins (integrins) in hemidesmosomes. This, too, causes severe blistering of epithelia.
Plasmodesmata
Although each plant cell is encased in a boxlike cell wall, it turns out that communication between cells is just as easy, if not easier, than between animal cells (Figure 3.15.4). Fine strands of cytoplasm, called plasmodesmata, extend through pores in the cell wall connecting the cytoplasm of each cell with that of its neighbors.
Plasmodesmata provide an easy route for the movement of ions, small molecules like sugars and amino acids, and even macromolecules like RNA and proteins, between cells. The larger molecules pass through with the aid of actin filaments. Plasmodesmata are sheathed by a plasma membrane that is simply an extension of the plasma membrane of the adjoining cells. This raises the intriguing question of whether a plant tissue is really made up of separate cells or is, instead, a syncytium: a single, multinucleated cell distributed throughout hundreds of tiny compartments. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.15%3A_Junctions_between_Cells.txt |
Plant cells are eukaryotic and have many of the structures found in animal cells.
• Plasma membrane
• Nucleus and nucleolus
• Mitochondria
• Ribosomes
• Endoplasmic reticulum
• Golgi apparatus
• Peroxisomes (the crystal in the electron micrograph is enclosed within a peroxisome)
• Microtubules
Plant cells differ from animal cells as they lack centioles and intermediate filament; they also do not have plastids and a cell wall and large vacuoles.
Plastids
Chloroplasts are the most familiar plastids. They are usually disk-shaped and about 5-8 µm in diameter and 2-4 µm thick. A typical plant cell has 20-40 of them. Chloroplasts are green because they contain chlorophylls — the pigments that harvest the light used in photosynthesis. Chloroplasts are probably the descendants of cyanobacteria that took up residence in the ancestor of the plants. Plant cells that are not engaged in photosynthesis also have plastids that serve other functions such as storing starch (when they are called leucoplasts) and storing the carotenoids that give flowers and fruits their color (when they are called chromoplasts)
The Cell Wall
The rigid cell wall of plants is made of fibrils of cellulose embedded in a matrix of several other kinds of polymers such as pectin and lignin. The linear nature of cellulose molecules and the many opportunities for side-to-side intermolecular hydrogen bonding provide just what one would want to build long and stiff fibrils.
• Primary cell walls: The cell walls of parenchyma and meristems are uniform in thickness and are primary cell walls. Although each cell appears encased within a box, in fact primary cell walls are perforated permitting plasmodesmata to connect adjacent cells.
• Secondary cell walls: The cells of sclerenchyma, collenchyma and xylem have secondary deposits of lignified cellulose which provide mechanical strength to the tissue.
Vacuoles
Vacuoles are enclosed by a single membrane. Young plant cells often contain many small vacuoles, but as the cells mature, these unite to form a large central vacuole. Vacuoles serve several functions such as:
• storing foods (e.g., proteins in seeds)
• storing wastes
• storing malic acid in CAM plants
• storing various ions (e.g., calcium, sodium, iron) which, among other functions, helps to
• maintain turgor in the cell.
Plant cells avoid bursting in hypotonic surroundings by their strong cell walls. These allow the build-up of turgor within the cell. Loss of turgor causes wilting.
Plasmolysis
When a freshwater (or terrestrial) plant is placed in sea water, its cells quickly lose turgor and the plant wilts. This is because sea water is hypertonic to the cytoplasm. As water diffuses from the cytoplasm into the sea water, the cells shrink — drawing their plasma membrane away from the cell wall.
3.17: Chloroplasts
A typical plant cell (e.g., in the palisade layer of a leaf) might contain as many as 50 chloroplasts.
The chloroplast is made up of 3 types of membrane:
• A smooth outer membrane which is freely permeable to molecules.
• A smooth inner membrane which contains many transporters: integral membrane proteins that regulate the passage in an out of the chloroplast of
• small molecules like sugars
• proteins synthesized in the cytoplasm of the cell but used within the chloroplast
• A system of thylakoid membranes
Thylakoids
The thylakoid membranes enclose a lumen: a system of vesicles (that may all be interconnected). At various places within the chloroplast these are stacked in arrays called grana (resembling a stack of coins). Four types of protein assemblies are embedded in the thylakoid membranes: These carry out the so-called light reactions of photosynthesis including:
1. Photosystem I which includes chlorophyll and carotenoid molecules
2. Photosystem II which also contains chlorophyll and carotenoid molecules
3. Cytochromes b and f
4. ATP synthase
The thylakoid membranes are surrounded by a fluid stroma, which contains all the enzymes, e.g., RUBISCO, needed to carry out the "dark" reactions of photosynthesis; that is, the conversion of CO2 into organic molecules like glucose. A number of identical molecules of DNA, each of which carries the complete chloroplast genome. The genes encode some — but not all of the molecules needed for chloroplast function. The others are
• transcribed from genes in the nucleus of the cell
• translated in the cytoplasm and
• transported into the chloroplast.
The electron micrograph in Figure 3.17.3 shows the inner surface of a thylakoid membrane. Each particle may represent one photosystem II complex. In the functioning chloroplast, these particles may not be as highly ordered as seen here.
3.18: Chlorophylls and Carotenoids
Chlorophylls
Two types of chlorophyll are found in plants and the green algae: chlorophyll a and chlorophyll b. The difference in their structures is shown in the above figure (red disks).
In the chloroplast, both types are associated with integral membrane proteins in the thylakoid membrane. Note the system of alternating single and double bonds (white bars) that run around the porphyrin ring. Although I am forced to draw the single and double bonds in fixed positions, actually the "extra" electrons responsible for the double bonds are not fixed between any particular pair of carbon atoms but instead are free to migrate around the ring. This property enables these molecules to absorb light. Both chlorophylls absorb light most strongly in the red and violet parts of the spectrum. Green light is absorbed poorly. Thus when white light shines on chlorophyll-containing structures like leaves, green light is transmitted and reflected and the structures appear green.
Carotenoids
Chloroplasts also contain carotenoids. These are also pigments with colors ranging from red to yellow. Carotenoids absorb light most strongly in the blue portion of the spectrum. They thus enable the chloroplast to trap a larger fraction of the radiant energy falling on it. Carotenoids are often the major pigments in flowers and fruits. The red of a ripe tomato and the orange of a carrot are produced by their carotenoids. In leaves, the carotenoids are usually masked by the chlorophylls. In the autumn, as the quantity of chlorophyll in the leaf declines, the carotenoids become visible and produce the yellows and reds of autumn foliage.
Figure 3.18.2 shows the structure of beta-carotene, one of the most abundant carotenoids. Note again the system of alternating single and double bonds that in this molecule runs along the hydrocarbon chain that connects the two benzene rings. As in chlorophyll, the electrons of the double bonds actually migrate though the chain and also make this molecule an efficient absorber of light. Many animals use ingested beta-carotene as a precursor for the synthesis of vitamin A. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.16%3A_Plant_Cells.txt |
A mature vascular plant (any plant other than mosses and liverworts), contains several types of differentiated cells. These are grouped together in tissues. Some tissues contain only one type of cell. Others consist of several cells.
Meristematic
The main function of meristematic tissue is mitosis. The cells are small, thin-walled, with no central vacuole and no specialized features. Meristematic tissue is located in the apical meristems at the growing points of roots and stems, the secondary meristems (lateral buds) at the nodes of stems (where branching occurs), and meristematic tissue, called the cambium, that is found within mature stems and roots. The cells produced in the meristems soon become differentiated into one or another of several types.
Protective
Protective tissue covers the surface of leaves and the living cells of roots and stems. Its cells are flattened with their top and bottom surfaces parallel. The upper and lower epidermis of the leaf are examples of protective tissue.
Parenchyma
The cells of parenchyma are large, thin-walled, and usually have a large central vacuole. They are often partially separated from each other and are usually stuffed with plastids. In areas not exposed to light, colorless plastids predominate and food storage is the main function. The cells of the white potato are parenchyma cells. Where light is present, e.g., in leaves, chloroplasts predominate and photosynthesis is the main function.
Sclerenchyma
The walls of these cells are very thick and built up in a uniform layer around the entire margin of the cell. Often, the cell dies after its cell wall is fully formed. Sclerenchyma cells are usually found associated with other cells types and give them mechanical support. Sclerenchyma is found in stems and also in leaf veins. Sclerenchyma also makes up the hard outer covering of seeds and nuts.
Collenchyma
Collenchyma cells have thick walls that are especially thick at their corners. These cells provide mechanical support for the plant. They are most often found in areas that are growing rapidly and need to be strengthened. The petiole ("stalk") of leaves is usually reinforced with collenchyma.
Xylem
Xylem conducts water and dissolved minerals from the roots to all the other parts of the plant. In angiosperms, most of the water travels in the xylem vessels. These are thick-walled tubes that can extend vertically through several feet of xylem tissue. Their diameter may be as large as 0.7 mm. Their walls are thickened with secondary deposits of cellulose and are usually further strengthened by impregnation with lignin. The secondary walls of the xylem vessels are deposited in spirals and rings and are usually perforated by pits.
Xylem vessels arise from individual cylindrical cells oriented end to end. At maturity the end walls of these cells dissolve away, and the cytoplasmic contents die. The result is the xylem vessel, a continuous nonliving duct. Xylem also contains tracheids. These are individual cells tapered at each end so the tapered end of one cell overlaps that of the adjacent cell. Like xylem vessels, they have thick, lignified walls and, at maturity, no cytoplasm. Their walls are perforated so that water can flow from one tracheid to the next. The xylem of ferns and conifers contains only tracheids. In woody plants, the older xylem ceases to participate in water transport and simply serves to give strength to the trunk. Wood is xylem. When counting the annual rings of a tree, one is counting rings of xylem.
Phloem
The main components of phloem are sieve elements and companion cells. Sieve elements are so-named because their end walls are perforated. This allows cytoplasmic connections between vertically-stacked cells. The result is a sieve tube that conducts the products of photosynthesis — sugars and amino acids — from the place where they are manufactured (a "source"), e.g., leaves, to the places ("sinks") where they are consumed or stored; such as
• roots
• growing tips of stems and leaves
• flowers
• fruits, tubers, corms, etc.
Sieve elements have no nucleus and only a sparse collection of other organelles. They depend on the adjacent companion cells for many functions.
Companion cells move sugars, amino acids and a variety of macromolecules into and out of the sieve elements. In "source" tissue, such as a leaf, the companion cells use transmembrane proteins to take up — by active transport — sugars and other organic molecules from the cells manufacturing them. Water follows by osmosis. These materials then move into adjacent sieve elements through plasmodesmata. The pressure created by osmosis drives the flow of materials through the sieve tubes.
In "sink" tissue, the sugars and other organic molecules leave the sieve elements through plasmodesmata connecting the sieve elements to their companion cells and then pass on to the cells of their destination. Again, water follows by osmosis where it may
• leave the plant by transpiration or
• increase the volume of the cells or
• move into the xylem for recycling through the plant | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.19%3A_Plant_Tissues.txt |
Apoptosis is a process of programmed cell death that occurs in multicellular organisms. There are two ways in which cells die: (1) They are killed by injurious agents or (2) they are induced to commit suicide.
Death by injury
Cells that are damaged by injury, such as by mechanical damage or exposure to toxic chemicals undergo a characteristic series of changes. They (and their organelles like mitochondria) swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted). The cell contents leak out, leading to inflammation of surrounding tissues.
Death by Suicide
Cells that are induced to commit suicide:
• shrink
• develop bubble-like blebs on their surface
• have the chromatin (DNA and protein) in their nucleus degraded
• have their mitochondria break down with the release of cytochrome c
• break into small, membrane-wrapped, fragments
• release (at least in mammalian cells) ATP and UTP
• These nucleotides bind to receptors on wandering phagocytic cells like macrophages and dendritic cells and attract them to the dying cells (a "find-me" signal")
• The phospholipid phosphatidylserine, which is normally hidden in the inner layer of the plasma membrane, is exposed on the surface
• This "eat me" signal is bound by other receptors on the phagocytes which then engulf the cell fragments
• The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)
The pattern of events in death by suicide is so orderly that the process is often called programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death is also called apoptosis. (There is no consensus yet on how to pronounce it; some say APE oh TOE sis; some say uh POP tuh sis.)
Why should a cell commit suicide?
There are two different reasons.
1. Programmed cell death is as needed for proper development as mitosis is.
Examples:
• The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis.
• The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them.
• The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation occurs by apoptosis.
• The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis.
• The elimination of T cells that might otherwise mount an autoimmune attack on the body occurs by apoptosis.
• During the pupal stage of insects that undergo complete metamorphosis, most of the cells of the larva die by apoptosis thus providing the nutrients for the development of the structures of the adult.
2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism.
Examples:
Cells infected with viruses
One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis and some viruses mount countermeasures to thwart it.
Cells of the immune system
As cell-mediated immune responses wane, the effector cells must be removed to prevent them from attacking body constituents. CTLs induce apoptosis in each other and even in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.
Cells with DNA damage
Damage to its genome can cause a cell
• to disrupt proper embryonic development leading to birth defects
• to become cancerous.
Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis. Is it any wonder that mutations in the p53 gene, producing a defective protein, are so often found in cancer cells (that represent a lethal threat to the organism if permitted to live)?
Cancer cells
Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells.
What makes a cell decide to commit suicide?
The balance between the withdrawal of positive signals; that is, signals needed for continued survival, and the receipt of negative signals.
Withdrawal of positive signals
The continued survival of most cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals: growth factors for neurons and Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes
Receipt of negative signals
• increased levels of oxidants within the cell
• damage to DNA by these oxidants or other agents like ultraviolet light, X-rays and chemotherapeutic drugs
• accumulation of proteins that failed to fold properly into their proper tertiary structure
• molecules that bind to specific receptors on the cell surface and signal the cell to begin the apoptosis program. These death activators include:
• Tumor necrosis factor-alpha (TNF-α) that binds to the TNF receptor
• Lymphotoxin (also known as TNF-β) that also binds to the TNF receptor
• Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called CD95)
The Mechanisms of Apoptosis
There are 3 different mechanisms by which a cell commits suicide by apoptosis.
1. Generated by signals arising within the cell
2. Triggered by death activators binding to receptors at the cell surface:
• TNF-α
• Lymphotoxin
• Fas ligand (FasL)
3. Triggered by dangerous reactive oxygen species
Apoptosis triggered by internal signals
• In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on their surface. Bcl-2 inhibits apoptosis.
• Internal damage to the cell
• causes a related protein, Bax, to migrate to the surface of the mitochondrion where it inhibits the protective effect of Bcl-2 and inserts itself into the outer mitochondrial membrane punching holes in it and causing
• cytochrome c to leak out.
• The released cytochrome c binds to the protein Apaf-1 ("apoptotic protease activating factor-1").
• Using the energy provided by ATP, these complexes aggregate to form apoptosomes. The apoptosomes bind to and activate caspase-9. Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get their name because they cleave proteins — mostly each other — at aspartic acid (Asp) residues.
• Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7).
• The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity (rather like that in blood clotting and complement activation) which leads to
• digestion of structural proteins in the cytoplasm,
• degradation of chromosomal DNA
• phagocytosis of the cell
Apoptosis triggered by external signals
• Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell
• Binding of the complementary death activator (FasL and TNF respectively) transmits a signal to the cytoplasm that leads to the activation of caspase 8
• Caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to phagocytosis of the cell.
• Example: When cytotoxic T cells recognize (bind to) their target,
• They produce more FasL at their surface.
• This binds with the Fas on the surface of the target cell leading to its death by apoptosis.
The early steps in apoptosis are reversible — at least in C. elegans. In some cases, final destruction of the cell is guaranteed only with its engulfment by a phagocyte.
Apoptosis-Inducing Factor (AIF)
Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases. Apoptosis-inducing factor (AIF) is a protein that is normally located in the intermembrane space of mitochondria. When the cell receives a signal telling it that it is time to die, AIF is released from the mitochondria (like the release of cytochrome c in the first pathway). It migrates into the nucleus and binds to DNA, which triggers the destruction of the DNA and cell death.
Apoptosis and Cancer
Some viruses associated with cancers use tricks to prevent apoptosis of the cells they have transformed.
• Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53.
• Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas
• produces a protein similar to Bcl-2
• produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).
Even cancer cells produced without the participation of viruses may have tricks to avoid apoptosis.
• Some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production.
• Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.
• Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells by the mechanism shown above.
• Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).
Apoptosis in the Immune System
The immune response to a foreign invader involves the proliferation of lymphocytes — T and/or B cells. When their job is done, they must be removed leaving only a small population of memory cells. This is done by apoptosis. Very rarely humans are encountered with genetic defects in apoptosis. The most common one is a mutation in the gene for Fas, but mutations in the gene for FasL or even one of the caspases are occasionally seen. In all cases, the genetic problem produces autoimmune lymphoproliferative syndrome or ALPS.
Features
• an accumulation of lymphocytes in the lymph nodes and spleen greatly enlarging them.
• the appearance of clones that are autoreactive; that is, attack "self" components producing such autoimmune disorders as
• hemolytic anemia
• thrombocytopenia
• the appearance of lymphoma — a cancerous clone of lymphocytes.
In most patients with ALPS, the mutation is present in the germline; that is, every cell in their body carries it. In a few cases, however, the mutation is somatic; that is, has occurred in a precursor cell in the bone marrow. These later patients are genetic mosaics — with some lymphocytes that undergo apoptosis normally and others that do not. The latter tend to out-compete the former and grow to become the major population in the lymph nodes and blood.
Apoptosis and Organ Transplants
For many years it has been known that certain parts of the body such as the anterior chamber of the eye and the testes are "immunologically privileged sites". Antigens within these sites fail to elicit an immune response. It turns out that cells in these sites differ from the other cells of the body in that they express high levels of FasL at all times. Thus antigen-reactive T cells, which express Fas, would be killed when they enter these sites. (This is the reverse of the mechanism described above.)
This finding raises the possibility of a new way of preventing graft rejection. If at least some of the cells on a transplanted kidney, liver, heart, etc. could be made to express high levels of FasL, that might protect the graft from attack by the T cells of the host's cell-mediated immune system. If so, then the present need for treatment with immunosuppressive drugs for the rest of the transplant recipient's life would be reduced or eliminated. So far, the results in animal experiments have been mixed. Allografts engineered to express FasL have shown increased survival for kidneys, but not for hearts or islets of Langerhans.
Apoptosis in Plants
Plants, too, can turn on a system of programmed cell death; for example, in an attempt to halt the spread of virus infection. The mechanism differs from that in animals although it, too, involves a protease that — like caspases — cleaves other proteins at Asp (and Asn) residues. Activation of this enzyme destroys the central vacuole, which is followed by disintegration of the rest of the cell. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.20%3A_Apoptosis.txt |
Collagens are insoluble, extracellular glycoproteins found in all animals. They are the most abundant proteins in the human body and are essential structural components of all connective tissues such as cartilage, bone, tendons, ligaments, fascia, skin. Gelatin is solubilized collagen. 29 types of collagens have been found in humans and the major ones are:
• Type I. The chief component of tendons, ligaments, and bones.
• Type II. Represents more than 50% of the protein in cartilage and is the major component of the vitreous body of the eye. It is also used to build the notochord of vertebrate embryos.
• Type III. Strengthens the walls of hollow structures like arteries, the intestine, and the uterus.
• Type IV. Forms the basal lamina of epithelia. (The basal lamina is often called the basement membrane, but is not related to lipid bilayer membranes.) A meshwork of Type IV collagens provides the filter for the blood capillaries and the glomeruli of the kidneys.
The other 25 types are probably equally important, but they are much less abundant.
Collagens Structure
The basic unit of collagens is a polypeptide consisting of the repeating sequence
n
where X is often proline (Pro) and Y is often hydroxyproline (proline to which an -OH group is added after synthesis of the polypeptide). The resulting molecule twists into an elongated, left-handed helix (NOT an alpha helix).
A single collagen molecule, tropocollagen, is used to make up larger collagen aggregates, such as fibrils. It is approximately 300 nm long and 1.5 nm in diameter, and it is made up of three polypeptide strands (called alpha peptides, see step 2), each of which has the conformation of a left-handed helix (Figure 3.21.1). These three left-handed helices are twisted together into a right-handed triple helix or "super helix", a cooperative quaternary structure stabilized by many hydrogen bonds.
When synthesized, the N- terminal and C- terminal of the polypeptide have globular domains, which keep the molecule soluble. As they pass through the endoplasmic reticulum (ER) and Golgi apparatus,
• The molecules are glycosylated.
• Hydroxyl (-OH) groups are added to the "Y" amino acid.
• S-S bonds link three chains covalently.
• The three molecules twist together to form a triple helix.
In some collagens (e.g., Type II), the three molecules are identical (the product of a single gene). In other collagens (e.g., Type I), two polypeptides of one kind (gene product) assemble with a second, quite similar, polypeptide, that is the product of a second gene.
When the triple helix is secreted from the cell (usually by a fibroblast), the globular ends are cleaved off. The resulting linear, insoluble molecules assemble into collagen fibers. They assemble in a staggered pattern that gives rise to the striations seen in the above electron micrograph. Type IV collagens are an exception; they form a meshwork rather than striated fibers.
Inherited Diseases Caused by Mutant Collagen Genes
• Brittle-bone disease ("osteogenesis imperfecta"): Caused by a mutation in one or the other of the two genes whose products are used to make Type I collagen. Like all the inherited collagen diseases, this one is inherited as a dominant trait. The reason: even though one collagen allele is normal, the assembly of the normal gene product with the mutant product produces defective collagen fibers. Bone marrow stem cells from patients with this disease have had their mutant gene knocked out by gene targeting and gained the ability to make good collagen and bone (when the cells were placed in immunodeficient mice). So this disease now seems to be a promising candidate for gene therapy.
• Forms of dwarfism: Caused by mutations in a Type II collagen gene.
• Rubber-man syndrome: Caused by a mutations in a Type I collagen gene. The subject has hyperextensible joints, tendons, and skin. (This inherited disorder represents one type of Ehlers-Danlos syndrome.)
• Ehlers-Danlos syndrome: It is caused by mutations in the gene for Type III collagen. Patients are at risk of rupture of major arteries or the intestine.
• Alport's syndrome: Most cases involve mutations in the gene on the X chromosome for one of the chains of Type IV collagen. So it shows the typical pattern of X-linked inheritance. Other cases are caused by two mutant autosomal genes for another of the Type IV collagen chains. Patients usually have damage to their glomeruli, leading to blood in their urine and, often, become deaf as well.
• Herniated discs between the vertebrae?: A study in Finland has found that some families that share a tendency to develop herniated discs (leading to sciatica) have an inherited point mutation in the gene (COL9A2) encoding one of the alpha chains in collagen IX. This collagen is one component of the extracellular matrix in the padding (discs) between our vertebrae.
Other Collagen Diseases
• Scurvy: Caused by a deficiency of vitamin C. The sufferer is unable to add hydroxyl (-OH) groups to proline to convert it into hydroxyproline.
• Goodpasture's Syndrome: Some people develop antibodies against an epitope on their Type IV collagen molecules. These attach to the basal lamina of epithelial cells and "fix" complement which damages the basal lamina. So Goodpasture's syndrome is an example of an autoimmune disorder.
The basal lamina of the lung epithelia and the glomeruli of the kidney are especially likely to be affected. In this photo (courtesy of Dr. Frank J. Dixon), a fluorescent antibody against human IgG shows the autoantibodies coating the basement membranes of the glomeruli in a patient with Goodpasture's syndrome.
3.22: Chromatophores
Chromatophores are irregularly shaped, pigment-containing cells. If the pigment is melanin, they are called melanophores. Chromatophores are common in crustaceans, cephalopod mollusks, lizards and amphibians, and some fishes.
Chromatophores are often used for camouflage. Figure 3.22.1 shows a winter flounder resting on a checkerboard pattern. The chromatophores of cephalopods change size (expand and contract) as a result of activity of muscle fibers and the motor neurons that terminate at them. In crustaceans and amphibians, the chromatophores have a fixed shape. Color change comes about through the dispersal (darkening) or aggregation (lightening) of granules within the cell. This is under hormonal control. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.21%3A_Collagens.txt |
Transport Across Cell Membranes
All cells acquire the molecules and ions they need from their surrounding extracellular fluid (ECF). There is an unceasing traffic of molecules and ions in and out of the cell through its plasma membrane (Examples: glucose, \(Na^+\), \(Ca^{2+}\)). In eukaryotic cells, there is also transport in and out of membrane-bounded intracellular compartments such as the nucleus, endoplasmic reticulum, and mitochondria (Examples: proteins, mRNA, \(Ca^{2+}\), and ATP).
The following problems can occur during transport:
1. Relative concentrations
Molecules and ions move spontaneously down their concentration gradient (i.e., from a region of higher to a region of lower concentration) by diffusion. Molecules and ions can be moved against their concentration gradient, but this process, called active transport, requires the expenditure of energy (usually from ATP).
2. Lipid bilayers are impermeable to most essential molecules and ions.
The lipid bilayer is permeable to water molecules and a few other small, uncharged, molecules like oxygen (O2) and carbon dioxide (CO2). These diffuse freely in and out of the cell. The diffusion of water through the plasma membrane is of such importance to the cell that it is given a special name - osmosis. Lipid bilayers are not permeable to ions such as K+, Na+, Ca2+ (called cations because when subjected to an electric field they migrate toward the cathode [the negatively-charged electrode]) and Cl-, HCO3- (called anions because they migrate toward the anode [the positively-charged electrode]). They are also not permeable to small hydrophilic molecules like glucose and macromolecules like proteins and RNA. The cells solve the problem of transporting ions and small molecules across their membranes with the help of the following two mechanisms:
• Facilitated diffusion: Transmembrane proteins create a water-filled pore through which ions and some small hydrophilic molecules can pass by diffusion. The channels can be opened (or closed) according to the needs of the cell.
• Active transport: Transmembrane proteins, called transporters, use the energy of ATP to force ions or small molecules through the membrane against their concentration gradient.
Facilitated Diffusion of Ions
Facilitated diffusion of ions takes place through proteins, or assemblies of proteins, embedded in the plasma membrane. These transmembrane proteins form a water-filled channel through which the ion can pass down its concentration gradient. The transmembrane channels that permit facilitated diffusion can be opened or closed. They are said to be "gated"; some types of gated ion channels:
• ligand-gated
• mechanically-gated
• voltage-gated
• light-gated
Ligand-gated ion channels
Many ion channels open or close in response to binding a small signaling molecule or "ligand". Some ion channels are gated by extracellular ligands; some by intracellular ligands. In both cases, the ligand is not the substance that is transported when the channel opens.
External ligands
External ligands (shown here in green) bind to a site on the extracellular side of the channel.
Examples:
• Acetylcholine (ACh). The binding of the neurotransmitter acetylcholine at certain synapses opens channels that admit Na+ and initiate a nerve impulse or muscle contraction.
• Gamma amino butyric acid (GABA). Binding of GABA at certain synapses — designated GABAA — in the central nervous system admits Cl- ions into the cell and inhibits the creation of a nerve impulse
Internal ligands
Internal ligands bind to a site on the channel protein exposed to the cytosol. Examples:
• "Second messengers", like cyclic AMP (cAMP) and cyclic GMP (cGMP), regulate channels involved in the initiation of impulses in neurons responding to odors and light respectively.
• ATP is needed to open the channel that allows chloride (Cl-) and bicarbonate (HCO3-) ions out of the cell. This channel is defective in patients with cystic fibrosis. Although the energy liberated by the hydrolysis of ATP is needed to open the channel, this is not an example of active transport; the ions diffuse through the open channel following their concentration gradient.
Mechanically-gated ion channels
Sound waves bending the cilia-like projections on the hair cells of the inner ear open up ion channels leading to the creation of nerve impulses that the brain interprets as sound. Mechanical deformation of the cells of stretch receptors opens ion channels leading to the creation of nerve impulses.
Voltage-gated ion channels
In so-called "excitable" cells like neurons and muscle cells, some channels open or close in response to changes in the charge (measured in volts) across the plasma membrane. For example, as an impulse passes down a neuron, the reduction in the voltage opens sodium channels in the adjacent portion of the membrane. This allows the influx of \(Na^+\) into the neuron and thus the continuation of the nerve impulse. Some 7000 sodium ions pass through each channel during the brief period (about 1 millisecond) that it remains open. This was learned by use of the patch clamp technique.
The Patch Clamp Technique
The properties of ion channels can be studied by means of the patch clamp technique. A very fine pipette (with an opening of about 0.5 µm) is pressed against the plasma membrane of either an intact cell or the plasma membrane can be pulled away from the cell and the preparation placed in a test solution of desired composition. Current flow through a single ion channel can then be measured.
Such measurements reveal that each channel is either fully open or fully closed; that is, facilitated diffusion through a single channel is "all-or-none". This technique has provided so much valuable information about ion channels that its inventors, Erwin Neher and Bert Sakmann, were awarded a Nobel Prize in 1991.
Facilitated Diffusion of Molecules
Some small, hydrophilic organic molecules, like sugars, can pass through cell membranes by facilitated diffusion. Once again, the process requires transmembrane proteins. In some cases, these — like ion channels — form water-filled pores that enable the molecule to pass in (or out) of the membrane following its concentration gradient.
Example:
Maltoporin. This homotrimer in the outer membrane of E. coli forms pores that allow the disaccharide maltose and a few related molecules to diffuse into the cell.
Example: The plasma membrane of human red blood cells contain transmembrane proteins that permit the diffusion of glucose from the blood into the cell.
Note that in all cases of facilitated diffusion through channels, the channels are selective; that is, the structure of the protein admits only certain types of molecules through. Whether all cases of facilitated diffusion of small molecules use channels is yet to be proven. Perhaps some molecules are passed through the membrane by a conformational change in the shape of the transmembrane protein when it binds the molecule to be transported.
In either case, the interaction between the molecule being transported and its transporter resembles in many ways the interaction between an enzyme and its substrate.
Active Transport
Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires a transmembrane protein (usually a complex of them) called a transporter and energy. The source of this energy is ATP.
The energy of ATP may be used directly or indirectly.
• Direct Active Transport. Some transporters bind ATP directly and use the energy of its hydrolysis to drive active transport.
• Indirect Active Transport. Other transporters use the energy already stored in the gradient of a directly-pumped ion. Direct active transport of the ion establishes a concentration gradient. When this is relieved by facilitated diffusion, the energy released can be harnessed to the pumping of some other ion or molecule.
Direct Active Transport
The Na+/K+ ATPase
The cytosol of animal cells contains a concentration of potassium ions (K+) as much as 20 times higher than that in the extracellular fluid. Conversely, the extracellular fluid contains a concentration of sodium ions (Na+) as much as 10 times greater than that within the cell. These concentration gradients are established by the active transport of both ions. And, in fact, the same transporter, called the Na+/K+ ATPase, does both jobs. It uses the energy from the hydrolysis of ATP to
• actively transport 3 Na+ ions out of the cell
• for each 2 K+ ions pumped into the cell.
This accomplishes several vital functions:
• It helps establish a net charge across the plasma membrane with the interior of the cell being negatively charged with respect to the exterior. This resting potential prepares nerve and muscle cells for the propagation of action potentials leading to nerve impulses and muscle contraction.
• The accumulation of sodium ions outside of the cell draws water out of the cell and thus enables it to maintain osmotic balance (otherwise it would swell and burst from the inward diffusion of water).
• The gradient of sodium ions is harnessed to provide the energy to run several types of indirect pumps.
The crucial roles of the Na+/K+ ATPase are reflected in the fact that almost one-third of all the energy generated by the mitochondria in animal cells is used just to run this pump.
The H+/K+ ATPase
The parietal cells of your stomach use this pump to secrete gastric juice. These cells transport protons (H+) from a concentration of about 4 x 10-8 M within the cell to a concentration of about 0.15 M in the gastric juice (giving it a pH close to 1). Small wonder that parietal cells are stuffed with mitochondria and uses huge amounts of ATP as they carry out this three-million fold concentration of protons.
The Ca2+ ATPases
A Ca2+ ATPase is located in the plasma membrane of all eukaryotic cells. It uses the energy provided by one molecule of ATP to pump one Ca2+ ion out of the cell. The activity of these pumps helps to maintain the ~20,000-fold concentration gradient of Ca2+ between the cytosol (~ 100 nM) and the ECF (~ 20 mM). In resting skeletal muscle, there is a much higher concentration of calcium ions (Ca2+) in the sarcoplasmic reticulum than in the cytosol. Activation of the muscle fiber allows some of this Ca2+ to pass by facilitated diffusion into the cytosol where it triggers contraction.
After contraction, this Ca2+ is pumped back into the sarcoplasmic reticulum. This is done by another Ca2+ ATPase that uses the energy from each molecule of ATP to pump 2 Ca2+ ions.
Pumps 1. - 3. are designated P-type ion transporters because they use the same basic mechanism: a conformational change in the proteins as they are reversibly phosphorylated by ATP. And all three pumps can be made to run backward. That is, if the pumped ions are allowed to diffuse back through the membrane complex, ATP can be synthesized from ADP and inorganic phosphate.
ABC Transporters
ABC ("ATP-Binding Cassette") transporters are transmembrane proteins that
• expose a ligand-binding domain at one surface and a
• ATP-binding domain at the other surface.
The ligand-binding domain is usually restricted to a single type of molecule.
The ATP bound to its domain provides the energy to pump the ligand across the membrane.
The human genome contains 48 genes for ABC transporters. Some examples:
• CFTR — the cystic fibrosis transmembrane conductance regulator
• TAP, the transporter associated with antigen processing
• The transporter that liver cells use to pump the salts of bile acids out into the bile.
• ABC transporters that pump chemotherapeutic drugs out of cancer cells thus reducing their effectiveness.
ABC transporters must have evolved early in the history of life. The ATP-binding domains in archaea, eubacteria, and eukaryotes all share a homologous structure, the ATP-binding "cassette".
Indirect Active Transport
Indirect active transport uses the downhill flow of an ion to pump some other molecule or ion against its gradient. The driving ion is usually sodium (Na+) with its gradient established by the Na+/K+ ATPase.
Symport Pumps
In this type of indirect active transport, the driving ion (Na+) and the pumped molecule pass through the membrane pump in the same direction. Examples:
• The Na+/glucose transporter. This transmembrane protein allows sodium ions and glucose to enter the cell together. The sodium ions flow down their concentration gradient while the glucose molecules are pumped up theirs. Later the sodium is pumped back out of the cell by the Na+/K+ ATPase. The Na+/glucose transporter is used to actively transport glucose out of the intestine and also out of the kidney tubules and back into the blood.
• All the amino acids can be actively transported, for example out of the kidney tubules and into the blood, by sodium-driven symport pumps.
• Sodium-driven symport pumps also return neurotransmitters to the presynaptic neuron.
• The Na+/iodide transporter. This symporter pumps iodide ions into the cells of the thyroid gland (for the manufacture of thyroxine) and also into the cells of the mammary gland (to supply the baby's need for iodide).
• The permease encoded by the lac operon of E. coli that transports lactose into the cell.
Antiport Pumps
In antiport pumps, the driving ion (again, usually sodium) diffuses through the pump in one direction providing the energy for the active transport of some other molecule or ion in the opposite direction. Example:
Ca2+ ions are pumped out of cells by sodium-driven antiport pumps. Antiport pumps in the vacuole of some plants harness the outward facilitated diffusion of protons (themselves pumped into the vacuole by a H+ ATPase) to the active inward transport of sodium ions. This sodium/proton antiport pump enables the plant to sequester sodium ions in its vacuole. Transgenic tomato plants that overexpress this sodium/proton antiport pump are able to thrive in saline soils too salty for conventional tomatoes. Antiport pumps to the active inward transport of nitrate ions (NO3)
Some inherited ion-channel diseases
A growing number of human diseases have been discovered to be caused by inherited mutations in genes encoding channels.
Examples:
• Chloride-channel diseases
• cystic fibrosis
• inherited tendency to kidney stones (caused by a different kind of chloride channel than the one involved in cystic fibrosis)
• Potassium-channel diseases
• the majority of cases of long QT syndrome, an inherited disorder of the heartbeat
• a rare, inherited tendency to epileptic seizures in the newborn
• several types of inherited deafness
• Sodium-channel diseases
• inherited tendency to certain types of muscle spasms
• Liddle's syndrome. Inadequate sodium transport out of the kidneys, because of a mutant sodium channel, leads to elevated osmotic pressure of the blood and resulting hypertension (high blood pressure)
Osmosis
Osmosis is a special term used for the diffusion of water through cell membranes. Although water is a polar molecule, it is able to pass through the lipid bilayer of the plasma membrane. Aquaporins — transmembrane proteins that form hydrophilic channels — greatly accelerate the process, but even without these, water is still able to get through. Water passes by diffusion from a region of higher to a region of lower concentration. Note that this refers to the concentration of water, NOT the concentration of any solutes present in the water. Water is never transported actively; that is, it never moves against its concentration gradient. However, the concentration of water can be altered by the active transport of solutes and in this way the movement of water in and out of the cell can be controlled. Example: the reabsorption of water from the kidney tubules back into the blood depends on the water following behind the active transport of \(Na^+\).
• Hypotonic solutions: If the concentration of water in the medium surrounding a cell is greater than that of the cytosol, the medium is said to be hypotonic. Water enters the cell by osmosis. A red blood cell placed in a hypotonic solution (e.g., pure water) bursts immediately ("hemolysis") from the influx of water. Plant cells and bacterial cells avoid bursting in hypotonic surroundings by their strong cell walls. These allow the buildup of turgor within the cell. When the turgor pressure equals the osmotic pressure, osmosis ceases.
• Isotonic solutions: When red blood cells are placed in a 0.9% salt solution, they neither gain nor lose water by osmosis. Such a solution is said to be isotonic. The extracellular fluid (ECF) of mammalian cells is isotonic to their cytoplasm. This balance must be actively maintained because of the large number of organic molecules dissolved in the cytosol but not present in the ECF. These organic molecules exert an osmotic effect that, if not compensated for, would cause the cell to take in so much water that it would swell and might even burst. This fate is avoided by pumping sodium ions out of the cell with the Na+/K+ ATPase.
• Hypertonic solutions: If red cells are placed in sea water (about 3% salt), they lose water by osmosis and the cells shrivel up. Sea water is hypertonic to their cytosol. Similarly, if a plant tissue is placed in sea water, the cell contents shrink away from the rigid cell wall. This is called plasmolysis. Sea water is also hypertonic to the ECF of most marine vertebrates. To avoid fatal dehydration, these animals (e.g., bony fishes like the cod) must continuously drink sea water and then desalt it by pumping ions out of their gills by active transport.
Marine birds, which may pass long periods of time away from fresh water, and sea turtles use a similar device. They, too, drink salt water to take care of their water needs and use metabolic energy to desalt it. In the herring gull, shown here, the salt is extracted by two glands in the head and released (in a very concentrated solution — it is saltier than the blood) to the outside through the nostrils. Marine snakes use a similar desalting mechanism. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.23%3A_Diffusion_Active_Transport_and_Membrane_Channels.txt |
In endocytosis, the cell engulfs some of its extracellular fluid (ECF) including material dissolved or suspended in it. A portion of the plasma membrane is invaginated, coated with molecules of the protein clathrin, and pinched off forming a membrane-bounded vesicle called an endosome (Figure \(1\)).
Phagocytosis
Phagocytosis ("cell eating") results in the ingestion of particulate matter (e.g., bacteria) from the ECF. The endosome is so large that it is called a phagosome or vacuole. Phagocytosis occurs only in certain specialized cells (e.g., neutrophils, macrophages, the amoeba) and occurs sporadically.
This electron micrograph shows a guinea pig phagocyte ingesting polystyrene beads. Several beads are already enclosed in phagosomes while the others are in the process of being engulfed. In due course, phagosomes deliver their contents to lysosomes. The membranes of the two organelles fuse. Once inside the lysosome, the contents of the phagosome, e.g. ingested bacteria, are destroyed by the degradative enzymes of the lysosome.
Games parasites play
Phagocytic cells, like macrophages and neutrophils, are an early line of defense against invading bacteria. However, some bacteria have evolved mechanisms to avoid destruction even after they have been engulfed by phagocytes.
Examples:
• Salmonella enterica is a bacterium that causes food poisoning in humans. Once engulfed by phagocytosis, it secretes a protein that prevents the fusion of its phagosome with a lysosome.
• Mycobacteria (e.g., the tubercle bacillus that causes tuberculosis) use a different trick.
• When the phagosome is first pinched off from the plasma membrane, it is coated with a protein called "TACO" (for tryptophan-aspartate-containing coat protein).
• This must be removed before the phagosome can fuse with a lysosome.
• Mycobacteria taken into a phagosome are able, in some way, to keep the TACO coat from being removed.
• Thus there is no fusion with lysosomes and the mycobacteria can continue to live in this protected intracellular location.
Some intracellular parasites exploit receptor-mediated endocytosis to sneak their way into their host cell. They have evolved surface molecules that serve as decoy ligands for receptors on the target cell surface. Binding to these receptors tricks the cell into engulfing the parasite.
Examples:
• Epstein-Barr Virus (EBV). This virus causes mononucleosis and is a contributing factor in the development of Burkitt's lymphoma, a cancer of B lymphocytes. It binds to a receptor present on the surface of B cells.
• Influenza virus. The hemagglutinin on the surface of the virus binds to carbohydrate on the surface of the target cell tricking the cell into engulfing it.
• Listeria monocytogenes. This food-borne bacterium can be dangerous to people with defective immune systems as well as to pregnant women and their newborn babies. It has two kinds of surface molecules each a ligand for a different receptor on the target cell surface.
• Streptococcus pneumoniae. Epithelial cells like those in the nasopharynx have receptors that are responsible for transporting IgA and IgM antibodies from the blood to the apical surface of the cell. The pneumococcus piggybacks on this receptor on its return trip into the cell. This is the organism that led to the discovery that genes are DNA.
Pinocytosis
In pinocytosis ("cell drinking"), the drop engulfed is relatively small (Figure \(1\)). Pinocytosis occurs in almost all cells and continuously.
This electron micrograph shows a section of the wall of a capillary (the smallest of the blood vessels). On the right is the interior or lumen of the capillary. In the middle is the tissue space separating the capillary wall from a nearby muscle cell (left). The small inpocketings of the plasma membrane are clearly seen (arrows). Most of these are open to the tissue space but some can also be seen on the other side of the cell apparently engulfing fluid from within the capillary. Perhaps most of the vesicles facing the tissue space are not taking up material by endocytosis but are instead discharging material by exocytosis. If so, the pinocytic vesicles formed at one surface of the cell may, after being detached, move through the cell to the opposite surface and there discharge their contents. In this way materials can be moved efficiently through the capillary wall. The pinocytosis vesicles in this image represent a subtype called caveolae. In addition to their function in endo- and exocytosis, they also can serve as a reservoir of plasma membrane. When a cell expands (e.g., by osmotic swelling) or is stretched, the caveolae flatten out providing more plasma membrane.
A cell sipping away at the ECF by pinocytosis acquires a representative sample of the molecules and ions dissolved in the ECF. But cells also have a much more elegant method for picking up critical components of the ECF that may be in scant supply as we shall now see.
Receptor-Mediated Endocytosis
Some of the integral membrane proteins that a cell displays at its surface are receptors for particular components of the ECF (Figure \(\PageIndex{1c}\)). For example, iron is transported in the blood complexed to a protein called transferrin. Cells have receptors for transferrin on their surface. When these receptors encounter a molecule of transferrin, they bind tightly to it. The complex of transferrin and its receptor is then engulfed by endocytosis. Ultimately, the iron is released into the cytosol. The strong affinity of the transferrin receptor for transferrin (its ligand) ensures that the cell will get all the iron it needs even if transferrin represents only a small fraction of the protein molecules present in the ECF. Receptor-mediated endocytosis is many thousand times more efficient than simple pinocytosis in enabling the cell to acquire the macromolecules it needs.
Low-Density Lipoprotein (LDL) Receptor
Cells take up cholesterol by receptor-mediated endocytosis. Cholesterol is an essential component of all cell membranes. Most cells can, as needed, either synthesize cholesterol or acquire it from the ECF. Human cells get much of their cholesterol from the liver and, if your diet is not strictly "100% cholesterol-free", by absorption from the intestine.
Cholesterol is a hydrophobic molecule and quite insoluble in water. Thus it cannot pass from the liver and/or the intestine to the cells simply dissolved in blood and ECF. Instead it is carried in tiny droplets of lipoprotein. The most abundant cholesterol carriers in humans are the low-density lipoproteins or LDLs.
LDL particles are spheres covered with a single layer of phospholipid molecules with their hydrophilic heads exposed to the watery fluid (e.g., blood) and their hydrophobic tails directed into the interior. Some 1,500 molecules of cholesterol (each bound to a fatty acid) occupy the hydrophobic interior of LDL particles. One molecule of a protein called apolipoprotein B (apoB) is exposed at the surface of each LDL particle.
The first step in acquiring LDL particles is for them to bind to LDL receptors exposed at the cell surface. These transmembrane proteins have a site that recognizes and binds to the apolipoprotein B on the surface of the LDL. The portion of the plasma membrane with bound LDL is internalized by endocytosis. A drop in the pH (from ~7 to ~5) causes the LDL to separate from its receptor. The vesicle then pinches apart into two smaller vesicles: one containing free LDLs; the other containing now-empty receptors. The vesicle with the LDLs fuses with a lysosome to form a secondary lysosome. The enzymes of the lysosome then release free cholesterol into the cytosol. The vesicle with unoccupied receptors returns to and fuses with the plasma membrane, turning inside out as it does so (exocytosis). In this way the LDL receptors are returned to the cell surface for reuse.
People who inherit two defective (mutant) genes for the LDL receptor have receptors that function poorly or not at all. This creates excessively high levels of LDL in their blood and predisposes them to atherosclerosis and heart attacks. The ailment is called familial (because it is inherited) hypercholesterolemia.
Mutations in APOB, the apoB gene, cause another form of inherited hypercholesterolemia.
Other small hydrophobic molecules are also transported in the blood while bound to soluble proteins:
• the retinoid vitamin A (retinol) bound to the retinol-binding protein
• the steroids
• 25[OH] vitamin D3 bound to the vitamin D binding protein
• cortisol bound to the corticosteroid binding globulin
• testosterone and estrogens bound to the sex hormone binding globulin
There is growing evidence that, like cholesterol, they are taken into the cell by receptor-mediated endocytosis.
Endocytosis removes portions of the plasma membrane and takes them inside the cell. To keep in balance, membrane must be returned to the plasma membrane. This occurs by exocytosis. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.24%3A_Endocytosis.txt |
Exocytosis is the reverse of endocytosis and that is just as well. In 30 minutes an active cell like a macrophage can endocytose an amount of plasma membrane equal to its complete plasma membrane. The electron micrograph in Figure 3.25.1 shows a guinea pig phagocyte ingesting polystyrene beads. Several beads are already enclosed in vacuoles while the others are in the process of being engulfed. So the cell must have a mechanism to restore the normal amount of plasma membrane. Exocytosis is that mechanism.
The Secretion Mechanism
Membrane-enclosed vesicles move to the cell surface where they fuse with the plasma membrane. This restores the normal amount of plasma membrane and any molecules dissolved in the fluid contents of these vesicles are discharged into the extracellular fluid - this is called secretion (e.g., the various components of the extracellular matrix are secreted by exocytosis). Any integral membrane proteins exposed to the interior surface of the vesicles will now be displayed at the cell surface because the vesicles turn inside out as they fuse with the plasma membrane. Thus exocytosis does not simply replace plasma membrane, but ensures that the plasma membrane will display its characteristic cell-surface proteins.
Exocytic vesicles are created from several sources. Some are simply endosomes traversing the cell and others are pinched off from endosomes before they fuse with lysosomes. Others bud off from the endoplasmic reticulum and Golgi apparatus taking their products to the surface of the cell. The exocytosis of lysosomes supplies the membrane needed to repair wounds in the plasma membrane.
Some cells specialize in secretion. In cells that secrete large amounts of protein, for example, the protein accumulates in specialized secretory granules formed by the Golgi apparatus. These move to the cell surface and discharge their contents to the outside. For example, exocrine cells in the pancreas synthesize and secrete pancreatic digestive enzymes. The electron micrograph in Figure 3.25.4 shows four cells in the pancreas of a bat. The lumen where their apical surfaces meet leads eventually to the pancreatic duct draining into the small intestine. The spherical bodies (budded off from the Golgi apparatus) contain precursors of digestive enzymes. One is discharging its contents into the lumen by exocytosis (red arrow).
The cells lining our intestine synthesize tiny droplets of fat and discharge them into the lacteals by exocytosis.
The Kiss-and-Run Mechanism
The process described above involves the fusion of the exocytotic vesicle with the plasma membrane. In some cells, such as at synapses, a second type of exocytosis also takes place: (1) the vesicles make a brief contact at the plasma membrane, (2) release their contents (neurotransmitters in this case) to the exterior and (3) retreat back into the cytosol. This "kiss-and-run" version of exocytosis does not restore plasma membrane to the cell.
The Exosome Mechanism
A third type of exocytosis is found in some cells and involves endosomes themselves invaginating their membrane. As the invaginations break off they produce vesicles within vesicles, called multivesicular bodies. When these fuse with the cell's plasma membrane, these tiny (40–100 nm) internal vesicles — called exosomes — are secreted. Exosomes are produced in abundance by dendritic cells and B-cells and enhance their antigen-presenting function. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/03%3A_The_Cellular_Basis_of_Life/3.25%3A_Exocytosis.txt |
Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms. The three main purposes of metabolism are the conversion of food/fuel to energy to run cellular processes, the conversion of food/fuel to building blocks for proteins, lipids, nucleic acids, and some carbohydrates, and the elimination of nitrogenous wastes. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments.
• 4.1: Enzymes
Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part. Link to discussion of these ribozymes). Enzymes bind temporarily to one or more of the reactants — the substrate(s) — of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.
• 4.2: ATP
ATP (Adenosine triphosphate) is a nucleotide that performs many essential roles in the cell. It is the major energy currency of the cell, providing the energy for most of the energy-consuming activities of the cell. It is one of the monomers used in the synthesis of RNA and, after conversion to deoxyATP (dATP), DNA. It regulates many biochemical pathways.
• 4.3: NAD and NADP
Nicotinamide adenine dinucleotide (NAD) and its relative nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. NAD participates in many redox reactions in cells, including those in glycolysis and most of those in the citric acid cycle of cellular respiration. NADP is the reducing agent produced by the light reactions of photosynthesis and is consumed in the Calvin cycle of photosynthesis and used in many other anabolic reactions.
• 4.4: Glycolysis
Glycolysis is the anaerobic catabolism of glucose and occurs in virtually all cells. In eukaryotes, it occurs in the cytosol, where it converts a molecule of glucose into 2 molecules of pyruvic acid.
• 4.5: Cellular Respiration
Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water.
• 4.6: ATP Synthase
ATP synthase is a huge molecular complex (>500,000 daltons) embedded in the inner membrane of mitochondria. Its function is to convert the energy of protons (H+) moving down their concentration gradient into the synthesis of ATP. 3 to 4 protons moving through this machine is enough to convert a molecule of ADP and Pi (inorganic phosphate) into a molecule of ATP. One ATP synthase complex can generate >100 molecules of ATP each second.
• 4.7: Photosynthesis - Pathway of Carbon Fixation
Photosynthesis is the synthesis of organic molecules using the energy of light.
• 4.8: Photosynthesis - The Role of Light
• 4.9: Photosynthesis - Dicovering the Secrets
This chapter talks about various scientists and their path towards discovering photosynthesis.
• 4.10: Chemiosmosis
Several kinds of evidence support the chemiosmotic theory of ATP synthesis in chloroplasts. When isolated chloroplasts are illuminated, the medium in which they are suspended becomes alkaline — as we would predict if protons were being removed from the medium and pumped into the thylakoids (where they reduce the pH to about 4.0 or so). The interior of thylakoids can be deliberately made acid (low pH) by suspending isolated chloroplasts in an acid medium (pH 4.0) for a period of time.
• 4.11: Metabolism
All living things must have an unceasing supply of energy and matter. The transformation of this energy and matter within the body is called metabolism.
• 4.12: Intermediary Metabolism
The immediate source of energy for most cells is glucose. But glucose is not the only fuel on which cells depend. Other carbohydrates, fats and proteins may in certain cells or at certain times be used as a source of ATP. The complexity of the mechanism by which cells use glucose may make you fervently hope that a similarly-constructed system is not needed for each kind of fuel. And indeed it is not.
• 4.13: G Proteins
G proteins are so-called because they bind the guanine nucleotides GDP and GTP. They are heterotrimers (i.e., made of three different subunits) associated with the inner surface of the plasma membrane and transmembrane receptors of hormones, etc. These are called G protein-coupled receptors (GPCRs).
• 4.14: Secondary Messengers
Second messengers are molecules that relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc. — to target molecules in the cytosol and/or nucleus. But in addition to their job as relay molecules, second messengers serve to greatly amplify the strength of the signal. Binding of a ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.
• 4.15: Bioluminescence
Bioluminescence is the ability of living things to emit light. It is found in many marine animals, both invertebrate (e.g., some cnidarians, crustaceans, squid) and vertebrate (some fishes); some terrestrial animals (e.g., fireflies, some centipedes); and some fungi and bacteria.
04: Cell Metabolism
Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part. Link to discussion of these ribozymes). Enzymes bind temporarily to one or more of the reactants — the substrate(s) — of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.
Examples:
• Catalase catalyzes the decomposition of hydrogen peroxide into water and oxygen. $2H_2O_2 \rightarrow 2H_2O + O_2$ One molecule of catalase can break 40 million molecules of hydrogen peroxide each second.
• Carbonic anhydrase is found in red blood cells where it catalyzes the reaction. $CO_2 + H_2O \rightleftharpoons H^+ + HCO_3^−$ It enables red blood cells to transport carbon dioxide from the tissues to the lungs. One molecule of carbonic anhydrase can process one million molecules of CO2 each second.
• Acetylcholinesterase catalyzes the breakdown of the neurotransmitter acetylcholine at several types of synapses as well as at the neuromuscular junction — the specialized synapse that triggers the contraction of skeletal muscle. One molecule of acetylcholinesterase breaks down 25,000 molecules of acetylcholine each second. This speed makes possible the rapid "resetting" of the synapse for transmission of another nerve impulse.
Enzyme activity can be analyzed quantitatively. To do its work, an enzyme must unite — even if ever so briefly — with at least one of the reactants. In most cases, the forces that hold the substrate in the active site of the enzyme are noncovalent, an assortment of hydrogen bonds, ionic interactions, and hydrophobic interactions.
Most of these interactions are weak and especially so if the atoms involved are farther than about one angstrom from each other. So successful binding of the substrate in the active site of the enzyme requires that the two molecules be able to approach each other closely over a fairly broad surface. Thus the analogy that a substrate molecule binds its enzyme like a key in a lock. This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.
Competitive inhibition
The necessity for a close, if brief, fit between enzyme and substrate explains the phenomenon of competitive inhibition. One of the enzymes needed for the release of energy within the cell is succinic dehydrogenase. It catalyzes the oxidation (by the removal of two hydrogen atoms) of succinic acid. If one adds malonic acid to cells, or to a test tube mixture of succinic acid and the enzyme, the action of the enzyme is strongly inhibited. This is because the structure of malonic acid allows it to bind to the same site on the enzyme. But there is no oxidation so no speedy release of products. The inhibition is called competitive because if you increase the ratio of succinic to malonic acid in the mixture, you will gradually restore the rate of catalysis. At a 50:1 ratio, the two molecules compete on roughly equal terms for the binding (=catalytic) site on the enzyme.
Enzyme cofactors
Many enzymes require the presence of an additional nonprotein - a cofactor.
• Some of these are metal ions such as Zn2+ (the cofactor for carbonic anhydrase), Cu2+, Mn2+, K+, and Na+.
• Some cofactors are small organic molecules called coenzymes. The B vitamins thiamine (B1), riboflavin (B2) and nicotinamide
are precursors of coenzymes.
Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its catalytic act.
Lysozyme: a model of enzyme action
A number of lysozymes are found in nature; in human tears and egg white, for examples. The enzyme is antibacterial because it degrades the polysaccharide that is found in the cell walls of many bacteria. It does this by catalyzing the insertion of a water molecule at forming a glycosidic bond. This hydrolysis breaks the chain at that point.
The bacterial polysaccharide consists of long chains of alternating amino sugars:
• N-acetylglucosamine (NAG)
• N-acetylmuramic acid (NAM)
These hexose units resemble glucose except for the presence of the side chains containing amino groups.
Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft. With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101. Some hydrogen bonds also form with the C=O groups of several peptide bonds. In addition, hydrophobic interactions may help hold the substrate in position.
X-ray crystallography has shown that as lysozyme and its substrate unite, each is slightly deformed. The fourth hexose in the chain (ring #4) becomes twisted out of its normal position. This imposes a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings 4 and 5. It is just at this point that the polysaccharide is broken. A molecule of water is inserted between these two hexoses, which breaks the chain. Here, then, is a structural view of what it means to lower activation energy. The energy needed to break this covalent bond is lower now that the atoms connected by the bond have been distorted from their normal position.
As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the "lock" as well as the "key" changes shape as the two are brought together. (This is sometimes called "induced fit".)
The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.
Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H+ and OH- ions. The hydrogen ion (H+) left over can replace that lost by Glu-35. In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.
Factors Affecting Enzyme Action
The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of tertiary structure (i.e. shape) in enzyme function and noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape. Examples:
• the protease pepsin works best as a pH of 1–2 (found in the stomach)
• the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).
Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease. Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes.
Regulation of Enzyme Activity
Several mechanisms work to make enzyme activity within the cell efficient and well-coordinated.
Anchoring enzymes in membranes
Many enzymes are inserted into cell membranes, for examples,
• the plasma membrane
• the membranes of mitochondria and chloroplasts
• the endoplasmic reticulum
• the nuclear envelope
These are locked into spatial relationships that enable them to interact efficiently.
Inactive precursors
Enzymes, such as proteases, that can attack the cell itself are inhibited while within the cell that synthesizes them. For example, pepsin is synthesized within the chief cells (in gastric glands) as an inactive precursor, pepsinogen. Only when exposed to the low pH outside the cell is the inhibiting portion of the molecule removed and active pepsin produced.
Feedback Inhibition
If the product of a series of enzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus further production of the enzyme is halted.
Precursor Activation
The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.
In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect. In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate and in precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.
Regulation of Enzyme Synthesis
The four mechanisms described above regulate the activity of enzymes already present within the cell. What about enzymes that are not needed or are needed but not present? Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on or off — the transcription of genes.
If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down. Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose, and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase — by transcribing and translating the necessary gene(s) — and so can begin to metabolize the sugar. E. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.01%3A_Enzymes.txt |
ATP (Adenosine triphosphate) is a nucleotide that performs many essential roles in the cell.
• It is the major energy currency of the cell, providing the energy for most of the energy-consuming activities of the cell.
• It is one of the monomers used in the synthesis of RNA and, after conversion to deoxyATP (dATP), DNA.
• It regulates many biochemical pathways.
Energy
When the third phosphate group of ATP is removed by hydrolysis, a substantial amount of free energy is released. The exact amount depends on the conditions, but we shall use a value of 7.3 kcal per mole.
\[\ce{ATP + H2O → ADP + P_i}\]
where \(\ce{ADP}\) is adenosine diphosphate and \(\ce{P_i}\) is inorganic phosphate.
Because of the substantial amount of energy that is liberated when it is broken, the bond between the second and third phosphates is commonly described as a "high-energy" bond and is depicted in the figure by a wavy red line. (The bond between the first and second phosphates is also "high-energy".) (But please note that the term is not being used in the same sense as the term "bond energy". In fact, these bonds are actually weak bonds with low bond energies.)
Cells contain a wide variety of enzymes — called ATPases — that catalyze the hydrolysis of ATP and couple the energy released to particular energy-consuming reactions in the cell (see examples below).
Synthesis of ATP
• ADP + Pi → ATP + H2O
• requires energy: 7.3 kcal/mole
• occurs in the cytosol by glycolysis
• occurs in mitochondria by cellular respiration
• occurs in chloroplasts by photosynthesis
Consumption of ATP
ATP powers most of the energy-consuming activities of cells, such as:
• Most anabolic reactions such as
• joining transfer RNAs to amino acids for assembly into proteins
• synthesis of nucleoside triphosphates for assembly into DNA and RNA
• synthesis of polysaccharides
• synthesis of fats
• active transport of molecules and ions
• nerve impulses
• maintenance of cell volume by osmosis
• adding phosphate groups (phosphorylation) to many different proteins, e.g., to alter their activity in cell signaling
• muscle contraction
• beating of cilia and flagella (including sperm)
• bioluminescence
Extracellular ATP
In mammals, ATP also functions outside of cells. Its release
• from damaged cells can elicit inflammation and pain
• from the carotid body signals a shortage of oxygen in the blood
• from taste receptor cells triggers action potentials in the sensory nerves leading back to the brain
• from the stretched wall of the urinary bladder signals when the bladder needs emptying
4.03: NAD and NADP
Nicotinamide adenine dinucleotide (NAD) and its relative nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. NADP is simply NAD with a third phosphate group attached as shown at the bottom of the figure.
Because of the positive charge on the nitrogen atom in the nicotinamide ring (upper right), the oxidized forms of these important redox reagents are often depicted as NAD+ and NADP+ respectively.
In cells, most oxidations are accomplished by the removal of hydrogen atoms. Both of these coenzymes play crucial roles in this. Each molecule of NAD+ (or NADP+) can acquire two electrons; that is, be reduced by two electrons. However, only one proton accompanies the reduction. The other proton produced as two hydrogen atoms are removed from the molecule being oxidized is liberated into the surrounding medium. For NAD, the reaction is thus:
\[\ce{NAD^{+} + 2H -> NADH + H^{+}}\]
NAD and NADP uses
NAD participates in many redox reactions in cells, including those in glycolysis and most of those in the citric acid cycle of cellular respiration.
NADP is the reducing agent produced by the light reactions of photosynthesis and is consumed in the Calvin cycle of photosynthesis and used in many other anabolic reactions in both plants and animals.
Under the conditions existing in a normal cell, the hydrogen atoms shown in red are dissociated from these acidic substances.
4.04: Glycolysis
Glycolysis is the anaerobic catabolism of glucose and occurs in virtually all cells (Figure $1$). In eukaryotes, it occurs in the cytosol, where it converts a molecule of glucose into 2 molecules of pyruvic acid.
$C_6H_{12}O_6 + 2NAD^+ \rightarrow 2C_3H_4O_3 + 2NADH + 2H^+$
The free energy stored in 2 molecules of pyruvic acid is somewhat less than that in the original glucose molecule;some of this difference is captured in 2 molecules of ATP.
The Fates of Pyruvic Acid
In Yeasts, Pyruvic acid is decarboxylated and reduced by NADH to form a molecule of carbon dioxide and one of ethanol.
$C_3H_4O_3 + NADH + H^+ → CO_2 + C_2H_5OH + NAD^+$
This accounts for the bubbles and alcohol in, for examples, beer and champagne via a process called alcoholic fermentation. The process is energetically wasteful because so much of the free energy of glucose (some 95%) remains in the alcohol (a good fuel).
In Red Blood Cells and active Muscles, Pyruvic acid is reduced by NADH forming a molecule of lactic acid.
$C_3H_4O_3 + NADH + H^+ → C_3H_6O_3 + NAD^+$
The process is called lactic acid fermentation. The process is energetically wasteful because so much free energy remains in the lactic acid molecule. (It can also be debilitating because of the drop in pH as the lactic acid produced in overworked muscles is transported out into the blood.)
In Mitochondria, Pyruvic acid is oxidized completely to form carbon dioxide and water via a process called cellular respiration. Approximately 40% of the energy in the original glucose molecule is trapped in molecules of ATP. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.02%3A_ATP.txt |
Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water.
\[C_6H_{12}O_{6} + 6O_2 + 6H_2O → 12H_2O + 6 CO_2 \]
The energy released is trapped in the form of ATP for use by all the energy-consuming activities of the cell. The process occurs in two phases:
• glycolysis, the breakdown of glucose to pyruvic acid
• the complete oxidation of pyruvic acid to carbon dioxide and water
In eukaryotes, glycolysis occurs in the cytosol and the remaining processes take place in mitochondria.
Mitochondria
Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. Their number within the cell ranges from a few hundred to, in very active cells, thousands. Their main function is the conversion of the potential energy of food molecules into ATP.
Mitochondria have:
• an outer membrane that encloses the entire structure
• an inner membrane that encloses a fluid-filled matrix
• between the two is the intermembrane space
• the inner membrane is elaborately folded with shelflike cristae projecting into the matrix.
• a small number (some 5–10) circular molecules of DNA
This electron micrograph in Figure \(1\), shows a single mitochondrion from a bat pancreas cell. Note the double membrane and the way the inner membrane is folded into cristae. The dark, membrane-bounded objects above the mitochondrion are lysosomes. The number of mitochondria in a cell can increase either by their fission (e.g. following mitosis) or decrease by their fusing together. Defects in either process can produce serious, even fatal, illness.
The Outer Membrane
The outer membrane contains many complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out of the mitochondrion.
The Inner Membrane
The inner membrane contains 5 complexes of integral membrane proteins:
• NADH dehydrogenase (Complex I)
• succinate dehydrogenase (Complex II)
• cytochrome c reductase (Complex III; also known as the cytochrome b-c1 complex)
• cytochrome c oxidase (Complex IV)
• ATP synthase (Complex V)
The Matrix
The matrix contains a complex mixture of soluble enzymes that catalyze the respiration of pyruvic acid and other small organic molecules. Here pyruvic acid is
• oxidized by NAD+ producing NADH + H+
• decarboxylated producing a molecule of
• carbon dioxide (CO2) and
• a 2-carbon fragment of acetate bound to coenzyme A forming acetyl-CoA
The Citric Acid Cycle
This 2-carbon fragment is donated to a molecule of oxaloacetic acid. The resulting molecule of citric acid (which gives its name to the process) undergoes the series of enzymatic steps shown in the diagram. The final step regenerates a molecule of oxaloacetic acid and the cycle is ready to turn again.
A brief summary of the cycle is as follows:
• Each of the 3 carbon atoms present in the pyruvate that entered the mitochondrion leaves as a molecule of carbon dioxide (CO2).
• At 4 steps, a pair of electrons (2e-) is removed and transferred to NAD+ reducing it to NADH + H+.
• At one step, a pair of electrons is removed from succinic acid and reduces the prosthetic group flavin adenine dinucleotide (FAD) to FADH2.
• The electrons of NADH and FADH2 are transferred to the electron transport chain.
The Electron Transport Chain
The electron transport chain consists of 3 complexes of integral membrane proteins
• the NADH dehydrogenase complex (I)
• the cytochrome c reductase complex (III)
• the cytochrome c oxidase complex (IV)
and two freely-diffusible molecules ubiquinone, cytochrome c, that shuttle electrons from one complex to the next.
The electron transport chain accomplishes:
• The stepwise transfer of electrons from NADH (and FADH2) to oxygen molecules to form (with the aid of protons) water molecules (H2O). Cytochrome c can only transfer one electron at a time, so cytochrome c oxidase must wait until it has accumulated 4 of them before it can react with oxygen.
• Harnessing the energy released by this transfer to the pumping of protons (H+) from the matrix to the intermembrane space.
• Approximately 20 protons are pumped into the intermembrane space as the 4 electrons needed to reduce oxygen to water pass through the respiratory chain.
• The gradient of protons formed across the inner membrane by this process of active transport forms a miniature battery.
• The protons can flow back down this gradient only by reentering the matrix through ATP synthase, another complex (complex V) of 16 integral membrane proteins in the inner membrane. The process is called chemiosmosis.
Chemiosmosis in mitochondria
Fig.4.5.5 Chemiosmosis in Mitochondria
The energy released as electrons pass down the gradient from NADH to oxygen is harnessed by three enzyme complexes of the respiratory chain (I, III, and IV) to pump protons (H+) against their concentration gradient from the matrix of the mitochondrion into the intermembrane space.
As their concentration increases there (which is the same as saying that the pH decreases), a strong diffusion gradient is set up. The only exit for these protons is through the ATP synthase complex. As in chloroplasts, the energy released as these protons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis and is an example of facilitated diffusion. One-half of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their discovery of how ATP synthase works.
How many ATPs?
It is tempting to try to view the synthesis of ATP as a simple matter of stoichiometry (the fixed ratios of reactants to products in a chemical reaction). But (with 3 exceptions) it is not. Most of the ATP is generated by the proton gradient that develops across the inner mitochondrial membrane. The number of protons pumped out as electrons drop from NADH through the respiratory chain to oxygen is theoretically large enough to generate, as they return through ATP synthase, 3 ATPs per electron pair (but only 2 ATPs for each pair donated by FADH2).
With 12 pairs of electrons removed from each glucose molecule,
• 10 by NAD+ (so 10x3=30); and
• 2 by FADH2 (so 2x2=4),
this could generate 34 ATPs. Add to this the 4 ATPs that are generated by the 3 exceptions and one arrives at 38. However, the energy stored in the proton gradient is also used for the active transport of several molecules and ions through the inner mitochondrial membrane into the matrix. NADH is also used as reducing agent for many cellular reactions. So the actual yield of ATP as mitochondria respire varies with conditions and probably seldom exceeds 30.
The three exceptions
A stoichiometric production of ATP does occur at:
• one step in the citric acid cycle yielding 2 ATPs for each glucose molecule. This step is the conversion of alpha-ketoglutaric acid to succinic acid.
• at two steps in glycolysis yielding 2 ATPs for each glucose molecule.
Mitochondrial DNA (mtDNA)
The human mitochondrion contains 5–10 identical, circular molecules of DNA. Each consists of 16,569 base pairs carrying the information for 37 genes which encode:
• 2 different molecules of ribosomal RNA (rRNA)
• 22 different molecules of transfer RNA (tRNA) (at least one for each amino acid)
• 13 polypeptides
The rRNA and tRNA molecules are used in the machinery that synthesizes the 13 polypeptides.
The 13 polypeptides participate in building several protein complexes embedded in the inner mitochondrial membrane.
• 7 subunits that make up the mitochondrial NADH dehydrogenase (complex I)
• cytochrome b, a subunit of cytochrome c reductase (complex III)
• 3 subunits of cytochrome c oxidase (complex IV)
• 2 subunits of ATP synthase (complex V)
Each of these protein complexes also requires subunits that are encoded by nuclear genes, synthesized in the cytosol, and imported from the cytosol into the mitochondrion. Nuclear genes also encode ~1,000 other proteins that must be imported into the mitochondrion.
Mutations in mtDNA cause human diseases
Mutations in 12 of the 13 polypeptide-encoding mitochondrial genes have been found to cause human disease. Although many different organs may be affected, disorders of the muscles and brain are the most common. Perhaps this reflects the great demand for energy of both these organs. (Although representing only ~2% of our body weight, the brain consumes ~20% of the energy produced when we are at rest.)
Some of these disorders are inherited in the germline. In every case, the mutant gene is received from the mother because none of the mitochondria in sperm survives in the fertilized egg. Other disorders are somatic; that is, the mutation occurs in the somatic tissues of the individual.
Example: Exercise Intolerance
A number of humans who suffer from easily-fatigued muscles turn out to have a mutations in their cytochrome b gene. Curiously, only the mitochondria in their muscles have the mutation; the mtDNA of their other tissues is normal. Presumably, very early in their embryonic development, a mutation occurred in a cytochrome b gene in the mitochondrion of a cell destined to produce their muscles.
The severity of mitochondrial diseases varies greatly. The reason for this is probably the extensive mixing of mutant DNA and normal DNA in the mitochondria as they fuse with one another. A mixture of both is called heteroplasmy. The higher the ratio of mutant to normal, the greater the severity of the disease. In fact by chance alone, cells can on occasion end up with all their mitochondria carrying all-mutant genomes — a condition called homoplasmy (a phenomenon resembling genetic drift).
Mitochondrial Replacement Techniques
As I noted above, only mothers can pass mutant mtDNA on to their offspring. Two techniques are under intense investigation, either of which could enable a mother to have children free of defective mitochondria. Mutations in some 228 nuclear genes have also been implicated in human mitochondrial diseases, but mitochondrial replacement techniques will not be able to help with these.
Why do mitochondria have their own genome?
Many of the features of the mitochondrial genetic system resemble those found in bacteria. This has strengthened the theory that mitochondria are the evolutionary descendants of a bacterium that established an endosymbiotic relationship with the ancestors of eukaryotic cells early in the history of life on earth. However, many of the genes needed for mitochondrial function have since moved to the nuclear genome. The recent sequencing of the complete genome of Rickettsia prowazekii has revealed a number of genes closely related to those found in mitochondria. Perhaps rickettsias are the closest living descendants of the endosymbionts that became the mitochondria of eukaryotes. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.05%3A_Cellular_Respiration.txt |
ATP synthase is a huge molecular complex (>500,000 daltons) embedded in the inner membrane of mitochondria. Its function is to convert the energy of protons (H+) moving down their concentration gradient into the synthesis of ATP. 3 to 4 protons moving through this machine is enough to convert a molecule of ADP and Pi (inorganic phosphate) into a molecule of ATP. One ATP synthase complex can generate >100 molecules of ATP each second.
ATP synthase can be separated into 2 parts:
• Fo - the portion embedded in the inner mitochondrial membrane
• F1-ATPase — the portion projecting into the matrix of the mitochondrion
This is why the intact ATP synthase is also called the FoF1-ATPase.
When the F1-ATPase is isolated in vitro, it catalyzes the hydrolysis of ATP to ADP and Pi (which is why it is called the F1-ATPase). While it is doing so, the central portion of Fo attached to the stalk rotates rapidly in a counter-clockwise direction (as viewed from above).
In the intact mitochondrion, the protons that have accumulated in the intermembrane space enter the Fo complex and exit from it into the matrix. The energy they give up as they travel down their concentration gradient rotates Fo and its stalk (at ~6000 rpm) in a clockwise direction. As it does so, it induces repeating conformational changes in the head proteins that enable them to convert ADP and Pi into ATP. (In the figure, two of the three dimers that make up the head proteins have been pulled aside to reveal the stalk inserted in their center.)
In both these cases, the machine is converting chemical energy from the hydrolysis of ATP in the in vitro case and the flow of protons down their concentration gradient in the intact mitochondrion into mechanical energy — the turning of the motor. But this remarkable device can be made to do the reverse, converting mechanical energy (turning of the motor) into chemical energy.
A group of Japanese scientists interested in nano-machines have succeeded in attaching magnetic beads to the stalks of the F1-ATPase isolated in vitro. Then using a rotating magnetic field they were able to make the stalks rotate. When rotated in a clockwise direction, the F1-ATPase synthesized ATP from ADP and Pi in the surrounding medium — at a rate of about 5 molecules per second! (When rotating the stalks in the counter-clockwise direction, or not rotating them at all, ATP was hydrolyzed into ADP and Pi.)
Their achievement was reported in Itoh, H., et al., Nature, 29 January 2004.
4.07: Photosynthesis - Pathway of Carbon Fixation
Photosynthesis is the synthesis of organic molecules using the energy of light. For the sugar glucose (one of the most abundant products of photosynthesis) the equation is:
\[\ce{6CO2 + 12H2O -> C6H12O6 + 6H2O + 6O2}\]
Light provides the energy to transfer electrons from water to nicotinamide adenine dinucleotide phosphate (\(\ce{NADP^{+}}\)) forming \(\ce{NADPH}\) and to generate ATP. Both ATP and NADPH provide the energy and electrons to reduce carbon dioxide (\(\ce{CO2}\)) to organic molecules.
The Steps that lead to Photosynthesis
• CO2 combines with the phosphorylated 5-carbon sugar ribulose bisphosphate.
• This reaction is catalyzed by the enzyme ribulose bisphosphate carboxylase oxygenase (RUBISCO)(an enzyme which can fairly claim to be the most abundant protein on earth).
• The resulting 6-carbon compound breaks down into two molecules of 3-phosphoglyceric acid (PGA).
• The PGA molecules are further phosphorylated (by ATP) and are reduced (by NADPH) to form phosphoglyceraldehyde (PGAL).
• Phosphoglyceraldehyde serves as the starting material for the synthesis of glucose and fructose.
• Glucose and fructose make the disaccharide sucrose, which travels in solution to other parts of the plant (e.g., fruit, roots).
• Glucose is also the monomer used in the synthesis of the polysaccharides starch and cellulose.
The Figure 4.7.1 shows the steps in the fixation of carbon dioxide during photosynthesis. All of these reactions occur in the stroma of the chloroplast. These steps were worked out by Melvin Calvin and his colleagues at the University of California and, for this reason, are named the Calvin cycle.
Calvin's Experiment
The experimental apparatus is shown above. After various intervals of illumination, a suspension of unicellular algae is inactivated and the contents of the cells extracted. The compounds in a drop of the extract are then separated by paper chromatography.
The identity of each substance may be determined simply by comparing its position with the positions occupied by known substances under the same conditions. Or, a fragment containing the spot can be cut from the sheet and chemically analyzed.
To determine which, if any, of the substances separated on the chromatogram are radioactive, a sheet of X-ray film is placed next to the chromatogram. If dark spots appear on the film (because of radiation emitted by the 14C atoms), their position can be correlated with the positions of the chemicals in the chromatogram. Using this technique of autoradiography, Calvin found that 14C turned up in glucose molecules within 30 seconds after the start of photosynthesis. When he permitted photosynthesis to proceed for only 5 seconds, however, the radioactivity was concentrated in several other, smaller, molecules.
The dark spots show the radioactive compounds produced after 10 secs (left) and 2 minutes (right) of photosynthesis by the green alga Scenedesmus. The alga was supplied with carbon dioxide labeled with 14C, a radioactive isotope of carbon. At 10 seconds, most of the radioactivity is found in 3-phosphoglyceric acid ("P-Glyceric"). At 2 minutes, phosphorylated 6-carbon sugars (glucose and fructose) have been synthesized as well as a number of amino acids. The small rectangle and circle (lower right-hand corners) mark the spots where the cell extract was applied. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.06%3A_ATP_Synthase.txt |
The heart of photosynthesis as it occurs in most autotrophs consists of two key processes:
• the removal of hydrogen (H) atoms from water molecules
• the reduction of carbon dioxide (CO2) by these hydrogen atoms to form organic molecules.
The second process involves a cyclic series of reactions named (after its discoverer) the Calvin Cycle.
The electrons (e) and protons (H+) that make up hydrogen atoms are stripped away separately from water molecules.
\[\ce{2H2O -> 4e^{-} + 4H^{+} + O2}\]
The electrons serve two functions:
• They reduce NADP+ to NADPH for use in the Calvin Cycle.
• They set up an electrochemical charge that provides the energy for pumping protons from the stroma of the chloroplast into the interior of the thylakoid.
The protons also serve two functions:
• They participate in the reduction of NADP+ to NADPH.
• As they flow back out from the interior of the thylakoid (by facilitated diffusion), passing down their concentration gradient), the energy they give up is harnessed to the conversion of ADP to ATP.
• Because it is drive by light, this process is called photophosphorylation.
\[\ce{ADP + P_i -> ATP}\]
The ATP provides the second essential ingredient for running the Calvin Cycle.
The removal of electrons from water molecules and their transfer to NADP+ requires energy. The electrons are moving from a redox potential of about +0.82 volt in water to −0.32 volt in NADPH. Thus enough energy must be available to move them against a total potential of 1.14 volts. Where does the needed energy come from? The answer: Light.
The Thylakoid Membrane
Chloroplasts contain a system of thylakoid membranes surrounded by a fluid stroma. Six different complexes of integral membrane proteins are embedded in the thylakoid membrane. The exact structure of these complexes differs from group to group (e.g., plant vs. alga) and even within a group (e.g., illuminated in air or underwater). They are as follows:
Photosystem I
The structure of photosystem I in a cyanobacterium ("blue-green alga") has been completely worked out. It probably closely resembles that of plants as well. It is a homotrimer with each subunit in the trimer containing:
• 12 different protein molecules bound to
• 96 molecules of chlorophyll a
• 2 molecules of the reaction center chlorophyll P700
• 4 accessory molecules closely associated with them
• 90 molecules that serve as antenna pigments
• 22 carotenoid molecules
• 4 lipid molecules
• 3 clusters of Fe4S4
• 2 phylloquinones
Photosystem II
Photosystem II is also a complex of
• > 20 different protein molecules bound to
• 50 or more chlorophyll a molecules
• 2 molecules of the reaction center chlorophyll P680
• 2 accessory molecules close to them
• 2 molecules of pheophytin (chlorophyll without the Mg++)
• the remaining molecules of chlorophyll a serve as antenna pigments.
• some half dozen carotenoid molecules. These also serve as antenna pigments.
• 2 molecules of plastoquinone
Light-Harvesting Complexes (LHC)
• LHC-I associated with photosystem I
• LHC-II associated with photosystem II
These LHCs also act as antenna pigments harvesting light and passing its energy on to their respective photosystems.
The LHC-II of spinach is a homotrimer, with each monomer containing
• a single polypeptide
• 8 molecules of chlorophyll a
• 6 molecules of chlorophyll b
• 4 carotenoid molecules
How the System Works
• Light is absorbed by the antenna pigments of photosystems II and I.
• The absorbed energy is transferred to the reaction center chlorophylls, P680 in photosystem II, P700 in photosystem I.
• Absorption of 1 photon of light by Photosystem II removes 1 electron from P680.
• With its resulting positive charge, P680 is sufficiently electronegative that it can remove 1 electron from a molecule of water.
• When these steps have occurred 4 times, requiring 2 molecules of water, 1 molecule of oxygen and 4 protons (H+) are released
• The electrons are transferred (by way of plastoquinonePQ in the figure) to the cytochrome b6/f complex where they provide the energy for chemiosmosis.
• Activation of P700 in photosystem I enables it to pick up electrons from the cytochrome b6/f complex (by way of plastocyanin — PC in the figure) and raise them to a sufficiently high redox potential that, after passing through ferredoxin (Fd in the figure),
• they can reduce NADP+ to NADPH.
The sawtooth shifts in redox potential as electrons pass from P680 to NADP+ have caused this system to be called the Z-Scheme (although as I have drawn the diagram, it looks more like an "N"). It is also called noncyclic photophosphorylation because it produces ATP in a one-way process (unlike cyclic photophosphorylation and pseudocyclic photophosphorylation described below).
Chemiosmosis in Chloroplasts
The energy released as electrons pass down the gradient between photosystem II and plastocyanin (PC) is harnessed by the cytochrome b6/f complex to pump protons (H+) against their concentration gradient from the stroma of the chloroplast into the interior of the thylakoid (an example of active transport). As their concentration increases inside (which is the same as saying that the pH of the interior decreases), a strong diffusion gradient is set up. The only exit for these protons is through the ATP synthase complex. As in mitochondria, the energy released as these protons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis and is an example of facilitated diffusion.
Cyclic Photophosphorylation
• Each CO2 taken up by the Calvin cycle) requires 2 NADPH molecules and 3 ATP molecules
• Each molecule of oxygen released by the light reactions supplies the 4 electrons needed to make 2 NADPH molecules.
• The chemiosmosis driven by these 4 electrons as they pass through the cytochrome b6/f complex liberates only enough energy to pump 12 protons into the interior of the thylakoid.
• But in order to make 3 molecules of ATP, the ATPase in chloroplasts appears to have 14 protons (H+) pass through it.
• So there appears to be a deficit of 2 protons.
• How is this deficit to be made up?
• One likely answer: cyclic photophosphorylation.
In cyclic photophosphorylation,
• the electrons expelled by the energy of light absorbed by photosystem I pass, as normal, to ferredoxin (Fd).
• But instead of going on to make NADPH,
• they pass to plastoquinone (PQ) and on back into the cytochrome b6/f complex.
• Here the energy each electron liberates pumps 2 protons (H+) into the interior of the
• thylakoid — enough to make up the deficit left by noncyclic photophosphorylation.
This process is truly cyclic because no outside source of electrons is required. Like the photocell in a light meter, photosystem I is simply using light to create a flow of current. The only difference is that instead of using the current to move the needle on a light meter, the chloroplast uses the current to help synthesize ATP.
Pseudocyclic Photophosphorylation
Another way to make up the deficit is by a process called pseudocyclic photophosphorylation in which some of the electrons passing to ferredoxin then reduce molecular oxygen back to H2O instead of reducing NADP+ to NADPH.
At first glance, this might seem a fruitless undoing of all the hard work of photosynthesis. But look again. Although the electrons cycle from water to ferredoxin and back again, part of their pathway is through the chemiosmosis-generating stem of cytochrome b6/f. Here, then, is another way that simply by turning on a light, enough energy is imparted to electrons that they can bring about the synthesis of ATP.
Antenna Pigments
Chlorophylls a and b differ slightly in the wavelengths of light that they absorb best (although both absorb red and blue much better than yellow and green). Carotenoids help fill in the gap by strongly absorbing green light. The entire complex ensures that most of the energy of light will be trapped and passed on to the reaction center chlorophylls. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.08%3A_Photosynthesis_-_The_Role_of_Light.txt |
This chapter talks about various scientists and their path towards discovering photosynthesis.
van Helmont
Perhaps the first experiment designed to explore the nature of photosynthesis was that reported by the Dutch physician van Helmont in 1648. Some years earlier, van Helmont had placed in a large pot exactly 200 pounds (91 kg) of soil that had been thoroughly dried in an oven. Then he moistened the soil with rain water and planted a 5-pound (2.3 kg) willow shoot in it. He then placed the pot in the ground and covered its rim with a perforated iron plate. The perforations allowed water and air to reach the soil but lessened the chance that dirt or other debris would be blown into the pot from the outside.
For five years, van Helmont kept his plant watered with rain water or distilled water. At the end of that time, he carefully removed the young tree and found that it had gained 164 pound, 3 ounces (74.5 kg). (This figure did not include the weight of the leaves that had been shed during the previous four autumns.) He then redried the soil and found that it weighed only 2 ounces (57 g) less that the original 200 pounds (91 kg). Faced with these experimental facts, van Helmont theorized that the increase in weight of the willow arose from the water alone. He did not consider the possibility that gases in the air might be involved.
Joseph Priestley
The first evidence that gases participate in photosynthesis was reported by Joseph Priestley in 1772. He knew that if a burning candle is placed in a sealed chamber, the candle soon goes out. If a mouse is then placed in the chamber, it soon suffocates because the process of combustion has used up all the oxygen in the air — the gas on which animal respiration depends. However, Priestley discovered that if a plant is placed in an atmosphere lacking oxygen, it soon replenishes the oxygen, and a mouse can survive in the resulting mixture. Priestley thought (erroneously) that it was simply the growth of the plant that accounted for this.
Ingen-Housz
It was another Dutch physician, Ingen-Housz, who discovered in 1778 that the effect observed by Priestley occurred only when the plant was illuminated. A plant kept in the dark in a sealed chamber consumes oxygen just as a mouse (or candle) does.
Ingen-Housz also demonstrated that only green parts of plants liberated oxygen during photosynthesis. Nongreen plant structure, such as woody stems, roots, flowers, and fruits actually consume oxygen in the process of respiration. We now know that this is because photosynthesis can go on only in the presence of the green pigment chlorophyll.
Jean Senebier
The growth of plants is accompanied by an increase in their carbon content. A Swiss minister, Jean Senebier, discovered that the source of this carbon is carbon dioxide and that the release of oxygen during photosynthesis accompanies the uptake of carbon dioxide. Senebier concluded (erroneously as it turned out) that in photosynthesis carbon dioxide is decomposed, with the carbon becoming incorporated in the organic matter of the plant and the oxygen being released.
CO2 + H2O → (CH2O) + O2
(The parentheses around the CH2O signify that no specific molecule is being indicated but, instead, the ratio of atoms in some carbohydrate, e.g., glucose, C6H12O6.) The equation also indicates that the ratio of carbon dioxide consumed to oxygen release is 1:1, a finding that was carefully demonstrated in the years following Senebier's work. Using glucose as the carbohydrate product, we can write the equation for photosynthesis as
6CO2 + 6H2O → C6H12O6 + 6O2
F. F. Blackman
The above equation shows the relationship between the substances used in and produced by the process. It tells us nothing about the intermediate steps. That photosynthesis does involve at least two quite distinct processes became apparent from the experiments of the British plant physiologist F. F. Blackman. His results can easily be duplicated by using the setup in Figure 4.9.1. The green water plant Elodea (available wherever aquarium supplies are sold) is the test organism. When a sprig is placed upside down in a dilute solution of NaHCO3 (which serves as a source of CO2) and illuminated with a flood lamp, oxygen bubbles are soon given off from the cut portion of the stem. One then counts the number of bubbles given off in a fixed interval of time at each of several light intensities. Plotting these data produces a graph like the one in Figure 4.9.2.
Since the rate of photosynthesis does not continue to increase indefinitely with increased illumination, Blackman concluded that at least two distinct processes are involved: one, a reaction that requires light and the other, a reaction that does not. This latter is called a "dark" reaction although it can go on in the light. Blackman theorized that at moderate light intensities, the "light" reaction limits or "paces" the entire process. In other words, at these intensities the dark reaction is capable of handling all the intermediate substances produced by the light reaction. With increasing light intensities, however, a point is eventually reached when the dark reaction is working at maximum capacity. Any further illumination is ineffective, and the process reaches a steady rate.
This interpretation is strengthened by repeating the experiment as a somewhat higher temperature. Most chemical reactions proceed more rapidly at higher temperatures (up to a point). At 35°C, the rate of photosynthesis does not level off until greater light intensities are present. This suggest that the dark reaction is now working faster. The fact that at low light intensities the rate of photosynthesis is no greater at 35°C than at 20°C also supports the idea that it is a light reaction that is limiting the process in this range. Light reactions depend, not on temperature, but simply on the intensity of illumination.
The increased rate of photosynthesis with increased temperature does not occur if the supply of CO2 is limited. As the figure shows, the overall rate of photosynthesis reaches a steady value at lower light intensities if the amount of CO2 available is limited. Thus CO2 concentration must be added as a third factor regulating the rate at which photosynthesis occurs. As a practical matter, however, the concentration available to terrestrial plants is simply that found in the atmosphere: 0.035%.
Van Niel
It was the American microbiologist Van Niel who first glimpsed the role that light plays in photosynthesis. He studied photosynthesis in purple sulfur bacteria. These microorganisms synthesize glucose from CO2 as do green plants, and they need light to do so. Water, however, is not the starting material. Instead they use hydrogen sulfide (H2S). Furthermore, no oxygen is liberated during this photosynthesis but rather elemental sulfur. Van Niel reasoned that the action of light caused a decomposition of H2S into hydrogen and sulfur atoms. Then, in a series of dark reactions, the hydrogen atoms were used to reduce CO2 to carbohydrate:
\[\ce{CO2 + 2H2S → (CH2O) + H2O + 2S}\]
Van Niel envisioned a parallel to the process of photosynthesis as it occurs in green plants. There the energy of light causes water to break up into hydrogen and oxygen. The hydrogen atoms are then used to reduce CO2 in a series of dark reactions:
\[\ce{CO2 + 2H2O → (CH2O) + H2O + O2}\]
If this theory is correct, then it follows that all of the oxygen released during photosynthesis comes from water just as all the sulfur produced by the purple sulfur bacteria comes from H2S. This conclusion directly contradicts Senebier's theory that the oxygen liberated in photosynthesis comes from the carbon dioxide. If Van Niel's theory is correct, then the equation for photosynthesis would have to be rewritten:
\[\ce{6CO2 + 12H2O → C6H12O6 + 6 H2O + 6O2}\]
In science, a theory should be testable. By deduction, one can make a prediction of how a particular experiment will come out if the theory is sound. In this case, the crucial experiments needed to test the two theories had to await the time when the growth of atomic research made it possible to produce isotopes other than those found naturally or in greater concentrations than are found naturally.
Samuel Ruben
In air, water and other natural materials containing oxygen, 99.76% of the oxygen atoms are 16O and only 0.20% of them are the heavier isotope 18O. In 1941, Samuel Ruben and his coworkers at the University of California were able to prepare specially "labeled" water in which the 0.85% of the molecules contained 18O atoms. When this water was supplied to a suspension of photosynthesizing algae, the proportion of 18O in the oxygen gas that was evolved was 0.85%, the same as that of the water supplied, and not simply the 0.20% found in all natural samples of oxygen (and its compounds like CO2).
% 18O FOUND IN
EXPERIMENT H2O CO2 O2
1. START 0.85 0.20
FINISH 0.85 0.61* 0.86
2. START 0.20 0.68
FINISH 0.20 0.57 0.20
* A non-biochemical exchange of oxygen atoms between the water and the bicarbonate ions used as a source of CO2 explains the uptake of the isotope by CO2 in the first experiment.
These results clearly demonstrated that Senebier's interpretation was in error. If all the oxygen liberated during photosynthesis comes from the carbon dioxide, we would expect the oxygen evolved in Ruben's experiment to contain simply the 0.20% found naturally. If, on the other hand, both the carbon dioxide and the water contribute to the oxygen released, we would expect its isotopic composition to have been some intermediate figure. In fact, the isotopic composition of the evolved oxygen was the same as that of the water used.
Ruben and his colleagues also prepared a source of carbon dioxide that was enriched in 18O atoms. When algae carried out photosynthesis using this material and natural water, the oxygen that was given off was not enriched in 18O. It contained simply the 0.20% 18O found in the natural water used. The heavy atoms presumably became incorporated in the other two products (carbohydrate and by-product water).
These experiments lent great support to Van Niel's idea that one function of light in photosynthesis was the separation of the hydrogen and oxygen atoms of water molecules. But there remained to work out just how the hydrogen atoms were made available to the dark reactions. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.09%3A_Photosynthesis_-_Dicovering_the_Secrets.txt |
Several kinds of evidence support the chemiosmotic theory of ATP synthesis in chloroplasts. When isolated chloroplasts are illuminated, the medium in which they are suspended becomes alkaline — as we would predict if protons were being removed from the medium and pumped into the thylakoids (where they reduce the pH to about 4.0 or so). The interior of thylakoids can be deliberately made acid (low pH) by suspending isolated chloroplasts in an acid medium (pH 4.0) for a period of time. When these chloroplasts are then transferred to a slightly alkaline medium (pH 8.5), that is, one with a lower concentration of protons and given a supply of ADP and inorganic phosphate (Pi), they spontaneously synthesize ATP. No light is needed.
This is a direct evidence that a gradient of protons can be harnessed to the synthesis of ATP.
4.11: Metabolism
All living things must have an unceasing supply of energy and matter. The transformation of this energy and matter within the body is called metabolism.
Catabolism and Anabolism
Catabolism is destructive metabolism. Typically, in catabolism, larger organic molecules are broken down into smaller constituents. This usually occurs with the release of energy (usually as ATP). Anabolism is constructive metabolism. Typically, in anabolism, small precursor molecules are assembled into larger organic molecules. This always requires the input of energy (often as ATP).
Autotrophic vs. Heterotrophic Nutrition
Green plants, algae, and some bacteria are autotrophs ("self-feeders"). Most of them use the energy of sunlight to assemble inorganic precursors, chiefly carbon dioxide and water, into the array of organic macromolecules of which they are made. The process is photosynthesis. Photosynthesis makes the ATP needed for the anabolic reactions in the cell. All other organisms, including ourselves, are heterotrophs. We secure all our energy from organic molecules taken in from our surroundings ("food"). Although heterotrophs may feed partially (as most of us do) or exclusively on other heterotrophs, all the food molecules come ultimately from autotrophs. We may eat beef but the steer ate grass. Heterotrophs degrade some of the organic molecules they take in (catabolism) to make the ATP that they need to synthesize the others into the macromolecules of which they are made (anabolism).
How humans (and other animals) do it
Humans are heterotrophs. We are totally dependent on ingested preformed organic molecules to meet all our energy needs. We are also dependent on preformed organic molecules as the building blocks to meet our anabolic needs.
The steps for converting food to energy in animals:
1. Ingestion: taking food within the body (although as the figure shows, it is still topologically in the external world, not the internal).
2. Digestion: The enzyme-catalyzed hydrolysis of polysaccharides (e.g., starch) to sugars, proteins to amino acids, fats to fatty acids and glycerol, and nucleic acids to nucleotides.
3. Absorption into the body and transport to the cells.
4. Absorption into cells.
Within cells, these molecules are further degraded into still simpler molecules containing two to four carbon atoms. These fragments (acetyl-CoA for example) face one of two alternatives. They may proceed up various metabolic pathways and serve as the building blocks of, for example, sugars and fatty acids. From these will be assembled the macromolecules of the cell (e.g., polysaccharides, fats, proteins, and nucleic acids). Alternatively, the molecules in this pool of two- to four-carbon fragments may be still further degraded — ultimately to simple inorganic molecules such as carbon dioxide (CO2), H2O, and ammonia (NH3). This phase of catabolism releases large amounts of energy (in the form of ATP). One use to which this energy is put is to run the anabolic activities of the cell. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.10%3A_Chemiosmosis.txt |
The immediate source of energy for most cells is glucose. But glucose is not the only fuel on which cells depend. Other carbohydrates, fats and proteins may in certain cells or at certain times be used as a source of ATP. The complexity of the mechanism by which cells use glucose may make you fervently hope that a similarly-constructed system is not needed for each kind of fuel. And indeed it is not.
One of the great advantages of the step-by-step oxidation of glucose into CO2 and H2O is that several of the intermediate compounds formed in the process link glucose metabolism to the metabolism of other food molecules.
For example, when fats are used as fuel, the glycerol portion of the molecule is converted into PGAL and enters the glycolytic pathway at that point. Fatty acids are converted into molecules of acetyl-CoA and enter the respiratory pathway to be oxidized in the mitochondria. The amino acids liberated by the hydrolysis of proteins can also serve as fuel.
• First, the nitrogen is removed, a process called deamination.
• The remaining fragments then enter the respiratory pathway at several points.
For examples,
• the amino acids Gly, Ser, Ala, and Cys are converted into pyruvic acid and enter the mitochondria to be respired.
• acetyl-CoA and several intermediates in the citric acid cycle serve as entry points for other amino acid fragments (shown in blue).
These links thus permit the respiration of excess fats and proteins in the diet. No special mechanism of cellular respiration is needed by those animals that depend largely on ingested fats (e.g., many birds) or proteins (e.g., carnivores) for their energy supply.
Much of the protein we consume is ultimately converted into glucose (a process called gluconeogenesis) to provide fuel for the brain and other tissues. Although all our foods are interconvertible to some extent, they are not completely so. In other words, no single food can supply all our anabolic needs. We can indeed synthesize many fats from glucose, but certain unsaturated fats cannot be synthesized and must be taken in directly in our diet. These are linoleic acid, linolenic acid, and arachidonic acid. All are unsaturated; that is, have double bonds. Although we can synthesize 11 of the amino acids from carbohydrate precursors, we must obtain 9 others (the "essential amino acids") directly.
Many of the points that connect carbohydrate metabolism to the catabolism of fats and proteins serve as two-way valves (indicated in the figure by double-headed arrows). They provide points of entry not only for the catabolism (cellular respiration) of fatty acids, glycerol, and amino acids, but for their synthesis (anabolism) as well. Thus the catabolic breakdown of starches can lead (through acetyl-CoA and PGAL) to the synthesis of fat.
Fructose presents a special problem
Fructose is produced by the digestion of the disaccharide sucrose (common table sugar) into the monosaccharides glucose and fructose. Both glucose and fructose share the same empirical formula (C6H12O6) and the same caloric content (686 kcal/mole). But the body treats them very differently.
Glucose is taken up and metabolized by all cells to generate ATP by glycolysis and cellular respiration, and excess glucose is preferentially converted into glycogen rather than fats. Fructose is taken up only by liver cells, and excess fructose is converted in fats (fatty acids and glycerol). In the U.S., most soft drinks and prepared foods are now sweetened with high-fructose (60% fructose, 40% glucose) corn syrup. Excessive consumption of these products may well be linked to the growing prevalence in the U.S. of obesity and type 2 diabetes.
4.13: G Proteins
G proteins are so-called because they bind the guanine nucleotides GDP and GTP. They are heterotrimers (i.e., made of three different subunits) associated with the inner surface of the plasma membrane and transmembrane receptors of hormones, etc. These are called G protein-coupled receptors (GPCRs).
The three subunits are:
• Gα, which carries the binding site for the nucleotide. At least 20 different kinds of Gα molecules are found in mammalian cells.
How G Proteins Work
• In the inactive state, Gα has GDP in its binding site.
• When a hormone or other ligand binds to the associated GPCR, an allosteric change takes place in the receptor (that is, its tertiary structure changes).
• This triggers an allosteric change in Gα causing
• GDP to leave and be replaced by GTP.
• GTP activates Gα causing it to dissociate from GβGγ (which remain linked as a dimer).
• Activated Gα in turn activates an effector molecule.
In a common example (shown here), the effector molecule is adenylyl cyclase - an enzyme in the inner face of the plasma membrane which catalyzes the conversion of ATP into the "second messenger" cyclic AMP (cAMP).
Activated Gα is a GTPase so it quickly converts its GTP to GDP. This conversion, coupled with the return of the Gβ and Gγ subunits, restores the G protein to its inactive state.
Some Types of Gα Subunits
Gαs
This type stimulates (s = "stimulatory") adenylyl cyclase. It is the one depicted here. It is associated with the receptors for many hormones such as:
• adrenaline
• glucagon
• luteinizing hormone (LH)
• parathyroid hormone (PTH)
• adrenocorticotropic hormone (ACTH)
s is the target of the toxin liberated by Vibrio cholerae, the bacterium that causes cholera. Binding of cholera toxin to Gαs keeps it turned "on". The resulting continuous high levels of cAMP causes a massive loss of salts from the cells of the intestinal epithelium. Massive amounts of water follow by osmosis causing a diarrhea that can be fatal if the salts and water are not quickly replaced.
Gαq
This activates phospholipase C (PLC) which generates the second messengers:
• inositol trisphosphate (IP3)
• diacylglycerol (DAG)
q is found in G proteins coupled to receptors for vasopressin, thyroid-stimulating hormone (TSH), and angiotensin.
Gαi
This inhibits (i = "inhibitory") adenylyl cyclase lowering the level of cAMP in the cell. Gai is activated by the receptor for somatostatin.
Gαt
The "t" is for transducin, the molecule responsible for generating a signal in the rods of the retina in response to light. Gαt triggers the breakdown of cyclic GMP (cGMP). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.12%3A_Intermediary_Metabolism.txt |
Second messengers are molecules that relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc. — to target molecules in the cytosol and/or nucleus. But in addition to their job as relay molecules, second messengers serve to greatly amplify the strength of the signal. Binding of a ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.
There are 3 major classes of second messengers:
1. cyclic nucleotides (e.g., cAMP and cGMP)
2. inositol trisphosphate (IP3) and diacylglycerol (DAG)
3. calcium ions (Ca2+)
Cyclic Nucleotides
Cyclic AMP (cAMP)
Some of the hormones that achieve their effects through cAMP as a second messenger:
• adrenaline
• glucagon
• luteinizing hormone (LH)
Cyclic AMP is synthesized from ATP by the action of the enzyme adenylyl cyclase.
• Binding of the hormone to its receptor activates
• a G protein which, in turn, activates
• adenylyl cyclase.
• The resulting rise in cAMP turns on the appropriate response in the cell by either (or both):
• changing the molecular activities in the cytosol, often using Protein Kinase A (PKA) — a cAMP-dependent protein kinase that phosphorylates target proteins
• turning on a new pattern of gene transcription
Cyclic GMP (cGMP)
Cyclic GMP is synthesized from the nucleotide GTP using the enzyme guanylyl cyclase. Cyclic GMP serves as the second messenger for
• atrial natriuretic peptide (ANP)
• nitric oxide (NO)
• the response of the rods of the retina to light
Some of the effects of cGMP are mediated through Protein Kinase G (PKG) — a cGMP-dependent protein kinase that phosphorylates target proteins in the cell.
Inositol trisphosphate (IP3) and diacylglycerol (DAG)
Peptide and protein hormones like vasopressin, thyroid-stimulating hormone (TSH), and angiotensin and neurotransmitters like GABA bind to G protein-coupled receptors (GPCRs) that activate the intracellular enzyme phospholipase C (PLC).
As its name suggests, it hydrolyzes phospholipids — specifically phosphatidylinositol-4,5-bisphosphate (PIP2) which is found in the inner layer of the plasma membrane. Hydrolysis of PIP2 yields two products:
• diacylglycerol (DAG): DAG remains in the inner layer of the plasma membrane. It recruits Protein Kinase C (PKC) — a calcium-dependent kinase that phosphorylates many other proteins that bring about the changes in the cell. As its name suggests, activation of PKC requires calcium ions. These are made available by the action of the other second messenger — IP3.
• inositol-1,4,5-trisphosphate (IP3): This soluble molecule diffuses through the cytosol and binds to receptors on the endoplasmic reticulum causing the release of calcium ions (Ca2+) into the cytosol. The rise in intracellular calcium triggers the response.
Example:
The calcium rise is needed for NF-AT (the "nuclear factor of activated T cells") to turn on the appropriate genes in the nucleus.
The remarkable ability of tacrolimus and cyclosporine to prevent graft rejection is due to their blocking this pathway.
The binding of an antigen to its receptor on a B cell (the BCR) also generates the second messengers DAG and IP3.
Calcium ions (Ca2+)
As the functions of IP3 and DAG indicate, calcium ions are also important intracellular messengers. In fact, calcium ions are probably the most widely used intracellular messengers.
In response to many different signals, a rise in the concentration of Ca2+ in the cytosol triggers many types of events such as
• muscle contraction
• exocytosis, e.g.
• release of neurotransmitters at synapses (and essential for the long-term synaptic changes that produce Long-Term Potentiation (LTP) and Long-Term Depression (LTD);
• secretion of hormones like insulin
• activation of T cells and B cells when they bind antigen with their antigen receptors (TCRs and BCRs respectively)
• adhesion of cells to the extracellular matrix (ECM)
• apoptosis
• a variety of biochemical changes mediated by Protein Kinase C (PKC).
Normally, the level of calcium in the cell is very low (~100 nM). There are two main depots of Ca2+ for the cell:
• The extracellular fluid (ECF — made from blood), where the concentration is ~ 2 mM or 20,000 times higher than in the cytosol;
• the endoplasmic reticulum ("sarcoplasmic" reticulum in skeletal muscle).
However, its level in the cell can rise dramatically when channels in the plasma membrane open to allow it in from the extracellular fluid or from depots within the cell such as the endoplasmic reticulum and mitochondria.
Getting Ca2+ into (and out of) the cytosol
• Voltage-gated channels
• open in response to a change in membrane potential, e.g. the depolarization of an action potential
• are found in excitable cells:
• skeletal muscle
• smooth muscle (These are the channels blocked by drugs, such as felodipine [Plendil®], used to treat high blood pressure. The influx of Ca2+ contracts the smooth muscle walls of the arterioles, raising blood pressure. The drugs block this.)
• neurons. When the action potential reaches the presynaptic terminal, the influx of Ca2+ triggers the release of the neurotransmitter.
• the taste cells that respond to salt.
• allow some 106 ions to flow in each second following the steep concentration gradient.
• Receptor-operated channels
These are found in the post-synaptic membrane and open when they bind the neurotransmitter. Example: NMDA receptors.
• G-protein-coupled receptors (GPCRs). These are not channels but they trigger a release of Ca2+ from the endoplasmic reticulum as described above. They are activated by various hormones and neurotransmitters (as well as bitter substances on taste cells in the tongue).
Ca2+ ions are returned
• to the ECF by active transport using
• an ATP-driven pump called a Ca2+ ATPase;
• two Na+/Ca2+ exchangers. These antiport pumps harness the energy of
• 3 Na+ ions flowing DOWN their concentration gradient to pump one Ca2+ against its gradient and
• 4 Na+ ions flowing down to pump 1 Ca2+ and 1 K+ ion up their concentration gradients.
• to the endoplasmic (and sarcoplasmic) reticulum using another Ca2+ ATPase.
How can such a simple ion like Ca2+ regulate so many different processes? Some factors at work:
• localization within the cell (e.g., released at one spot — the T-system is an example — or spread throughout the cell)
• by the amount released (amplitude modulation, "AM")
• by releasing it in pulses of different frequencies (frequency modulation, "FM") | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.14%3A_Secondary_Messengers.txt |
Bioluminescence is the ability of living things to emit light. It is found in many marine animals, both invertebrate (e.g., some cnidarians, crustaceans, squid) and vertebrate (some fishes); some terrestrial animals (e.g., fireflies, some centipedes); and some fungi and bacteria. The molecular details vary from organism to organism, but each involves a luciferin (a light-emitting substrate), a luciferase (an enzyme that catalyzes the reaction), ATP (the source of energy) and molecular oxygen, O2.
The more ATP available, the brighter the light. In fact, firefly luciferin and luciferase are commercially available for measuring the amount of ATP in biological materials. Fireflies use their flashes to attract mates. The pattern differs from species to species. In one species, the females sometimes mimic the pattern used by females of another species. When the males of the second species respond to these "femmes fatales", they are eaten!
How Fireflies Control their Flashing
Barry Trimmer and his colleagues at Tufts University have recently discovered how fireflies turn their luminescent organs (called lanterns) ON.
• The luminescent cells of the lanterns are close to cells at the end of the tracheoles (that bring oxygen to — and take carbon dioxide away from — the insect's tissues).
• These cells contain nitric oxide synthase (NOS), the enzyme that liberates the gas nitric oxide (NO) from arginine.
• Nerve impulses activate the release of NO from these cells.
• The NO diffuses into the lantern cells and inhibits cellular respiration in the mitochondria (probably by blocking the action of cytochrome c oxidase)
• With cellular respiration inhibited, the oxygen content of the cells increases.
• This turns on light production in the peroxisomes that contain luciferase and luciferin-ATP (the ATP is generated when the lanterns are dark).
• The quick decay of NO probably contributes to the short duration of the flash.
Bioluminescence in Marine Animals
The widespread occurrence of luminescence among deep-sea animals reflects the perpetual darkness in which they live. At least one fish has its luminescent organ located at the tip of a protruding stalk and uses it as bait to lure prey within reach of its jaws. When disturbed, one species of squid emits a cloud of luminescent water instead of the ink that its shallow-water relatives use. Some marine animals that live near the surface have luminescent organs on their underside. These probably make it more difficult for predators beneath them to see them against the light background of the surface.
Fig \(2\): Bioluminescence in fish courtesy of Prof. J. W. Hastings
In the case of fishes, the light is emitted by luminescent bacteria that grow in luminescent organs. The photos show the flashlight fish, Photoblepharon palpebratus, with the lid of its luminescent organ open (left) and closed (right). The light is produced by continuously-emitting luminescent bacteria within the organs, but its display is controlled by the fish. These animals, which were photographed along reefs in the Gulf of Elat, Israel, appear to use their luminescent organs for such varied functions as: (1) attracting prey, (2) signaling other members of their species, and (3) confusing potential predators. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/04%3A_Cell_Metabolism/4.15%3A_Bioluminescence.txt |
• 5.1: Transformation in Bacteria
Bacteria have no sexual reproduction in the sense that eukaryotes do. The have no alternation of diploid and haploid generations, no gametes, and no meiosis. However, the essence of sex is genetic recombination, and bacteria do have three mechanisms to accomplish that: transformation, conjugation and transduction.
• 5.2: The Hershey - Chase Experiments
Hershey and Chase found that when bacteriophages containing 32P (radioactive), were allowed to infect nonradioactive bacteria, all the infected cells became radioactive and, in fact, much of the radioactivity was passed on to the next generation of bacteriophages. However, when the bacteria were infected with bacteriophages labeled with 35S, and then the virus coats removed (by whirling them in an electric blender), practically no radioactivity could be detected in the infected cells.
• 5.3: The Double Helix of DNA
This structure of DNA was worked out by Francis Crick and James D. Watson in 1953. It revealed how DNA - the molecule that Avery had shown was the physical substance of the genes. It could be replicated and so passed on from generation to generation. For this epochal work, they shared a Nobel Prize in 1962.
• 5.4: Base Pairing in DNA and RNA
The rules of base pairing tell us that if we can "read" the sequence of nucleotides on one strand of DNA, we can immediately deduce the complementary sequence on the other strand. The rules of base pairing explain the phenomenon that whatever the amount of adenine (A) in the DNA of an organism, the amount of thymine (T) is the same (called Chargaff's rule). Similarly, whatever the amount of guanine (G), the amount of cytosine (C) is the same.
• 5.5: DNA Replication
Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle. When the replication is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has remained intact as it served as the template for the synthesis of a complementary strand. This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new.
• 5.6: The Meselson - Stahl Experiment
While Watson and Crick had suggested that this was the way the DNA would turn out to be replicated, proof of the model came from the experiments of M. S. Meselson and F. W. Stahl. They grew E. coli is a medium using ammonium ions as the source of nitrogen for DNA (as well as protein) synthesis. 14N is the common isotope of nitrogen, but they could also use ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N.
• 5.7: Restriction Enzymes
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.
• 5.8: DNA Sequencing by the Dideoxy Method
The most popular chemical method for sequencing the nucleotides sequence in a sample of DNA this the dideoxy method which gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom. A dideoxynucleotide (dideoxythymidine triphosphate) can be added to the growing DNA strand, but stop chain elongation because there is no 3′ -OH for the next nucleotide to be attached to. For this reason, the dideoxy method is also called the chain termination method.
• 5.9: Genome Sizes
The genome of an organism is the complete set of genes specifying how its phenotype will develop (under a certain set of environmental conditions). In this sense, then, diploid organisms (like ourselves) contain two genomes, one inherited from our mother, the other from our father.
• 5.10: The Human Genome Projects
• 5.11: The Human and Chimpanzee Genomes
Now that the genomes of both the human and the chimpanzee have been determined, it is possible to make more direct comparisons between the two species. Their genomes are 98.8% identical (between any two humans — picked at random — the figure is closer to 99.5%).
• 5.12: Pyrosequencing
All of the sequenced genomes listed in Genome Sizes were determined using the dideoxy method invented by Frederick Sanger and described elsehwere. However, now a great effort is being expended to find ways to sequence DNA more rapidly (and more cheaply). Several new methods are being developed and many are already commercially available. Its method is called pyrosequencing or sequencing by synthesis.
• 5.13: DNA Repair
DNA in the living cell is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected. A failure to repair DNA produces a mutation.
• 5.14: Harlequin Chromosomes
Incredible though it may seem, each single human chromosome that you observe under 440x magnification of your laboratory microscope contains a single molecule of DNA. For some chromosomes, this molecule — if stretched out — would extend 5 cm (2 inches). Each chromosome contains but a single molecule of DNA and the replication of a chromosome is semi-conservative. The information encoded in each strand of DNA remains intact and serves as the template for the assembly of a complementary strand.
• 5.15: Metagenomics - Exploring the Microbial World
05: DNA
Bacteria have no sexual reproduction in the sense that eukaryotes do. The have no alternation of diploid and haploid generations, no gametes, and no meiosis. However, the essence of sex is genetic recombination, and bacteria do have three mechanisms to accomplish that: transformation, conjugation and transduction.
Transformation
Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surroundings. The ability to deliberately transform the bacterium E. coli has made possible the cloning of many genes, including human genes, and the development of the biotechnology industry. The first demonstration of bacterial transformation was done with Streptococcus pneumoniae and led to the discovery that DNA is the substance of the genes. The path leading to this epoch-making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith.
The cells of S. pneumoniae (also known as the pneumococcus) are usually surrounded by a gummy capsule made of a polysaccharide. When grown on the surface of a solid culture medium, the capsule causes the colonies to have a glistening, smooth appearance. These cells are called "S" cells. However, after prolonged cultivation on artificial medium, some cells lose the ability to form the capsule, and the surface of their colonies is wrinkled and rough ("R"). With the loss of their capsule, the bacteria also lose their virulence. Injection of a single S pneumococcus into a mouse will kill the mouse in 24 hours or so. But an injection of over 100 million (100 x 106) R cells is entirely harmless.
The reason? The capsule prevents the pneumococci from being engulfed and destroyed by scavenging cells, neutrophils and macrophages, in the body. The R forms are completely at the mercy of phagocytes. Pneumococci also occur in over 90 different types: I, II, III and so on. The types differ in the chemistry of their polysaccharide capsule. Unlike the occasional shift of S -> R, the type of the organism is constant. Mice injected with a few S cells of, say, Type II pneumococci, will soon have their bodies teeming with descendant cells of the same type.
However, Griffith found that when living R cells (which should have been harmless) and dead S cells (which also should have been harmless) were injected together, the mouse became ill and living S cells could be recovered from its body. Furthermore, the type of the cells recovered from the mouse's body was determined by the type of the dead S cells. In the experiment shown above, injection of living R-I cells and dead S-II cells produced a dying mouse with its body filled with living S-II pneumococci. The S-II cells remained true to their new type. Something in the dead S-II cells had made a permanent change in the phenotype of the R-I cells. The process was named transformation.
Oswald Avery and his colleagues at The Rockefeller Institute in New York City eventually showed that the "something" was DNA. In pursuing Griffith's discovery, they found that they could bring about the same kind of transformation in vitro using an extract of the bacterial cells. They treated this extract with:
• an enzyme to destroy the polysaccharide of the capsule (S-III in their experiments)
• solvents to remove all lipids
• trypsin and chymotrypsin to destroy any residual proteins
• RNase to destroy RNA
They discovered that it did not destroy the ability of their extracts to transform the bacteria. However, treating the extracts with DNase to destroy the DNA in them did abolish their transforming activity. So DNA was the only material in the dead cells capable of transforming cells from one type to another. DNA was the substance of genes.
Although the chemical composition of the capsule is determined by genes, the relationship is indirect. DNA is transcribed into RNA and RNA is translated into proteins. The phenotype of the pneumococci — the chemical composition of the polysaccharide capsule — is determined by the particular enzymes (proteins) used in polysaccharide synthesis. Avery and his colleagues Colin MacLeod and Maclyn McCarty published their epoch-making findings on February 1, 1944. Unfortunately, the importance of their discovery was not sufficiently appreciated by scientists in general and the Nobel Committee in particular, and Avery died before their work could be honored with a Nobel Prize. (Nobel prizes are never given posthumously.)
Conjugation
Some bacteria, E. coli is an example, can transfer a portion of their chromosome to a recipient with which they are in direct contact. As the donor replicates its chromosome, the copy is injected into the recipient. At any time that the donor and recipient become separated, the transfer of genes stops. Those genes that successfully made the trip replace their equivalents in the recipient's chromosome.
Conjugation can only occur between cells of opposite mating types: the donor (or "male") carries a fertility factor (F+) and the recipient ("female") does not (F). The fertility factor is a set of genes originally acquired from a plasmid and now integrated into the bacterial chromosome. It establishes the origin of replication for the chromosome. A portion of F is the "locomotive" that pulls the chromosome into the recipient cell and the rest of it is the "caboose".
In E. coli, about one gene gets across each second that the cells remain together. (So, it takes about 100 min for the entire genome (4377 genes) to make it. However, the process is easily interrupted so it is more likely that host genes close behind the leading F genes ("locomotive") will make it than those farther back. The "caboose" seldom makes it so failing to receive a complete F factor, the recipient cell continues to be "female".
The DNA that makes it across finds the homologous region on the female chromosome and replaces it (by a double crossover). By deliberately separating the cells (in a kitchen blender) at different times, the order and relative spacing of the genes can be determined. In this way, a genetic map — equivalent to the genetic maps of eukaryotes — can be made. However, here the map intervals are seconds, not centimorgans (cM).
Figure 5.1.4 shows the mechanism of conjugation in E. coli cells where the "male" lacks functional genes needed to synthesize the vitamin biotin and the amino acid methionine (Bio, Met) so these must be added to its culture medium. The "female" has those genes (Bio+, Met+) but has nonfunctional (mutant) genes that prevent it from being able to synthesize the amino acids threonine and leucine (Thr, Leu), so these must be added to its culture medium.
When cultured together, some female cells receive the functional Thr and Leu genes from the male donor. A double crossover enables them to replace the nonfunctional alleles. Now the cells now can grow on a "minimal" medium containing only glucose and salts.
Transduction
Bacteriophages are viruses that infect bacteria. In the process of assembling new virus particles, some host DNA may be incorporated in them. The virion head can hold only so much DNA so these viruses while still able to infect new host cells and may be unable to lyze them. Instead the hitchhiker bacterial gene (or genes) may be inserted into the DNA of the new host, replacing those already there and giving the host an altered phenotype. This phenomenon is called transduction.
Reductionist vs. holistic Approach
The understanding of complex systems almost always has to await unraveling the details of some simpler system. You may feel that trying to find out how one type of pneumococcus could be converted into another was an exceedingly specialized and esoteric pursuit. But Avery and his coworkers realized the broader significance of what they were observing and, in due course, the rest of the scientific world did as well. By electing to work with a well-defined system: the conversion of R forms of one type into S forms of a different type, these researchers made a discovery that has revolutionized biology and medicine. Attempting to understand the workings of complex systems by first understanding the workings of their parts is called reductionism. Some scientists (and many nonscientists) question the value of reductionism. They favor a holistic approach emphasizing the workings of the complete system.
However, the record speaks for itself. From skyscrapers to moon walks, to computer chips to the advances of modern medicine, progress comes from first understanding the properties of the parts that make up the whole. The late George Wald, who won the 1967 Nobel Prize in Physiology for his discoveries of the molecular basis of detecting light, once worried that his work was overly specialized — studying not vision, not the eye, not the whole retina, not even their rods and cones, but just the chemical reactions of their rhodopsins. But he came to realize "it is as though this were a very narrow window through which at a distance one can see only a crack of light. As one comes closer, the view grows wider and wider, until finally through this same window one is looking at the universe. I think this is the way it always goes in science, because science is all one. It hardly matters where one enters, provided one can come closer....".
Significance of Genetic Recombination in Bacteria
Transformation, conjugation, and transduction were discovered in the laboratory. How important are these mechanisms of genetic recombination in nature? The completion of the sequence of the entire genome of a variety of different bacteria (and archaea) suggest that genes have in the past moved from one species to another. This phenomenon is called lateral gene transfer (LGT). The remarkable spread of resistance to multiple antibiotics may have been aided by the transfer of resistance genes within populations and even between species. Many bacteria have enzymes that enable them to destroy foreign DNA that gets into their cells. It seem unlikely that these would be needed if that did not occur in nature. In any case, these restriction enzymes have provided the tools upon which the advances of molecular biology and the biotechnology industry depend. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.01%3A_Transformation_in_Bacteria.txt |
In 1952 (seven years after Avery's demonstration that genes were DNA), two geneticists: A. D. Hershey and Martha Chase, provided further proof. They worked with a DNA virus, called T2, which infects E. coli (and so is a bacteriophage). Figure 5.2.1 shows the essential elements of the infective cycle of DNA bacteriophages like T2. The virions attach to the surface of their host cell (a). The proteins of the capsid inject the DNA core into the cell (b). Once within the cell, some of the bacteriophage genes (the "early" genes) are transcribed (by the host's RNA polymerase) and translated (by the host's ribosomes, tRNA, etc.) to produce enzymes that will make many copies of the phage DNA and will turn off (even destroy) the host's DNA.
As fresh copies of phage DNA accumulate, other genes (the "late" genes) are transcribed and translated to form the proteins of the capsid (c). The stockpile of DNA cores and capsid proteins are assembled into complete virions (d). Another "late" gene is transcribed and translated into molecules of lysozyme. The lysozyme attacks the peptidoglycan wall (from the inside, of course). Eventually the cell ruptures and releases its content of virions ready to spread the infection to new host cells (e).
Bacteriophages produced within bacteria growing in radioactive culture medium will themselves be radioactive. If radioactive sulfur atoms (35S) are present, they will be incorporated into the protein coats of the bacteriophages since two of the amino acids — cysteine and methionine — contain sulfur (Figure 5.2.2). However, the DNA will be nonradioactive because there are no sulfur atoms in DNA. If radioactive phosphorus (32P) is used instead, the DNA become radioactive — because of its many phosphorus atoms — but not the proteins.
Hershey and Chase found that when bacteriophages containing 32P (radioactive), were allowed to infect nonradioactive bacteria, all the infected cells became radioactive and, in fact, much of the radioactivity was passed on to the next generation of bacteriophages. However, when the bacteria were infected with bacteriophages labeled with 35S, and then the virus coats removed (by whirling them in an electric blender), practically no radioactivity could be detected in the infected cells. From these experiments, it was clear that the DNA component of the bacteriophages is injected into the bacterial cell while the protein component remains outside. However, it is the injected component — DNA — that is able to direct the formation of new virus particles complete with protein coats. So here is further proof that genes are DNA.
5.03: The Double Helix of DNA
This structure of DNA was worked out by Francis Crick and James D. Watson in 1953. It revealed how DNA - the molecule that Avery had shown was the physical substance of the genes. It could be replicated and so passed on from generation to generation. For this epochal work, they shared a Nobel Prize in 1962.
5.04: Base Pairing in DNA and RNA
Rules of Base Pairing
The rules of base pairing (or nucleotide pairing) are:
• A with T: the purine adenine (A) always pairs with the pyrimidine thymine (T)
• C with G: the pyrimidine cytosine (C) always pairs with the purine guanine (G)
This is consistent with there not being enough space (20 Å) for two purines to fit within the helix and too much space for two pyrimidines to get close enough to each other to form hydrogen bonds between them. But why not A with C and G with T? The answer: only with A & T and with C & G are there opportunities to establish hydrogen bonds (shown here as dotted lines) between them (two between A & T; three between C & G). These relationships are often called the rules of Watson-Crick base pairing, named after the two scientists who discovered their structural basis.
Table 5.4.1: Relative Proportions (%) of Bases in DNA
Organism A T G C
Human 30.9 29.4 19.9 19.8
Chicken 28.8 29.2 20.5 21.5
Grasshopper 29.3 29.3 20.5 20.7
Sea Urchin 32.8 32.1 17.7 17.3
Wheat 27.3 27.1 22.7 22.8
Yeast 31.3 32.9 18.7 17.1
E. coli 24.7 23.6 26.0 25.7
The rules of base pairing tell us that if we can "read" the sequence of nucleotides on one strand of DNA, we can immediately deduce the complementary sequence on the other strand. The rules of base pairing explain the phenomenon that whatever the amount of adenine (A) in the DNA of an organism, the amount of thymine (T) is the same (called Chargaff's rule). Similarly, whatever the amount of guanine (G), the amount of cytosine (C) is the same. The C+G:A+T ratio varies from organism to organism, particularly among the bacteria, but within the limits of the experimental error, A=T and C=G. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.02%3A_The_Hershey_-_Chase_Experiments.txt |
With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.
Licensing: positive control of replication
In order to be replicated, each origin of replication must be bound by:
• an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
• Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include:
• Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
• MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
Once replication begins in S phase,
• Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
• The MCM proteins leave in front of the advancing replication fork.
5.06: The Meselson - Stahl Experiment
DNA Replication is Semiconservative
The structure of DNA suggested to Watson and Crick the mechanism by which DNA — hence genes — could be copied faithfully. They proposed that when the time came for DNA to be replicated, the two strands of the molecule
• separated from each other but
• remained intact as each served as the template for the synthesis of
• a complementary strand.
As this interpretative figure indicates, their results show that DNA molecules are not degraded and reformed from free nucleotides between cell divisions, but instead, each original strand remains intact as it builds a complementary strand from the nucleotides available to it. This is called semiconservative replication because each daughter DNA molecule is one-half "old" and one-half "new".
Immortal strands. Note that the "old" strand (the red one in the top half of the figure) is immortal because — barring mutations or genetic recombination — it will continue to serve as an unchanging template down through the generations.
E. coli is a bacterium, but semiconservative replication of DNA also occurs in eukaryotes. And because each DNA molecule in a eukaryote is incorporated in one chromosome, the replication of entire chromosomes is semiconservative as well. This also means that the eukaryotic chromosome contains one "immortal strand" of DNA.
5.07: Restriction Enzymes
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. To be able to sequence DNA, it is first necessary to cut it into smaller fragments. Many DNA-digesting enzymes (like those in your pancreatic fluid) can do this, but most of them are no use for sequence work because they cut each molecule randomly. This produces a heterogeneous collection of fragments of varying sizes. What is needed is a way to cleave the DNA molecule at a few precisely-located sites so that a small set of homogeneous fragments are produced. The tools for this are the restriction endonucleases. The rarer the site it recognizes, the smaller the number of pieces produced by a given restriction endonuclease.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence
5'GGCC3'
3'CCGG5'
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus φX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined. HaeIII and AluI cut straight across the double helix producing "blunt" ends. However, many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single-stranded DNA. These are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Any other source of DNA treated with the same enzyme will produce such molecules. Mixed together, these molecules can join with each other by the base pairing between their sticky ends. The union can be made permanent by another enzyme, a DNA ligase, that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA).
The ability to produce recombinant DNA molecules has not only revolutionized the study of genetics, but has laid the foundation for much of the biotechnology industry. The availability of human insulin (for diabetics), human factor VIII (for males with hemophilia A), and other proteins used in human therapy all were made possible by recombinant DNA.
Artificial Restriction Enzymes
In addition to the many natural restriction enzymes isolated from bacteria and archaea, it is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence. Examples:
• zinc-finger nucleases
• TALENs
• CRISPR RNA molecules
5.08: DNA Sequencing by the Dideoxy Method
The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with
• a mixture of all four normal (deoxy) nucleotides in ample quantities
• dATP
• dGTP
• dCTP
• dTTP
• a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
• ddATP
• ddGTP
• ddCTP
• ddTTP
• DNA polymerase I | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.05%3A_DNA_Replication.txt |
The genome of an organism is the complete set of genes specifying how its phenotype will develop (under a certain set of environmental conditions). In this sense, then, diploid organisms (like ourselves) contain two genomes, one inherited from our mother, the other from our father. The table below presents a selection of representative genome sizes from the rapidly-growing list of organisms whose genomes have been sequenced.
Table of Genome Sizes (haploid)
Base pairs Genes Notes
φX174 5,386 11 virus of E. coli
Human mitochondrion 16,569 37
Nasuia deltocephalinicola 112,091 137 smallest genome yet found in a bacterium. This β-proteobacterium lives in a mutualistic relationship within a special organ of an insect (a leaf hopper) which it supplies with essential amino acids.
Epstein-Barr virus (EBV) 172,282 80 causes mononucleosis
nucleomorph of Guillardia theta 551,264 511 all that remains of the nuclear genome of a red alga (a eukaryote) engulfed long ago by another eukaryote
Mycoplasma genitalium 580,073 525 two of the smallest true organisms
Mycoplasma pneumoniae 816,394 679
Rickettsia prowazekii 1,111,523 834 bacterium that causes epidemic typhus
Treponema pallidum 1,138,011 1,039 bacterium that causes syphilis
Pelagibacter ubique 1,308,759 1,354 smallest genome yet found in a free-living organism (marine α-proteobacterium)
Helicobacter pylori 1,667,867 1,589 chief cause of stomach ulcers (not stress and diet)
Methanocaldococcus jannaschii 1,664,970 1,783 These unicellular microbes look like typical bacteria but their genes are so different from those of either bacteria or eukaryotes that they are classified in a third kingdom: Archaea.
Aeropyrum pernix 1,669,695 1,885
Methanothermobacter thermoautotrophicus 1,751,377 2,008
Streptococcus pneumoniae 2,160,837 2,236 the pneumococcus
Pandoravirus 2,473,870 2556 A virus (of an amoeba) with a genome larger than that of the bacteria and archaea above and about the same as that of some parasitic eukaryotes.
Listeria monocytogenes 2,944,528 2,926 2,853 of these encode proteins; the rest RNAs
Synechocystis 3,573,470 4,003 a marine cyanobacterium ("blue-green alga")
E. coli K-12 4,639,221 4,377 4,290 of these genes encode proteins; the rest RNAs
E. coli O157:H7 5.44 x 106 5,416 strain that is pathogenic for humans; has 1,346 genes not found in E. coli K-12
Schizosaccharomyces pombe 12,462,637 4,929 Fission yeast. A eukaryote with fewer genes than the three bacteria below.
Agrobacterium tumefaciens 4,674,062 5,419 Useful vector for making transgenic plants; shares many genes with Sinorhizobium meliloti
Pseudomonas aeruginosa 6.3 x 106 5,570 Increasingly common cause of opportunistic infections in humans.
Sinorhizobium meliloti 6,691,694 6,204 The rhizobial symbiont of alfalfa. Genome consists of one chromosome and 2 large plasmids.
Saccharomyces cerevisiae 12,495,682 5,770 Budding yeast. A eukaryote.
Neurospora crassa 38,639,769 10,082 Plus 498 RNA genes.
Thalassiosira pseudonana 34.5 x 106 11,242 A diatom. Plus 144 chloroplast and 40 mitochondrial genes encoding proteins
Naegleria gruberi 41 x 106 15,727 This free-living unicellular organism lives as both an amoeboid and a flagellated form. 4,133 of its genes are also found in other eukaryotes suggesting that they were present in the common ancestor of all eukaryotes. The great variety of functions encoded by these genes also suggests that the common ancestor of all eukaryotes was itself as complex as many of the present-day unicellular members.
Drosophila melanogaster 122,653,977 ~17,000 the "fruit fly"
Caenorhabditis elegans 100,258,171 21,733
Humans 3.3 x 109 ~21,000
Tetraodon nigroviridis (a pufferfish) 3.42 x 108 27,918 Although Tetraodon seems to have more protein-encoding genes than we do, it has much less non-coding DNA so its total genome is about a tenth the size of ours.
Mouse 2.8 x 109 ~23,000
Amphibians 109–1011 ?
Arabidopsis thaliana 0.135 x 109 27,407 a flowering plant (angiosperm) with one of the smallest genomes known in the plant kingdom.
Picea abies 19.6 x 109 28,354 the Norway spruce, a conifer (gymnosperm). Even though it has only ~900 more genes than Arabidopsis, it has 145 times as much DNA. Most of this appears to be derived from transposons.
Psilotum nudum 2.5 x 1011 ?
Even though Psilotum nudum (sometimes called the "whisk fern") is a far simpler plant than Arabidopsis (it has no true leaves, flowers, or fruit), it has 3000 times as much DNA. No one knows why, but 80% or more of it is repetitive DNA containing no genetic information. This is also the case for some amphibians, which contain 30 times as much DNA as we do, but certainly are not 30 times as complex. The total amount of DNA in the haploid genome is called its C value. The lack of a consistent relationship between the C value and the complexity of an organism (e.g., amphibians vs. mammals) is called the C value paradox.
Not all genes are Indispensable
The scientists at The Institute for Genomic Research (now known as the J. Craig Venter Institute) who determined the Mycoplasma genitalium sequence have followed this work by systematically destroying its genes (by mutating them with insertions) to see which ones are essential to life and which are dispensable. Of the 485 protein-encoding genes, they have concluded that only 381 of them are essential to life. In other words, the loss of any one of the 381 is lethal; the loss of any one of the others is not. (This is not to say that all the organism needs are those 381 — see "A Minimal Genome?" below.)
Using similar techniques, three groups have recently found that only about 10% of the genes in the human genome (~2000 of them) must be present for human cells to grow successfully in culture. These genes encode proteins for such essential functions as controlling the cell cycle, DNA replication, DNA transcription and RNA translation. The cells can tolerate the loss of any one of the other ~18,000 genes. Thus the human genome appears to have redundant pathways that can often compensate for the loss of a single gene at least for cells growing in culture. Probably others will turn out to be essential for the development and functioning of the various types of differentiated cells in the intact body.
A Minimal Genome?
In March of 2016, workers at the J. Craig Venter Institute reported that they had created a strain of mycoplasma containing only 473 genes. This synthetic organism, which grows vigorously in culture, now holds the record for the smallest genome of a free-living organism. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.09%3A_Genome_Sizes.txt |
Shortly after their press conferences, the two groups that had been striving for several years to map the human genome published their findings:
• The International Human Genome Sequencing Consortium (IHGSC) in the 15 February 2001 issue of Nature
• Celera Genomics, a company in Rockville, Maryland, in the 16 February issue of Science
These achievements were monumental, but before we examine them, let us be clear as to what they were not.
What was not found
• Neither group had determined the complete sequence of the human genome. Each of our chromosomes is a single molecule of DNA. Some day the sequence of base pairs in each will be known from one end to the other. But in 2001, thousands of gaps remained to be filled. What they had done was present a series of draft sequences that represented about 90% (probably the most interesting 90%) of the genome.
• Even taken together, the results did not provide an accurate count of the number of protein-encoding genes in our genome (in contrast to such genomes as those of mitochondrial DNA, the Epstein-Barr virus and many of the bacterial genomes.
One reason: the large number and large size of the introns that split these genes make it difficult to recognize the open reading frames (ORFs) that encode proteins.
The number of genes were much smaller than predicted
The two groups came up with slightly different estimates of the number of protein-encoding genes, but both in the range of 30 to 38 thousand:
• barely two times larger than the genomes of
• Drosophila (~17,000 genes)
• C. elegans (<22,000 genes)
• and representing only 1– 2% of the total DNA in the cell;
• and a third of the 100,000 genes that many had predicted would be found.
• (By 2011, the number had been reduced to some 21,000.)
Are the tiny roundworm and fruit fly almost as complex as we are?
Probably not, although we share many homologous genes (called "orthologs") with both these animals. But many of our protein-encoding genes produce more than one protein product (e.g., by alternative splicing of the primary transcript of the gene). On average, each of our ORFs produces 2 to 3 different proteins. So the human "proteome" (our total number of proteins) may be 10 or more times larger than that of the fruit fly and roundworm.
A larger proportion of our genome encodes transcription factors and is dedicated to control elements (e.g., enhancers) to which these transcription factors bind. The combinatorial use of these elements probably provides much greater flexibility of gene expression than is found in Drosophila and C. elegans.
Gene diversity and density
There are some giants such as dystrophin with its 79 exons spread over 2.4 million base pairs of DNA and titin whose 363 exons can encode a single protein with as many as ~38,000 amino acids. The average human gene contains 4 exons totaling 1,350 base pairs and thus encodes an average protein of 450 amino acids. The density of genes on the different chromosomes varies from 23 genes per million base pairs on chromosome 19 (for a total of 1,400 genes) to only 5 genes per million base pairs on chromosome 13.
Humans have many genes not found in invertebrates
Humans, and presumably most vertebrates, have genes not found in invertebrate animals like Drosophila and C. elegans. These include genes encoding:
• antibodies and T cell receptors for antigen (TCRs)
• the transplantation antigens of the major histocompatibility complex (MHC) (HLA, the MHC of humans)
• cell-signaling molecules including the many types of cytokines
• the molecules that participate in blood clotting
• mediators of apoptosis. Although these proteins occur in Drosophila and C. elegans, we have a much richer assortment of them.
Gene Duplication
Both groups added to the list of human genes that have arisen by repeated duplication (e.g., by unequal crossing over) from a single precursor gene; for examples, the genes (several hundred) for olfactory receptors and the various globin genes.
Repetitive DNA
Both groups verified the presence of large amounts of repetitive DNA. In fact, this DNA — with similar sequences occurring over and over — is one of the main obstacles to assembling the DNA sequences in proper order.
• LINES (long interspersed elements)
• SINES (short interspersed elements) including Alu elements
• Retrotransposons
• DNA transposons
All told, repetitive DNA probably accounts for over 50% of our total genome.
What remains to be done?
• Keep looking for genes.
As of March 2010, 19,956 protein-encoding genes had been positively identified, but there probably are a thousand or more still to be found.
• Determine the human proteome; that is, the total complement of proteins we synthesize.
• Analyze how clusters of genes are coordinately expressed
• in various types of cells
• at different times in the life of a cell.
Such analysis will benefit greatly from the availability to gene chip technology and will also help us to understand how such a modest increase in gene number from Drosophila to humans could produce such a different outcome!
• Determine the genomes of other vertebrates.
This will not only help us recognize more human genes but will give us insight into what makes us unique.
Already we know that large sections of our genome have closely-related homologs in the mouse.
Examples:
• The collection of genes — and even their order — on human chromosome 17 matches closely those of mouse chromosome 11. The same is true of human chromosome 20 and mouse chromosome 2.
• Humans and mice (also rats) share several hundred absolutely identical stretches of DNA extending for 200–800 base pairs.
• Some are present in the exons of genes, especially genes involved in RNA processing.
• Some are found in or near the introns of genes, especially genes encoding proteins involved in DNA transcription.
• Some are found between genes — especially those, like Pax6, essential to embryonic development — and may serve as enhancers.
To have avoided any mutations for 60 million years since humans and rodents went their separate evolutionary ways suggest that these regions perform functions absolutely essential to mammalian life. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.10%3A_The_Human_Genome_Projects.txt |
Now that the genomes of both the human and the chimpanzee have been determined, it is possible to make more direct comparisons between the two species.
Comparison results
Their genomes are 98.8% identical (between any two humans — picked at random — the figure is closer to 99.5%).
• Another gene product that differs between the two species is the protein FoxP2. FoxP2 is a transcription factor. Rare humans with only one copy of the gene (FOXP2) have severe language defects.
The human FOXP2 gene differs from that of the chimp by 5 nucleotides, 2 of which result in non-synonymous codons encoding 2 different amino acids in the protein. The human protein differs from that in the mouse by only 3 amino acids. When you consider that we shared a common ancestor with mice over 60 million years ago but with the champanzee only about 6 million years ago, it is tempting to think that these recent changes in the human gene are related to the acquisition of language.
• While there are only small (~1%) coding differences in their genes, their genomes differ in other ways.
• Many insertions and deletions ("indels") and
• many gene duplications
are found in one species but not the other. Later work has revealed that of 510 chimpanzee sequences that are deleted in the human genome, only one occurs in the coding region of a gene. The others are found in introns or between genes, and at least some of these occur in gene-regulatory regions like enhancers.
• Many single-nucleotide differences create different splicing sites so alternative splicing can produce substantial differences in the proteins of two species.
5.12: Pyrosequencing
In laboratories around the world there is an intense desire to sequence more genomes.
• those of a wide variety of organisms to aid in establishing evolutionary relationships;
• those of pooled populations of microorganisms in, for examples, sea water, soil, the large intestine;
• other humans to look for genes that predispose to disease and genetic patterns in various ethnic groups.
All of the sequenced genomes listed in Genome Sizes were determined using the dideoxy method invented by Frederick Sanger and described elsehwere. However, now a great effort is being expended to find ways to sequence DNA more rapidly (and more cheaply).
The Genome Sequencer
Several new methods are being developed and one is already commercially available (the Genome Sequencer 20 System). Its method is called pyrosequencing or sequencing by synthesis. It works like this.
• The DNA to be sequenced is broken up into fragments of ~100 base pairs and denatured to form single-stranded DNA (ssDNA).
• Single ssDNA fragments are attached to microscopic beads, which are separated from each other.
• The polymerase chain reaction (PCR) is run on each bead so that each becomes coated with ~ 10 million identical copies of that fragment.
• The beads are placed singly into separate, microscopic wells (~200,000 of them).
• Each well receives a cocktail of reagents:
• DNA polymerase — for adding deoxyribonucleotides to the ssDNA
• adenosine phosphosulfate (APS)
• ATP sulfurylase — an enzyme that forms ATP from adenosine phosphosulfate (APS) and pyrophosphate (PPi)
• luciferin
• luciferase - an ATPase that catalyzes the conversion of luciferin to oxyluciferin with the liberation of light
The sequencing run:
• Each of the thousands of wells is flooded with one four deoxyribonucleotides, dTTP, dCTP, and dGTP, but instead of dATP (which would trigger the luciferin reaction), deoxyadenosine alpha-thiotriphosphate (dATPαS) is used instead. DNA polymerase ignores the difference and uses it whenever a T is encountered on the ssDNA template, but luciferase doesn't recognize to it.
• In any well where the complementary nucleotide is present at the 3' end of the template, the nucleotide is added and pyrophosphate is liberated.
• The amount of light is proportional to the number of that nucleotide added. So if, for example, the incoming nucleotide is dGTP, and there is a string of 3 Cs on the template, the light emitted will be 3 times brighter than if only one C is present.
• A detector picks up the light (if any) from each well and the data are recorded.
• Then each of the remaining 3 nucleotides are added in sequence.
• Then the sequence of 4 additions is repeated until synthesis is complete.
The above diagram also shows the type of data produced in a single well. The height of the peak of light production gives the number of additions that occurred when a particular nucleotide was added (bottom). Computer software then displays the template sequence (top) for each of the thousands of different fragments sequenced. With this technology, as many as 20 million base pairs of genome sequence can be learned in an instrument run of less than 6 hours. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.11%3A_The_Human_and_Chimpanzee_Genomes.txt |
DNA in the living cell is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected.
A failure to repair DNA produces a mutation.
The recent publication of the human genome has already revealed 130 genes whose products participate in DNA repair. More will probably be identified soon.
Agents that Damage DNA
• Certain wavelengths of radiation including ionizing radiation such as gamma rays and X-rays and ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield.
• Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways.
• Chemicals in the environment
• many hydrocarbons, including some found in cigarette smoke
• some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
• Chemicals used in chemotherapy, especially chemotherapy of cancers
Types of DNA Damage
1. All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.
• One of the most frequent is the loss of an amino group ("deamination") - resulting, for example, in a C being converted to a U.
2. Mismatches of the normal bases because of a failure of proofreading during DNA replication.
• Common example: incorporation of the pyrimidine U (normally found only in RNA) instead of T.
3. Breaks in the backbone.
• Can be limited to one of the two strands (a single-stranded break, SSB) or on both strands (a double-stranded break (DSB). Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
4. Crosslinks Covalent linkages can be formed between bases
• on the same DNA strand ("intrastrand") or
• on the opposite strand ("interstrand").
Several chemotherapeutic drugs used against cancers crosslink DNA
Repairing Damaged Bases
Damaged or inappropriate bases can be repaired by several mechanisms:
• Direct chemical reversal of the damage
• Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.
1. Base Excision Repair (BER)
2. Nucleotide Excision Repair (NER)
3. Mismatch Repair (MMR)
The 2015 Nobel Prize in chemistry was shared by three researchers for their pioneering work in DNA repair: Tomas Lindahl (BER), Aziz Sancar (NER), and Paul Modrich (MMR).
Direct Reversal of Base Damage
Perhaps the most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH3-) (an example of alkylation) to Cs followed by deamination to a T. Fortunately, most of these changes are repaired by enzymes, called glycosylases, that remove the mismatched T restoring the correct C. This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below).
Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation. Some of the methyl groups can be removed by a protein encoded by our MGMT gene. However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein.
This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful. Each of the myriad types of chemical alterations to bases requires its own mechanism to correct. What the cell needs are more general mechanisms capable of correcting all sorts of chemical damage with a limited toolbox. This requirement is met by the mechanisms of excision repair.
Base Excision Repair (BER)
The steps and some key players:
1. removal of the damaged base (estimated to occur some 20,000 times a day in each cell in our body!) by a DNA glycosylase. We have at least 8 genes encoding different DNA glycosylases each enzyme responsible for identifying and removing a specific kind of base damage.
2. removal of its deoxyribose phosphate in the backbone, producing a gap. We have two genes encoding enzymes with this function.
3. replacement with the correct nucleotide. This relies on DNA polymerase beta, one of at least 11 DNA polymerases encoded by our genes.
4. ligation of the break in the strand. Two enzymes are known that can do this; both require ATP to provide the needed energy.
Nucleotide Excision Repair (NER)
NER differs from BER in several ways.
• It uses different enzymes.
• Even though there may be only a single "bad" base to correct, its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage.
The steps and some key players:
1. The damage is recognized by one or more protein factors that assemble at the location.
2. The DNA is unwound producing a "bubble". The enzyme system that does this is Transcription Factor IIH, TFIIH, (which also functions in normal transcription).
3. Cuts are made on both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed.
4. A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct nucleotides. The DNA polymerases responsible are designated polymerase delta and epsilon.
5. A DNA ligase covalently inserts the fresh piece into the backbone.
Xeroderma Pigmentosum (XP)
XP is a rare inherited disease of humans which, among other things, predisposes the patient to
• pigmented lesions on areas of the skin exposed to the sun and
• an elevated incidence of skin cancer.
It turns out that XP can be caused by mutations in any one of several genes — all of which have roles to play in NER. Some of them:
• XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER.
• XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging. [Link]
• XPF, which cuts the backbone on the 5' side of the damage
• XPG, which cuts the backbone on the 3' side.
Transcription-Coupled NER
Nucleotide-excision repair proceeds most rapidly in cells whose genes are being actively transcribed on the DNA strand that is serving as the template for transcription. This enhancement of NER involves XPB, XPD, and several other gene products. The genes for two of them are designated CSA and CSB (mutations in them cause an inherited disorder called Cockayne's syndrome).
The CSB product associates in the nucleus with RNA polymerase II, the enzyme responsible for synthesizing messenger RNA (mRNA), providing a molecular link between transcription and repair. One plausible scenario: If RNA polymerase II, tracking along the template (antisense) strand), encounters a damaged base, it can recruit other proteins, e.g., the CSA and CSB proteins, to make a quick fix before it moves on to complete transcription of the gene.
Mismatch Repair (MMR)
Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A•T, C•G). It can enlist the aid of enzymes involved in both base-excision repair (BER) and nucleotide-excision repair (NER) as well as using enzymes specialized for this function.
• Recognition of a mismatch requires several different proteins including one encoded by MSH2.
• Cutting the mismatch out also requires several proteins, including one encoded by MLH1.
Mutations in either of these genes predisposes the person to an inherited form of colon cancer. So these genes qualify as tumor suppressor genes.
How does the MMR system know which is the incorrect nucleotide?
In E. coli, certain adenines become methylated shortly after the new strand of DNA has been synthesized. The MMR system works more rapidly, and if it detects a mismatch, it assumes that the nucleotide on the already-methylated (parental) strand is the correct one and removes the nucleotide on the freshly-synthesized daughter strand. How such recognition occurs in mammals is not yet known.
Synthesis of the repair patch is done by DNA polymerase delta. Cells also use the MMR system to enhance the fidelity of recombination; i.e., assure that only homologous regions of two DNA molecules pair up to cross over and recombine segments (e.g., in meiosis).
Repairing Strand Breaks
Ionizing radiation and certain chemicals can produce both single-strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA backbone.
Single-Strand Breaks (SSBs)
Breaks in a single strand of the DNA molecule are repaired using the same enzyme systems that are used in Base-Excision Repair (BER).
Double-Strand Breaks (DSBs)
There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule:
• Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ). A protein called Ku is essential for NHEJ. Ku is a heterodimer of the subunits Ku70 and Ku80. Errors in direct joining may be a cause of the various translocations that are associated with cancers. Some examples include Burkitt's lymphoma, the Philadelphia chromosome in chronic myelogenous leukemia (CML) and B-cell leukemia.
• Homologous Recombination (also known as Homology-Directed Repair — HDR). Here the broken ends are repaired using the information on the intact sister chromatid (available in G2 after chromosome duplication), or on the homologous chromosome (in G1; that is, before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog — a task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by NHEJ. or on the
• same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-to-back).
• Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast and ovarian cancers.
The Generation of Antibody Diversity
Some of the same enzymes used to repair DSBs by direct joining are also used to break and reassemble the gene segments used to make
• antibody variable regions; that is, to accomplish V(D)J joining — (mice whose Ku80 genes have been knocked out cannot do this);
• different antibody classes; that is, to accomplish class switching
Meiosis also involves DSBs
Recombination between homologous chromosomes in meiosis I also involves the formation of DSBs and their repair. So it is not surprising that this process uses the same enzymes.
Meiosis I with the alignment of homologous sequences provides a mechanism for repairing damaged DNA; that is, mutations. in fact, many biologists feel that the main function of sex is to provide this mechanism for maintaining the integrity of the genome.
However, most of the genes on the human Y chromosome have no counterpart on the X chromosome, and thus cannot benefit from this repair mechanism. They seem to solve this problem by having multiple copies of the same gene — oriented in opposite directions. Looping the intervening DNA brings the duplicates together and allowing repair by homologous recombination.
Gene Conversion
If the sequence used as a template for repairing a gene by homologous recombination differs slightly from the gene needing repair; that is, is an allele, the repaired gene will acquire the donor sequence. This nonreciprocal transfer of genetic information is called gene conversion.
The donor of the new gene sequence may be:
• the homologous chromosome (during meiosis)
• the sister chromatid (also during meiosis)
• a duplicate of the gene on the same chromosome (during mitosis)
Gene conversion during meiosis alters the normal mendelian ratios. Normally, meiosis in a heterozygous (A,a) parent will produce gametes or spores in a 1:1 ratio; e.g., 50% A; 50% a. However, if gene conversion has occurred, other ratios will appear. If, for example, an A allele donates its sequence as it repairs a damaged a allele, the repaired gene will become A, and the ratio will be 75% A; 25% a.
Cancer Chemotherapy
• The hallmark of all cancers is continuous cell division.
• Each division requires both
• the replication of the cell's DNA (in S phase) and
• transcription and translation of many genes needed for continued growth.
• So, any chemical that damages DNA has the potential to inhibit the spread of a cancer.
• Many (but not all) drugs used for cancer therapy do their work by damaging DNA.
The table lists (by trade name as well as generic name) some of the anticancer drugs that specifically target DNA.
6-mercaptopurine Purinethol® purine analog. One effect: substitutes for G, inducing abortive MMR and strand breaks
Gemcitabine Gemzar® pyrimidine analog substitutes for C blocking strand elongation
Cyclophosphamide Cytoxan® alkylating agents; form interstrand and/or intrastrand crosslinks
Melphalan Alkeran®
Busulfan Myleran®
Chlorambucil Leukeran®
Mitomycin Mutamycin®
Cisplatin Platinol® forms crosslinks
Bleomycin Blenoxane® cuts DNA strands between GT or GC
Irinotecan Camptosar® inhibit the proper functioning of enzymes (topoisomerases) needed to unwind DNA for replication and transcription
Mitoxantrone Novantrone®
Doxorubicin Adriamycin®
Dactinomycin Cosmegen® inserts into the double helix preventing its unwinding
The cancer patient has many other cell types that are also proliferating rapidly, e.g., cells of the intestinal endothelium, bone marrow and hair follicles. Anticancer drugs also damage these — producing many of the unpleasant side effects of "chemo". Agents that damage DNA are themselves carcinogenic, and chemotherapy poses a significant risk of creating a new cancer, often a leukemia. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.13%3A_DNA_Repair.txt |
Most eukaryotes have several to many pairs of chromosomes, and we might expect that at metaphase of mitosis the chromosomes would align at the metaphase plate at random so that some containing the immortal DNA strand would go to one pole; the remainder to the other. And this is generally the case. However, there may be some exceptions.
Stem cells divide to produce two daughter cells:
• one that will continue as a stem cell and
• one that will go on to differentiate.
There is evidence that when some types of stem cells divide, for example a subset found in skeletal muscle, the chromatids containing the immortal strand all line up on one side of the metaphase plate and the daughter cell receiving this set is the one that remains a stem cell. Although the mechanism by which this occurs is unknown, one can appreciate a potential value to the organism. Errors (mutations) in DNA occur most often during its replication. By keeping the original template in the stem cell population, introduced errors (mutations) disappear when the differentiated cell dies at the end of its useful life. Another possible advantage of nonrandom segregation of parental vs. newly-synthesized DNA: it may assure that epigenetic alterations of their respective DNA strands are transmitted to the appropriate daughter cells.
(The figure represents a haploid cell with n = 2. Each bar represents one strand of the DNA double helix.)
However, other experiments, with other types of stem cells, find that
• only certain chromosomes (e.g., the X and Y in Drosophila male germline stem cells) preferentially segregate the parental chromatids to the cell that will remain a stem cell;
• and for others, the distribution of immortal strands at metaphase is random, and thus the drawing on the right does not reflect what happens in those cases.
5.15: Metagenomics - Exploring the Microbial World
All the genomes listed on my page Genome Sizes describe the complete genome of a single species. For bacteria and archaeons, this means that the organism was grown in pure culture to provide the DNA for sequencing. But it is now clear that the microbial world contains vast numbers of both groups that have never been grown in the laboratory and thus have escaped study. Soil, water, and the contents of our large intestine are examples of habitats that teem with unknown microorganisms.
Thanks to the recent development of sequencing machines capable of rapidly (and inexpensively) sequencing huge amounts of DNA, it is now practical to sequence the DNA extracted from complex microbial ecosystems like that found in a soil sample. Several different approaches are used, but all depend on a first step of extracting the microbial DNA from the sample (and separating it from the far more complex DNA of any eukaryotes that may be present).
Assessing Microbial Diversity
The DNA encoding the small subunit (16S) of the ribosomes of both bacteria and archaeons contain some highly conserved regions; that is, regions of identical or almost identical sequence. Using primers that target these regions, one can then produce enough material by the polymerase chain reaction PCR to sequence the entire 16S rRNA gene.
Comparing the various sequences to a database of sequences from known organisms, one can estimate how many different types of microbes are present. Because of the substantial genetic diversity found between "strains" of a single species (e.g., E. coli K-12 and E.coli O157:H7), closely-related (> 97% identity) 16S rDNA sequences are assigned to a single "phylotype" because we cannot be sure whether they belong to separate species or to two strains of the same species. In either case, the collection of 16S rDNA sequences can be arranged to form a phylogenetic tree to show the patterns of relatedness.
Cataloging the Genes in a Microbial Ecosystem
Analyzing the 16S rDNA genes in a sample tells us who is there, but, of course, is not a complete genome and tells us nothing about the other genes present in the various members of the population. This information can be gained by "shotgun" sequencing of the environmental DNA sample.
The Steps:
• Break the DNA in short fragments.
• Insert these into a vector, e.g. a plasmid capable of growing in E. coli K-12.
• Expose E. coli cells to this random mix and grow the individual bacterial cells into colonies.
• The result: a library containing millions of random DNA fragments from the original sample.
• Isolate the plasmids and sequence them. Sequence "reads" average around 100 nucleotides — far shorter than a gene but often enough to move on to the next step.
• Use a powerful computer to attempt to assemble the fragments into a linear sequence of DNA. The computer looks for identical stretches of nucleotides in different fragments and uses the overlap to assemble them into a "contig".
• Look (have the computer look) for open reading frames (ORFs) of protein-encoding genes.
• Compare the ORFs with those of known microbes already in databases to see if a function can be deduced.
The sheer diversity of organisms in most microbial ecosystems makes it virtually impossible to find enough contigs to assemble a complete genome for any one organism like those listed in Genome Sizes. What you get instead is a window into the many kinds of genes present in one inhabitant or another of that ecosystem. For example, you may discover genes that encode proteins able to degrade environmental pollutants or genes able to synthesize a new antibiotic.
Finding New Functions in Microbial Populations
Another way of exploiting metagenomics is to look for new functions in the host (e.g. E. coli) if it can express the new gene with which it was transformed. For example, screening the library of E. coli clones for the ability to resist an antibiotic can reveal genes involved in antibiotic resistance — a worrisome development in recent years.
Some Applications of Metagenomics
• The Sargasso Sea: Metagenomic analysis of the DNA extracted from sea water in the Sargasso Sea revealed the presence of over a thousand different 16S rDNA genes (and thus approximately that number of different species) and over a million protein-encoding genes.
• The Human Colon: 0.3 g fecal samples from two healthy humans produced 78 million base pairs of sequence. Each subject produced some 25 thousand open reading frames (ORFs) of which about half could be recognized as already-known bacterial or archaeal genes. Included were genes encoding enzymes for the synthesis of vitamins (e.g., vitamin B1), amino acids, and enzymes for the digestion of complex polysaccharides in our diet which would otherwise be indigestible. Perhaps as much as 10% of the energy we extract from our food is made available to us by the activity of these microorganisms.
• Acid Mine Drainage: Metagenomic analysis of the acidic water (pH ~0.5) flowing from an abandoned metal mine in California revealed a much simpler ecosystem than those described above: only 3 species of bacteria and 2 of archaea. With such limited diversity, it was possible to assemble almost-complete genomes for two of these organisms.
• A South African Gold Mine: Simpler still was the ecosystem found in water 2.8 km (1.7 miles) down in a gold mine. Only one organism turned up: an autotrophic bacterium capable of extracting energy from inorganic substances in its environment and synthesizing all the molecules needed for its life from them. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/05%3A_DNA/5.14%3A_Harlequin_Chromosomes.txt |
Neurospora crassa
Neurospora crassa is an ascomycete, the red bread mold. Like all fungi, it reproduces by spores. It produces two kinds of spores:
• Conidia are spores produced by asexual reproduction. Mitosis of the haploid nuclei of the active, growing fungus generates the conidia.
• Ascospores, on the other hand, are formed following sexual reproduction. If two different mating types ("sexes") are allowed to grow together, they will fuse to form a diploid zygote. Meiosis of this zygote then gives rise to the haploid ascospores.
Neurospora is particularly well suited for genetic studies because
• It can be grown quickly on simple culture medium.
• It spends most of its life cycle in the haploid condition so any recessive mutations will show up in its phenotype.
• When the diploid zygote undergoes meiosis, the nuclei produced by
• Meiosis I, followed by
• Meiosis II, followed by
• one mitotic division
are confined to a narrow tube, the ascus.
• Because the nuclei cannot slip past one another, if
• the zygote nucleus is heterozygous for a gene (shown here as a and A) and
• no crossing over near that locus occurs during meiosis I,
the ascus will finally have four spores at one end containing one allele and four spores at the other end containing the other allele.
The One Gene - One Enzyme Theory
Sucrose, a few salts, and one vitamin — biotin — provide the nutrients that Neurospora needs to synthesize all the macromolecules of its cells.
Geneticists George W. Beadle and E. L. Tatum exposed some of the conidia of one mating type of Neurospora to ultraviolet rays in order to induce mutations.
• Then individual irradiated spores were allowed to germinate on a "complete" medium; that is, one enriched with various vitamins and amino acids.
• Once each had developed a mycelium, it was allowed to mate with the other mating type.
• The ascospores produced were dissected out individually and each one placed on complete medium.
• After growth had occurred, portions of each culture were subcultured on minimal medium.
• Sometimes growth continued; sometimes it didn't.
• When it did not ("1st" in the figure) , the particular strain was then supplied with a mixture of vitamins, amino acids, etc. until growth did occur ("2nd ").
• Eventually each mutated strain was found to have acquired a need for one nutrient; in the example illustrated here, the vitamin thiamine ("3rd").
Beadle and Tatum reasoned that radiation had caused a gene that permits the synthesis of thiamine from the simple ingredients in minimal medium to mutate to an allele that does not. The synthesis of thiamine from sucrose requires a number of chemical reactions, each one catalyzed by a specific enzyme.
By adding, one at a time, the different precursors of thiamine to the medium in which their mutant mold was placed, they were able to narrow down the defect to the absence of a single enzyme.
• If they added to the minimal medium any precursor further along in the process, growth occurred.
• Any precursor before the blocked step could not support growth.
Thus, in this example, the conversion of precursor C to precursor D was blocked because of the absence of the needed enzyme (c).
This led them to postulate the one gene - one enzyme theory: each gene in an organism controls the production of a specific enzyme. It is these enzymes that catalyze the reactions that lead to the phenotype of the organism.
Today, we know that, in fact, not only enzymes, but all the other proteins from which the organism is built are encoded by genes. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/06%3A_Gene_Expression/6.01%3A_One_Gene_-_One_Enzyme_Theory.txt |
The majority of genes are expressed as the proteins they encode. The process occurs in two steps:
• Transcription = DNA RNA
• Translation = RNA protein
Gene Transcription: DNA → RNA
DNA serves as the template for the synthesis of RNA much as it does for its own replication.
The Steps of Transcription
• Some 50 different protein transcription factors bind to promoter sites, usually on the 5′ side of the gene to be transcribed.
• An enzyme, an RNA polymerase, binds to the complex of transcription factors.
• Working together, they open the DNA double helix.
• The RNA polymerase proceeds to read one strand moving in it's 3'→ 5' direction.
• In eukaryotes, this requires — at least for protein-encoding genes — that the nucleosomes in front of the advancing RNA polymerase (Pol II) be removed. A complex of proteins is responsible for this. The same complex replaces the nucleosomes after the DNA has been transcribed and Pol II has moved on.
• As the RNA polymerase travels along the DNA strand, it assembles ribonucleotides (supplied as triphosphates, e.g., ATP) into a strand of RNA.
• Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. Thus for each C encountered on the DNA strand, a G is inserted in the RNA; for each G, a C; and for each T, an A. However, each A on the DNA guides the insertion of the pyrimidine uracil (U, from uridine triphosphate, UTP). There is no T in RNA.
.)
• Synthesis of the RNA proceeds in the 5′ → 3′ direction.
• As each nucleoside triphosphate is brought in to add to the 3′ end of the growing strand, the two terminal phosphates are removed.
• When transcription is complete, the transcript is released from the polymerase and, shortly thereafter, the polymerase is released from the DNA.
Note that at any place in a DNA molecule, either strand may be serving as the template; that is, some genes "run" one way, some the other (and in a few remarkable cases, the same segment of double helix contains genetic information on both strands!). In all cases, however, RNA polymerase transcribes the DNA strand in its 3'→ 5' direction.
Types of RNA
Sedimentation pattern produced by high-speed centrifugation of RNA extracted from the precursors of rabbit red blood cells. The discrete bands represent particular classes of RNA. The transfer RNAs band at about 4S. The ribosomal RNAs of eukaryotes sediment at 5S, 5.8S, 18S, and 28S. (The larger the sedimentation unit, S, the larger the molecule — but not proportionally.) The RNA forming the band at 9S is the messenger RNA for the synthesis of hemoglobin, the major protein synthesized in these cells. In most types of cells, the messenger RNAs are extremely heterogenous, with small amounts distributed from 6S to 25S.
Several types of RNA are synthesized in the nucleus of eukaryotic cells.
• messenger RNA (mRNA)
• ribosomal RNA (rRNA)
• transfer RNA (tRNA)
• small nuclear RNA (snRNA)
• small nucleolar RNA (snoRNA)
• microRNA (miRNA). These are tiny (~22 nucleotides) RNA molecules that regulate the expression of messenger RNA (mRNA) molecules.
• long non-coding RNA (lncRNA)
Messenger RNA (mRNA)
Messenger RNA will be translated into a polypeptide. Messenger RNA comes in a wide range of sizes reflecting the size of the polypeptide it encodes. Most cells produce small amounts of thousands of different mRNA molecules, each to be translated into a peptide needed by the cell. Many mRNAs are common to most cells, encoding "housekeeping" proteins needed by all cells (e.g., the enzymes of glycolysis). Other mRNAs are specific for only certain types of cells. These encode proteins needed for the function of that particular cell (e.g., the mRNA for hemoglobin in the precursors of red blood cells).
Ribosomal RNA (rRNA)
This will be used in the building of ribosomes: machinery for synthesizing proteins by translating mRNA. There are 4 kinds. In eukaryotes, these are
• 18S rRNA. One of these molecules, along with some 30 different protein molecules, is used to make the small subunit of the ribosome.
• 28S, 5.8S, and 5S rRNA. One each of these molecules, along with some 45 different proteins, are used to make the large subunit of the ribosome.
The S number given each type of rRNA reflects the rate at which the molecules sediment in the ultracentrifuge. The larger the number, the larger the molecule (but not proportionally). The 28S, 18S, and 5.8S molecules are produced by the processing of a single primary transcript from a cluster of identical copies of a single gene. The 5S molecules are produced from a different cluster of identical genes.
Transfer RNA (tRNA)
These are the RNA molecules that carry amino acids to the growing polypeptide. There are some 32 different kinds of tRNA in a typical eukaryotic cell.
• Each is the product of a separate gene.
• They are small (~4S), containing 73-93 nucleotides.
• Many of the bases in the chain pair with each other forming sections of double helix.
• The unpaired regions form 3 loops.
• Each kind of tRNA carries (at its 3′ end) one of the 20 amino acids (thus most amino acids have more than one tRNA responsible for them).
• At one loop, 3 unpaired bases form an anticodon.
• Base pairing between the anticodon and the complementary codon on a mRNA molecule brings the correct amino acid into the growing polypeptide chain.
Small Nuclear RNA (snRNA)
DNA transcription of the genes for mRNA, rRNA, and tRNA produces large precursor molecules ("primary transcripts") that must be processed within the nucleus to produce the functional molecules for export to the cytosol. Some of these processing steps are mediated by snRNAs.
Approximately a dozen different genes for snRNAs, each present in multiple copies, have been identified. The snRNAs have various roles in the processing of the other classes of RNA. For example, several snRNAs are part of the spliceosomes that participate in converting pre-mRNA into mRNA by excising the introns and splicing the exons.
Small Nucleolar RNA (snoRNA)
As the name suggests, these small (60–300 nucleotides) RNAs are found in the nucleolus where they are responsible for several functions:
• Some participate in making ribosomes by helping to cut up the large RNA precursor of the 28S, 18S, and 5.8S molecules.
• Others chemically modify many of the nucleotides in rRNA, tRNA, and snRNA molecules, e.g., by adding methyl groups to ribose.
• Some have been implicated in the alternative splicing of pre-mRNA to different forms of mature mRNA.
• One snoRNA serves as the template for the synthesis of telomeres.
In vertebrates, the snoRNAs are made from introns removed during RNA processing.
MicroRNAs (miRNAs)
MicroRNAs" ("miRNAs") are single-stranded RNA molecules containing about 22 nucleotides and are about the same size as siRNAs. MicroRNAs are found in all animals (humans generate some 1000 miRNAs) and plants but not in fungi. They contain 19–25 nucleotide. They are
• encoded in the genome
• some by stand-alone genes (that may encode several miRNAs)
• some by portions of an intron of the gene whose mRNA they will regulate.
• may be expressed in
• only certain cell types and
• at only certain times in the differentiation of a particular cell type.
While direct evidence of the function of many of these newly-discovered gene products remains to be discovered, they regulate gene expression by regulating messenger RNA (mRNA), either
• destroying the mRNA when the sequences match exactly (the usual situation in plants) or
• repressing its translation when the sequences are only a partial match.
MicroRNAs have two traits ideally suited for this:
• Being so small, they can be rapidly transcribed from their genes.
• They do not need to be translated into a protein product to act.
MicroRNAs regulate (repress) expression of genes in mammals as well. Genome analysis has revealed thousands of human genes whose transcripts (mRNAs) contain sequences to which one or more of our miRNAs might bind. Probably each miRNA can bind to as many as 200 different mRNA targets while each mRNA has binding sites for multiple miRNAs. Such a system provides many opportunities for coordinated mRNA translation
Long Non-coding RNA (lncRNA)
Only messenger RNA encodes polypeptides. All the other classes of RNA are thus called non-coding RNA. In addition to the rRNAs, snRNAs, and snoRNAs, there is a large (more than 10,000 in humans), heterogenous collection of transcripts longer than 200 nucleotides that are classified as lncRNAs. The function, if any, of most of these remains to be discovered.
However, some lncRNAs have been found to participate in the regulation of such diverse activities as splicing, translation, imprinting, and transcription. Two examples:
• XIST RNA, which contains thousands of nucleotides, inactivates one of the two X chromosomes in female vertebrates.
• Some lncRNAs participate in bringing the enhancer and promoter regions of genes close together ("looping") to regulate gene transcription.
While much remains to be learned about their functions, taken together non-coding RNAs probably account for three-quarters of the transcription going on in the nucleus.
The RNA polymerases
The RNA polymerases are huge multi-subunit protein complexes. Three kinds are found in eukaryotes.
• RNA polymerase I (Pol I). It transcribes the rRNA genes for the precursor of the 28S, 18S, and 5.8S molecules (and is the busiest of the RNA polymerases).
• RNA polymerase II (Pol II; also known as RNAP II). It transcribes protein-encoding genes into mRNA (and also the snRNA genes).
• RNA polymerase III (Pol III). It transcribes the 5S rRNA genes and all the tRNA genes.
RNA Processing: pre-mRNA → mRNA
All the primary transcripts produced in the nucleus must undergo processing steps to produce functional RNA molecules for export to the cytosol. We shall confine ourselves to a view of the steps as they occur in the processing of pre-mRNA to mRNA.
Most eukaryotic genes are split into segments. In decoding the open reading frame of a gene for a known protein, one usually encounters periodic stretches of DNA calling for amino acids that do not occur in the actual protein product of that gene. Such stretches of DNA, which get transcribed into RNA but not translated into protein, are called introns. Those stretches of DNA that do code for amino acids in the protein are called exons. Examples:
• The gene for one type of collagen found in chickens is split into 52 separate exons.
• The gene for dystrophin, which is mutated in boys with muscular dystrophy, has 79 exons.
• Even the genes for rRNA and tRNA are split by introns.
• The human genome is estimated to contain some 180,000 exons. With a current estimate of 21,000 genes, the average exon content of our genes is about 9.
• Synthesis of the cap. This is a modified guanine (G) which is attached to the 5′ end of the pre-mRNA as it emerges from RNA polymerase II (Pol II). The cap
• protects the RNA from being degraded by enzymes that degrade RNA from the 5′ end;
• serves as an assembly point for the proteins needed to recruit the small subunit of the ribosome to begin translation.
• Step-by-step removal of introns present in the pre-mRNA and splicing of the remaining exons. This step takes place as the pre-mRNA continues to emerge from Pol II.
• Synthesis of the poly(A) tail. This is a stretch of adenine (A) nucleotides. When a special poly(A) attachment site in the pre-mRNA emerges from Pol II, the transcript is cut there, and the poly(A) tail is attached to the exposed 3′ end. This completes the mRNA molecule, which is now ready for export to the cytosol. (The remainder of the transcript is degraded, and the RNA polymerase leaves the DNA.)
The above image is an electron micrograph of a mRNA-DNA hybrid molecule formed by mixing the messenger RNA (mRNA) from a clone of antibody-secreting cells with single-stranded DNA from the same kind of cells. The bar represents the length of 1000 bases. The lower diagram is an interpretation of the micrograph. The solid line represents the DNA; the dotted line the mRNA. The loops (IA, IB, etc.) represent the introns that separate the exons encoding the domains of an antibody heavy chain:
• V = variable region
• E1 = first constant region (CH1) domain
• EH = hinge region
• E2 and E3 = the nucleotides encoding the two C-terminal domains (CH2 and CH3)
The unhybridized portion of the mRNA is its poly(A) tail.
Alternative Splicing
The processing of pre-mRNA for many proteins proceeds along various paths in different cells or under different conditions. For example, early in the differentiation of a B cell (a lymphocyte that synthesizes an antibody) the cell first uses an exon that encodes a transmembrane domain that causes the molecule to be retained at the cell surface. Later, the B cell switches to using a different exon whose domain enables the protein to be secreted from the cell as a circulating antibody molecule.
Alternative splicing provides a mechanism for producing a wide variety of proteins from a small number of genes. While we humans may turn out to have only some 20 thousand genes, we probably make at least 10 times that number of different proteins. It is now estimated that 92–94% of our genes produce pre-mRNAs that are alternatively-spliced. There is evidence that the pattern of alternative splicing differs consistently in different tissues and so must be regulated. But whether all the products are functional or that many are simply the outcome of an error-prone process remains to be seen.
Alternative splicing not only provides different proteins from a single gene but also different 3' UTRs and 5' UTRs. Although not translated into protein, these untranslated regions contain signals that, for example, dictate where in the cell that protein will accumulate. Two examples:
• The 3' UTR of the bicoid gene in Drosophila directs the mRNA to the anterior of the embryo
• the same region in the VegT gene of Xenopus directs its mRNA to the vegetal pole of the embryo
One of the most dramatic examples of alternative splicing is the Dscam gene in Drosophila. This single gene contains some 116 exons of which 17 are retained in the final mRNA. Some exons are always included; others are selected from an array. Theoretically this system is able to produce 38,016 different proteins. And, in fact, over 18,000 different ones have been found in Drosophila hemolymph.
These Dscam proteins are used to establish a unique identity for each neuron. It works like this. Each developing neuron synthesizes a dozen or so Dscam mRNAs out of the thousands of possibilities. Which ones are selected appears to be simply a matter of chance, but because of the great number of possibilities, each neuron will most likely end up with a unique set of a dozen or so Dscam proteins. As each developing neuron in the central nervous system sprouts dendrites and an axon, these express its unique collection of Dscam proteins. If the various extensions of a single neuron should meet each other in the tangled web that is the hallmark of nervous tissue, they are repelled. In this way, thousands of different neurons can coexist in intimate contact without the danger of nonfunctional contacts between the various extensions of the same neuron.
neuron should encounter each other, they avoid establishing a synapse by the repulsion mediated by their identical collection of protocadherins. In this way, thousands of different neurons can coexist in intimate contact without the danger of nonfunctional contacts between the various extensions of the same neuron.
Whether a particular segment of RNA will be retained as an exon or excised as an intron can vary under different circumstances, such as
• what type of cell the gene is in
• what stage of differentiation that cell is passing through
• what extracellular signals that cell is receiving.
Clearly the switching to an alternate splicing pathway must be closely regulated.
Trans-splicing
Most genes are transcribed and their transcripts processed as described above. RNA polymerase travels down a single strand of a single gene locus to form pre-mRNA that is processed (including removal of introns) to form the mature mRNA. But there are exceptions. A number of cases have been found where two different precursor transcripts have been spliced together to form the final RNA molecule. The phenomenon is called trans-splicing.
Examples: synthesis of a single RNA molecule by splicing together transcripts from loci
• located far apart on the same chromosome or
• on opposite strands of the same gene locus or
• that are the two alleles of the gene on their separate (homologous) chromosomes.
The biological importance of these trans-spliced transcripts is still unknown for most of them. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/06%3A_Gene_Expression/6.02%3A_The_Transcription_of_DNA_into_RNA.txt |
The genetic code consists of 64 triplets of nucleotides. These triplets are called codons.With three exceptions, each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code: most of the amino acids being encoded by more than one codon.
One codon, AUG serves two related functions:
• It signals the start of translation
• It codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain
The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually "read" during the synthesis of polypeptides (the process called translation). But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene. Because DNA sequencing has become so rapid and because most genes are now being discovered at the level of DNA before they are discovered as mRNA or as a protein product, it is extremely useful to have a table of codons expressed as DNA. So here are both.
Note that for each table, the left-hand column gives the first nucleotide of the codon, the 4 middle columns give the second nucleotide, and the last column gives the third nucleotide.
The RNA Codons
Second nucleotide
U C A G
U UUU Phenylalanine (Phe) UCU Serine (Ser) UAU Tyrosine (Tyr) UGU Cysteine (Cys) U
UUC Phe UCC Ser UAC Tyr UGC Cys C
UUA Leucine (Leu) UCA Ser UAA STOP UGA STOP A
UUG Leu UCG Ser UAG STOP UGG Tryptophan (Trp) G
C CUU Leucine (Leu) CCU Proline (Pro) CAU Histidine (His) CGU Arginine (Arg) U
CUC Leu CCC Pro CAC His CGC Arg C
CUA Leu CCA Pro CAA Glutamine (Gln) CGA Arg A
CUG Leu CCG Pro CAG Gln CGG Arg G
A AUU Isoleucine (Ile) ACU Threonine (Thr) AAU Asparagine (Asn) AGU Serine (Ser) U
AUC Ile ACC Thr AAC Asn AGC Ser C
AUA Ile ACA Thr AAA Lysine (Lys) AGA Arginine (Arg) A
AUG Methionine (Met) or START ACG Thr AAG Lys AGG Arg G
G GUU Valine Val GCU Alanine (Ala) GAU Aspartic acid (Asp) GGU Glycine (Gly) U
GUC (Val) GCC Ala GAC Asp GGC Gly C
GUA Val GCA Ala GAA Glutamic acid (Glu) GGA Gly A
GUG Val GCG Ala GAG Glu GGG Gly G
The DNA Codons
These are the codons as they are read on the sense (5' to 3') strand of DNA. Except that the nucleotide thymidine (T) is found in place of uridine (U), they read the same as RNA codons. However, mRNA is actually synthesized using the antisense strand of DNA (3' to 5') as the template.
This table could well be called the Rosetta Stone of life.
The Genetic Code (DNA)
TTT Phe TCT Ser TAT Tyr TGT Cys
TTC Phe TCC Ser TAC Tyr TGC Cys
TTA Leu TCA Ser TAA STOP TGA STOP
TTG Leu TCG Ser TAG STOP TGG Trp
CTT Leu CCT Pro CAT His CGT Arg
CTC Leu CCC Pro CAC His CGC Arg
CTA Leu CCA Pro CAA Gln CGA Arg
CTG Leu CCG Pro CAG Gln CGG Arg
ATT Ile ACT Thr AAT Asn AGT Ser
ATC Ile ACC Thr AAC Asn AGC Ser
ATA Ile ACA Thr AAA Lys AGA Arg
ATG Met* ACG Thr AAG Lys AGG Arg
GTT Val GCT Ala GAT Asp GGT Gly
GTC Val GCC Ala GAC Asp GGC Gly
GTA Val GCA Ala GAA Glu GGA Gly
GTG Val GCG Ala GAG Glu GGG Gly
*When within gene; at beginning of gene, ATG signals where translation of the RNA will begin.
Codon Bias
All but two of the amino acids (Met and Trp) can be encoded by from 2 to 6 different codons. However, the genome of most organisms reveals that certain codons are preferred over others. In humans, for example, alanine is encoded by GCC four times as often as by GCG. This probably reflects a greater translation efficiency by the translation apparatus (e.g., ribosomes) for certain codons over their synonyms.
Exceptions to the Code
The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. However, some exceptions have been found. Most of these involve assigning one or two of the three STOP codons to an amino acid instead.
Mitochondrial genes
When mitochondrial mRNA from animals or microorganisms (but not from plants) is placed in a test tube with the cytosolic protein-synthesizing machinery (amino acids, enzymes, tRNAs, ribosomes) it fails to be translated into a protein.One of the reasons is because these mitochondria use UGA to encode tryptophan (Trp) rather than as a chain terminator. When translated by cytosolic machinery, synthesis stops where Trp should have been inserted. In addition, most animal mitochondria use AUA for methionine not isoleucine and all vertebrate mitochondria use AGA and AGG as chain terminators. Yeast mitochondria assign all codons beginning with CU to threonine instead of leucine (which is still encoded by UUA and UUG as it is in cytosolic mRNA).
Plant mitochondria use the universal code, and this has permitted angiosperms to transfer mitochondrial genes to their nucleus with great ease.
Nuclear genes
Violations of the universal code are far rarer for nuclear genes.
A few unicellular eukaryotes have been found that use one or two (of their three) STOP codons for amino acids instead.
Nonstandard Amino Acids
The vast majority of proteins are assembled from the 20 amino acids listed above even though some of these may be chemically altered, e.g. by phosphorylation, at a later time.
However, two cases have been found where an amino acid that is not one of the standard 20 is inserted by a tRNA into the growing polypeptide.
• selenocysteine. This amino acid is encoded by UGA. UGA is still used as a chain terminator, but the translation machinery is able to discriminate when a UGA codon should be used for selenocysteine rather than STOP. This codon usage has been found in certain Archaea, eubacteria, and animals (humans synthesize 25 different proteins containing selenium).
• pyrrolysine. In several species of Archaea and bacteria, this amino acid is encoded by UAG. How the translation machinery knows when it encounters UAG whether to insert a tRNA with pyrrolysine or to stop translation is not yet known. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/06%3A_Gene_Expression/6.03%3A_Genetic_Code.txt |
This image shows the structure of alanine transfer RNA (tRNAala) from yeast. It consists of a single strand of 77 ribonucleotides. The chain is folded on itself, and many of the bases pair with each other forming four helical regions. Loops are formed in the unpaired regions of the chain. (The bases circled in blue have been chemically-modified following synthesis of the molecule.)
At least one kind of tRNA is present for each of the 20 amino acids used in protein synthesis. (Some amino acids employ the services of two or three different tRNAs, so most cells contain as many as 32 different kinds of tRNA.) The amino acid is attached to the appropriate tRNA by an activating enzyme (one of 20 aminoacyl-tRNA synthetases) specific for that amino acid as well as for the tRNA assigned to it.
Each kind of tRNA has a sequence of 3 unpaired nucleotides — the anticodon — which can bind, following the rules of base pairing, to the complementary triplet of nucleotides — the codon — in a messenger RNA (mRNA) molecule. Just as DNA replication and transcription involve base pairing of nucleotides running in opposite direction, so the reading of codons in mRNA (5' -> 3') requires that the anticodons bind in the opposite direction.
Anticodon: 3' CGA 5'
Codon: 5' GCU 3'
The RNA Codons
Second nucleotide
U C A G
U UUU Phenylalanine (Phe) UCU Serine (Ser) UAU Tyrosine (Tyr) UGU Cysteine (Cys) U
UUC Phe UCC Ser UAC Tyr UGC Cys C
UUA Leucine (Leu) UCA Ser UAA STOP UGA STOP A
UUG Leu UCG Ser UAG STOP UGG Tryptophan (Trp) G
C CUU Leucine (Leu) CCU Proline (Pro) CAU Histidine (His) CGU Arginine (Arg) U
CUC Leu CCC Pro CAC His CGC Arg C
CUA Leu CCA Pro CAA Glutamine (Gln) CGA Arg A
CUG Leu CCG Pro CAG Gln CGG Arg G
A AUU Isoleucine (Ile) ACU Threonine (Thr) AAU Asparagine (Asn) AGU Serine (Ser) U
AUC Ile ACC Thr AAC Asn AGC Ser C
AUA Ile ACA Thr AAA Lysine (Lys) AGA Arginine (Arg) A
AUG Methionine (Met) or START ACG Thr AAG Lys AGG Arg G
G GUU Valine Val GCU Alanine (Ala) GAU Aspartic acid (Asp) GGU Glycine (Gly) U
GUC (Val) GCC Ala GAC Asp GGC Gly C
GUA Val GCA Ala GAA Glutamic acid (Glu) GGA Gly A
GUG Val GCG Ala GAG Glu GGG Gly G
Note:
• Most of the amino acids are encoded by synonymous codons that differ in the third position of the codon.
• In some cases, a single tRNA can recognize two or more of these synonymous codons.
• Example: phenylalanine tRNA with the anticodon 3' AAG 5' recognizes not only UUC but also UUU.
• The violation of the usual rules of base pairing at the third nucleotide of a codon is called "wobble"
• The codon AUG serves two related functions
• It begins every message; that is, it signals the start of translation placing the amino acid methionine at the amino terminal of the polypeptide to be synthesized.
• When it occurs within a message, it guides the incorporation of methionine.
• Three codons, UAA, UAG, and UGA, act as signals to terminate translation. They are called STOP codons.
The Steps of Translation
Initiation
• The small subunit of the ribosome binds to a site "upstream" (on the 5' side) of the start of the message.
• It proceeds downstream (5' -> 3') until it encounters the start codon AUG. (The region between the mRNA cap and the AUG is known as the 5'-untranslated region [5'-UTR].)
• Here it is joined by the large subunit and a special initiator tRNA.
• The initiator tRNA binds to the P site (shown in pink) on the ribosome.
• In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.)
Elongation
• An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with:
• an elongation factor (called EF-Tu in bacteria; EF-1 in eukaryotes)
• GTP (the source of the needed energy)
• The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red).
• The initiator tRNA is released from the P site.
• The ribosome moves one codon downstream.
• This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA.
• This last step is promoted by another protein elongation factor (called EF-G in bacteria; EF-2 in eukaryotes) and the energy of another molecule of GTP.
Note: the initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached.
The A site is so-named because it binds only to the incoming aminoacyl-tRNA; that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site.
Termination
• The end of translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA). (The nucleotides from this point to the poly(A) tail make up the 3'-untranslated region [3'-UTR] of the mRNA.)
• There are no tRNA molecules with anticodons for STOP codons.
• However, protein release factors recognize these codons when they arrive at the A site.
• Binding of these proteins —along with a molecule of GTP — releases the polypeptide from the ribosome.
• The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis.
Polysomes
A single mRNA molecule usually has many ribosomes traveling along it, in various stages of synthesizing the protein. This complex is called a polysome.
Codon Bias
All but two of the amino acids (Met and Trp) can be encoded by from 2 to 6 different codons. However, the genome of most organisms reveals that certain codons are preferred over others. In humans, for example, alanine is encoded by GCC four times as often as by GCG. This probably reflects a greater translation efficiency by the translation apparatus for certain codons over their synonyms.
• At the start of translation, two or more of a set of synonymous codons (e.g., the 6 codons that incorporate leucine in the growing protein) are used alternately. The need to locate first one and then another tRNA for that amino acid slows down the rate of translation.
• This may aid in keeping ribosomes from bumping into each other on the polysome.
• It may also provide more time for the nascent protein to begin to fold correctly as it emerges from the ribosome.
• Once translation is well underway (after 30–50 amino acids have been added), one particular codon tends to be chosen each time its amino acid is called for. Presumably this now increases the efficiency (speed) of translation.
• Most organisms have more than the 61 genes needed to encode a tRNA for each of the 61 codons (we have 270 tRNA genes). The presence of multiple genes for tRNAs with an identical anticodon increases the concentration of tRNAs able to bind a particular codon. Messenger RNAs — especially those of active genes — tend to favor codons that correspond to abundant tRNAs carrying the anticodon.
Codon bias even extends to pairs of codons: wherever a human protein contains the amino acids Ala-Glu, the gene encoding those amino acids is seven times as likely to use the codons GCAGAG rather than the synonymous GCCGAA. Codon bias is exploited by the biotechnology industry to improve the yield of the desired product. The ability to manipulate codon bias may also usher in a era of safer vaccines.
Quality Control
Defective mRNA molecules can be produced by mutations in the gene as well as errors introduced during transcription (albeit at a remarkably low rate). In addition to producing mRNAs with incorrect codons for amino acids, these errors can produce mRNA molecules that have
• Premature Termination Codons (PTCs); that is, the introduction of a STOP codon before the normal end of the message. Translation of these mRNAs produces a truncated protein that is probably ineffective and may be harmful. The problem can sometimes be solved by Nonsense-Mediated mRNA Decay (NMD).
• no STOP codon. These produce "nonstop" transcripts. The problem can be solved by Nonstop mRNA Decay.
Nonsense-Mediated mRNA Decay (NMD)
Premature termination codons (PTCs) may be generated by "nonsense" mutations, frameshifts, and RNA processing (intron removal) errors. They are also an inevitable consequence of creating antigen receptors on B cells and T cells.
Mechanisms
• During RNA processing within the nucleus, protein complexes are added at each spot where adjacent exons are spliced together. (These are important signals for exporting the mRNA to the cytoplasm.)
• In the cytoplasm, as the ribosome moves down the mRNA, these complexes are removed (and sent back to the nucleus for reuse).
• If the ribosome encounters a premature termination codon, the final exon-exon tag(s) are not removed, and this marks the defective mRNA for destruction (in P bodies).
Mutations that introduce premature termination codons are responsible for some cases of such inherited human diseases as cystic fibrosis and Duchenne muscular dystrophy (DMD).
A drug, designated PTC124 or ataluren, causes the ribosome to skip over PTCs while still enabling normal termination of translation. PTC124 has shown promise in animal models of cystic fibrosis and DMD and phase II clinical trials are now being conducted on humans.
Nonstop mRNA Decay
Nonstop transcripts occur when there is no STOP codon in the message. As a result the ribosome is unable to recruit the release factors needed to leave the mRNA. Nonstop transcripts are formed during RNA processing, e.g., by having the poly(A) tail put on before the STOP codon is reached.
Mechanisms
Eukaryotes and bacteria handle the problem of no STOP codon differently.
• In eukaryotes, when the ribosome stalls at the end of the poly(A) tail, proteins are recruited to release the ribosome for reuse and to degrade the faulty message.
• In bacteria, a special RNA molecule — called tmRNA saves the day. It is called tmRNA because it has the properties of both a transfer RNA and a messenger RNA. The transfer part adds alanine to the A site on the ribosome. The ribosome then moves on to the messenger part which encodes 10 amino acids that target the molecule for destruction (and releases the ribosome for reuse).
Regulation of Translation
The expression of most genes is controlled at the level of their transcription. Transcription factors (proteins) bind to promoters and enhancers turning on (or off) the genes they control. However, gene expression can also be controlled at the level of translation.
By General RNA-Degradation Machinery
P bodies
The cytosol of eukaryotes contains protein complexes that compete with ribosomes for access to mRNAs. As these increase their activity, they sequester mRNAs in larger aggregates called P bodies (for "processing bodies", but this processing should not be confused with the processing of pre-mRNA to mature mRNA that occurs in the nucleus).
These protein complexes break down the mRNA by
• removing its "cap"
• removing its poly(A) tail
• degrading the remaining message (nibbling away in the 5' -> 3' direction)
What controls the dynamic balance between ribosomes and P bodies for access to mRNAs remains to be learned. But this mechanism provides for
• destruction of "bad" mRNAs (e.g., those with premature STOP codons
• turnover of mRNAs thus increasing the flexibility of gene expression in the cell
Exosomes
These are hollow macromolecular complexes with two openings. They take in unfolded RNA molecules and degrade them in the 3' -> 5' direction. (In neither structure nor function do these exosomes resemble the exosomes involved in antigen presentation that unfortunately share the same name.)
By MicroRNAs (miRNAs)
Here small RNA molecules bind to a complementary portion in the 3'-UTR of the mRNA and prevent it from being translated by ribosomes and/or trigger its destruction. Both these activities take place in P bodies.
By Riboswitches
It turns out that the regulation of the level of certain metabolites is controlled by riboswitches. A riboswitch is a part of a molecule of messenger RNA (mRNA) with a specific binding site for the metabolite (or a close relative).
Examples:
• If thiamine pyrophosphate (the active form of thiamine [vitamin B1]) is available in the culture medium of E. coli,
• It binds to a messenger RNA whose protein product is an enzyme needed to synthesize thiamine from the ingredients in minimal medium.
• Binding induces an allosteric shift in the structure of the mRNA so that it can no longer bind to a ribosome and thus cannot be translated into the enzyme.
• E. coli no longer wastes resources on synthesizing a vitamin that is available preformed.
A thiamine pyrophosphate riboswitch has also been found in plants, archaea, and Neurospora. The one in Neurospora regulates genes involved in vitamin B1 metabolism by alternative splicing of their transcripts. (Other riboswitches act on transcription rather than translation
• If vitamin B12 is present in the cell, it binds to the mRNA which encodes a protein needed to import the vitamin from the culture medium. This, too, induces an allosteric shift in the mRNA that prevents it from binding a ribosome. E. coli no longer wastes resources on synthesizing a transporter for a vitamin that it already has enough of.
• Some Gram-positive bacteria (E. coli is Gram-negative) control the level of a sugar needed to synthesize their cell wall with a riboswitch. In this case, as the concentration of the sugar builds up, it binds to the messenger RNA (mRNA) whose product is the enzyme that makes the sugar. This causes the mRNA to self-destruct so production of the enzyme — and thus the sugar — ceases.
It has been suggested that these regulatory mechanisms, which do not involve any protein, are a relict from an "RNA world". | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/06%3A_Gene_Expression/6.04%3A_The_Translation_of_RNA_into_Proteins.txt |
Occasionally researches encounter a gene with a sequence of nucleotides that does not match exactly that in its RNA product:
• messenger RNA (mRNA)
• ribosomal RNA (rRNA)
• transfer RNA (tRNA)
• microRNA (miRNA)
If the product is mRNA, some of the codons in the open reading frame (ORF) of the gene specify different amino acids from those in the protein translated from the mRNA of the gene.
The reason is RNA editing: the alteration of the sequence of nucleotides in the RNA
• after it has been transcribed from DNA but
• before it is translated into protein
RNA editing occurs by two distinct mechanisms:
• Substitution Editing: chemical alteration of individual nucleotides (the equivalent of point mutations).
These alterations are catalyzed by enzymes that recognize a specific target sequence of nucleotides (much like restriction enzymes):
• cytidine deaminases that convert a C in the RNA to uracil (U);
• adenosine deaminases that convert an A to inosine (I), which the ribosome translates as a G. Thus a CAG codon (for Gln) can be converted to a CGG codon (for Arg).
• Insertion/Deletion Editing: insertion or deletion of nucleotides in the RNA.
These alterations are mediated by guide RNA molecules that
• base-pair as best they can with the RNA to be edited and
• serve as a template for the addition (or removal) of nucleotides in the target
Substitution Editing
The human APOB gene
Humans have a single locus encoding the APOB gene.
• It contains 29 exons (separated by 28 introns).
• The exons contain a total of 4564 codons.
• Codon 2153 is CAA, which is a codon for the amino acid glutamine (Gln).
• The gene is expressed in cells of both the liver and the intestine.
• In both locations, transcription produces a pre-messenger RNA that must be spliced to produce the mRNA to be translated into protein.
• In the Liver. Here the process occurs normally producing apolipoprotein B-100 — a protein containing 4,563 amino acids — that is essential for the transport of cholesterol and other lipids in the blood.
• In the Intestine
• In the cells of the intestine, an additional step of pre-mRNA processing occurs: the chemical modification of the C nucleotide in Codon 2153 (CAA) into a U.
• This RNA editing changes the codon from one encoding the amino acid glutamine (Gln) to a STOP codon (UAA)
• The modification is catalyzed by the enzyme cytidine deaminase that
• recognizes the sequence of the RNA at that one place in the molecule and
• catalyzes the deamination of C thus forming U.
• Translation of the mRNA stops at codon #2153 forming apolipoprotein B-48 — a protein containing 2152 amino acids — that aids in the absorption of dietary lipids from the contents of the intestine.
DNA can also be edited. B cells express another cytidine deaminase (called activation-induced deaminase or AID) that is essential for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. Humans with disabling mutations in the gene for this enzyme produce only IgM antibodies. However, here the enzyme is acting on DNA, not RNA. In attempting to repair the mismatch formed (dC•dG converted to dU•dG), the normal DNA repair machinery of the cell produces CSR or SHM as the situation warrants. (This process is also responsible for the occasional aberrant translocation of the heavy-chain gene segments to a proto-oncogene. The result is a B-cell cancer — a lymphoma or leukemia.)
Other examples of substitution editing
• Some mRNAs, tRNAs, and rRNAs in both the mitochondria and chloroplasts of plants;
• mRNAs encoding subunits of some receptors of neurotransmitters in the mammalian brain, e.g.,
• the AMPA receptor for Glu
• a serotonin receptor
• a tRNA in the mitochondria of the duckbill platypus
Insertion/Deletion Editing
The gene in mitochondria of Trypanosoma brucei
Several genes encoded in the mitochondrial DNA of this species (the cause of sleeping sickness in humans) encode transcripts that must be edited to make the mRNA molecules that will be translated into protein.
Editing requires a special class of RNA molecules called guide RNA (gRNA).
These small molecules have sequences that are complementary to the region around the site to be edited. The guide RNA base-pairs — as best it can — with this region. Note that in addition to the usual purine-pyrimidine pairing of C-G and A-U, G-U base-pairing can also occur.
Because of the lack of precise sequence complementarity, bulges occur either
• in the guide RNA where, usually, there are As not found in the transcript to be edited (as shown here) or
• in the transcript to be edited.
The bulges are eliminated by cutting the backbone of the shorter molecule and inserting complementary bases.
• In the first case (shown here) this produces insertions (here of Us)
• In the second case (not shown) this produces deletions.
Note that in the example shown here, the insertion of 4 nucleotides has created a frameshift so that the amino acids encoded downstream (after Val) in the edited RNA are entirely different from those specified by the gene itself.
Other examples of insertion/deletion editing
Insertion/deletion editing has also been found occur with
• mRNA, rRNA, and tRNA transcripts in the mitochondria of the slime mold Physarum polycephalum
• in measles virus transcripts
Why RNA Editing?
Good question. Some possibilities:
• So RNA editing appears to be here to stay. In fact, defects in RNA editing are associated with some human cancers as well as with amyotrophic lateral sclerosis (ALS — "Lou Gehrig's disease").
6.06: Expressed Sequence Tags
Only a very small percentage (1.2% in humans) of the DNA in vertebrate genomes encodes proteins (the "proteome") because the exons of most genes are separated by much-longer introns between our genes lie vast amounts of DNA much of which appears to regulate the expression of our genes but is not transcribed and translated into a protein product. So even when the complete sequence of a genome is known, it is often difficult to spot particular genes (open reading frames or ORFs).
One approach to solving the problem is to examine a transcriptome of the organism. Most commonly this is defined as: All the messenger RNA (mRNA) molecules transcribed from the genome. It is "a" transcriptome, not "the" transcriptome, because what genes are transcribed in a cell depends on the kind of cell (e.g., liver cell vs. lymphocyte) and what the cell is doing at that time, e.g.,
• getting ready to divide by mitosis;
• responding to the arrival of a hormone or cytokine;
• getting ready to secrete a protein product.
Expressed Sequence Tags (ESTs)
ESTs are short (200–500 nucleotides) DNA sequences that can be used to identify a gene that is being expressed in a cell at a particular time.
The Procedure:
• Isolate the messenger RNA (mRNA) from a particular tissue (e.g., liver)
• Treat it with reverse transcriptase. Reverse transcriptase is a DNA polymerase that uses RNA as its template. Thus it is able to make genetic information flow in the reverse (RNA ->DNA) of its normal direction (DNA -> RNA).
• This produces complementary DNA (cDNA). Note that cDNA differs from the normal gene in lacking the intron sequences.
• Sequence 200–500 nucleotides at both the 5′ and 3′ ends of each cDNA.
• Examine the database of the organism's genome to find a matching sequence.
6.07: Ribosomal RNA (rRNA) Gene Cluster
The above picture shows an electron micrograph (26,500x) showing transcription of the DNA encoding ribosomal RNA (rRNA) molecules in the nucleolus of a developing egg cell of the spotted newt.
Eukaryotes have several hundred identical genes encoding ribosomal RNA.
The long filaments (green arrow) are DNA molecules coated with proteins. The fibers extending in clusters from the main axes are molecules of ribosomal RNA which will be used in the construction of the cell's ribosomes.
Note how transcription begins at one end of each gene, with the RNA molecules getting longer (red arrow) as they proceed toward completion.
Note also the large number (up to 100) of RNA molecules that are transcribed simultaneously from each gene.
The portions of DNA bare of RNA appear to be genetically inactive. (Courtesy of O. L. Miller, Jr., and Barbara R. Beatty, Biology Division, Oak Ridge National Laboratory.) | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/06%3A_Gene_Expression/6.05%3A_RNA_Editing.txt |
• 7.2: The Cell Cycle
A eukaryotic cell cannot divide into two, the two into four, etc. unless two processes alternate: doubling of its genome (DNA) in S phase (synthesis phase) of the cell cycle; halving of that genome during mitosis (M phase).
• 7.3: Mitosis
When a eukaryotic cell divides into two, each daughter or progeny cell must receive a complete set of genes (for diploid cells, this means 2 complete genomes, (2n) a pair of centrioles (in animal cells) some mitochondria and, in plant cells, chloroplasts as well some ribosomes, a portion of the endoplasmic reticulum, and perhaps other organelles,
• 7.4: Polyploidy
Cells (and their owners) are polyploid if they contain more than two haploid (n) sets of chromosomes; that is, their chromosome number is some multiple of n greater than the 2n content of diploid cells. For example, triploid (3n) and tetraploid cell (4n) cells are polyploid.
• 7.5: Endoreplication
Endoreplication is the replication of DNA during the S phase of the cell cycle without the subsequent completion of mitosis and/or cytokinesis. Endoreplication is also known as endoreduplication. Endoreplication occurs in certain types of cells in both animals and plants.
• 7.6: Sex Chromosomes
The nuclei of human cells contain 22 autosomes and 2 sex chromosomes. In females, the sex chromosomes are the 2 X chromosomes. Males have one X chromosome and one Y chromosome. The presence of the Y chromosome is decisive for unleashing the developmental program that leads to a baby boy.
• 7.7: Meiosis
Mitosis produces two cells with the same number of chromosomes as the parent cell. Mitosis of a diploid cell (2n) produces two diploid daughter cells. If two diploid cells went on to participate in sexual reproduction, their fusion would produce a tetraploid (4n) zygote. The solution for this problem is Meiosis.
Thumbnail: Life cycle of the cell. (CC BY-SA 4.0; BruceBlaus).
07: Cell Division
In eukaryotes, chromosomes consist of a single molecule of DNA associated with many copies of 5 kinds of histones. Histones are proteins rich in lysine and arginine residues and thus positively-charged. For this reason they bind tightly to the negatively-charged phosphates in DNA. Cchromosomes have a small number of copies of many different kinds of non-histone proteins. Most of these are transcription factors that regulate which parts of the DNA will be transcribed into RNA.
Structure
For most of the life of the cell, chromosomes are too elongated and tenuous to be seen under a microscope. However, before a cell is ready to divide by mitosis, each chromosome is duplicated (during S phase of the cell cycle). As mitosis begins, the duplicated chromosomes condense into short (~ 5 µm) structures which can be stained and easily observed under the light microscope. These duplicated chromosomes are called dyads.
When first seen, the duplicates are held together at their centromeres. In humans, the centromere contains 1–10 million base pairs of DNA. Most of this is repetitive DNA: short sequences (e.g., 171 bp) repeated over and over in tandem arrays. While they are still attached, it is common to call the duplicated chromosomes sister chromatids, but this should not obscure the fact that each is a bona fide chromosome with a full complement of genes.
The kinetochore is a complex of >80 different proteins that forms at each centromere and serves as the attachment point for the spindle fibers that will separate the sister chromatids as mitosis proceeds into anaphase. The shorter of the two arms extending from the centromere is called the p arm; the longer is the q arm. Staining with the trypsin-giemsa method reveals a series of alternating light and dark bands called G bands. G bands are numbered and provide "addresses" for the assignment of gene loci.
Chromosome Numbers
All animals have a characteristic number of chromosomes in their body cells called the diploid (or 2n) number. These occur as homologous pairs, one member of each pair having been acquired from the gamete of one of the two parents of the individual whose cells are being examined. The gametes contain the haploid number (n) of chromosomes. In plants, the haploid stage takes up a larger part of its life cycle.
Table 7.1.1: Diploid numbers of some commonly studied organisms
Homo sapiens (human) 46
Mus musculus (house mouse) 40
Drosophila melanogaster (fruit fly) 8
Caenorhabditis elegans (microscopic roundworm) 12
Saccharomyces cerevisiae (budding yeast) 32
Arabidopsis thaliana (plant in the mustard family) 10
Xenopus laevis (South African clawed frog) 36
Canis familiaris (domestic dog) 78
Gallus gallus (chicken) 78
Zea mays (corn or maize) 20
Muntiacus reevesi (the Chinese muntjac, a deer) 23
Muntiacus muntjac (its Indian cousin) 6
Myrmecia pilosula (an ant) 2
Parascaris equorum var. univalens (parasitic roundworm) 2
Cambarus clarkii (a crayfish) 200
Equisetum arvense (field horsetail, a plant) 216
Karyotypes
The complete set of chromosomes in the cells of an organism is its karyotype. It is most often studied when the cell is at metaphase of mitosis when all the chromosomes are present as dyads. The karyotype of the human female contains 23 pairs of homologous chromosomes: 22 pairs of autosomes and an additional 1 pair of X chromosomes. In contrast, the karyotype of the human male contains the same 22 pairs of autosomes with one X chromosome and one Y chromosome. A gene on the Y chromosome designated SRY is the master switch for making a male. Both X and Y chromosomes are called the sex chromosomes.
Above is a human karyotype (of which sex?). It differs from a normal human karyotype in having an extra #21 dyad. As a result, this individual suffered from a developmental disorder called Down Syndrome. The inheritance of an extra chromosome, is called trisomy, in this case trisomy 21. It is an example of aneuploidy
Translocations
Karyotype analysis can also reveal translocations between chromosomes. A number of these are associated with cancers, for example
• the Philadelphia chromosome (Ph1) formed by a translocation between chromosomes 9 and 22 and a cause of Chronic Myelogenous Leukemia (CML)
• a translocation between chromosomes 8 and 14 that causes Burkitt's lymphoma
• a translocation between chromosomes 18 and 14 that causes B-cell leukemia
Fluorescence in situ Hybridization (FISH)
Figure 7.1.3 provides dramatic evidence of the truth of the story of chromosomes. A piece of single-stranded DNA was prepared that was complementary to the DNA of the human gene encoding the enzyme muscle glycogen phosphorylase. A fluorescent molecule was attached to this DNA. The dyads in a human cell were treated to denature their DNA; that is, to make the DNA single-stranded. When this preparation was treated with the fluorescent DNA, the complementary sequences found and bound each other. This produced a fluorescent spot close to the centromere of each sister chromatid of two homologous dyads (of chromosome 11, upper right). This analytical procedure, which here revealed the gene locus for the muscle glycogen phosphorylase gene, is called fluorescence in situ hybridization or FISH.
DNA Content
The molecule of DNA in a single human chromosome ranges in size from 50 x 106 nucleotide pairs in the smallest chromosome (stretched full-length this molecule would extend 1.7 cm) up to 250 x 106nucleotide pairs in the largest (which would extend 8.5 cm). Stretched end-to-end, the DNA in a single human diploid cell would extend over 2 meters. In the intact chromosome, however, this molecule is packed into a much more compact structure. The packing reaches its extreme during mitosis when a typical chromosome is condensed into a structure about 5 µm long (a 10,000-fold reduction in length). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/07%3A_Cell_Division/7.01%3A_Chromosomes.txt |
A eukaryotic cell cannot divide into two, the two into four, etc. unless two processes alternate:
• doubling of its genome (DNA) in S phase (synthesis phase) of the cell cycle;
• halving of that genome during mitosis (M phase).
Control of the Cell Cycle
The passage of a cell through the cell cycle is controlled by proteins in the cytoplasm. Among the main players in animal cells are:
• Their levels in the cell rise and fall with the stages of the cell cycle.
• Their levels in the cell remain fairly stable, but each must bind the appropriate cyclin (whose levels fluctuate) in order to be activated. They add phosphate groups to a variety of protein substrates that control processes in the cell cycle.
• The anaphase-promoting complex (APC). (The APC is also called the cyclosome, and the complex is often designated as the APC/C.) The APC/C
• triggers the events leading to destruction of cohesin (as described below) thus allowing the sister chromatids to separate
• degrades the mitotic (B) cyclins
Steps in the cycle
• A rising level of G1-cyclins bind to their Cdks and signal the cell to prepare the chromosomes for replication.
• A rising level of S-phase promoting factor (SPF) — which includes A cyclins bound to Cdk2 — enters the nucleus and prepares the cell to duplicate its DNA (and its centrosomes).
• As DNA replication continues, cyclin E is destroyed, and the level of mitotic cyclins begins to rise (in G2).
• Translocation of M-phase promoting factor (the complex of mitotic [B] cyclins with the M-phase Cdk [Cdk1]) into the nucleus initiates
• assembly of the mitotic spindle
• breakdown of the nuclear envelope
• cessation of all gene transcription
• condensation of the chromosomes
• These events take the cell to metaphase of mitosis.
• At this point, the M-phase promoting factor activates the anaphase-promoting complex (APC/C) which
• allows the sister chromatids at the metaphase plate to separate and move to the poles (= anaphase), completing mitosis.
Separation of the sister chromatids depends on the breakdown of the cohesin that has been holding them together. It works like this.
• Cohesin breakdown is caused by a protease called separase (also known as separin).
• Separase is kept inactive until late metaphase by an inhibitory chaperone called securin.
• Anaphase begins when the anaphase promoting complex (APC/C) destroys securin (by tagging it with ubiquitin for deposit in a proteasome) thus ending its inhibition of separase and allowing
• separase to break down cohesin
• destroys B cyclins. This is also done by attaching them to ubiquitin which targets them for destruction by proteasomes.
• turns on synthesis of G1 cyclins (D) for the next turn of the cycle.
• degrades geminin, a protein that has kept the freshly-synthesized DNA in S phase from being re-replicated before mitosis.
This is only one of the mechanisms by which the cell ensures that every portion of its genome is copied once — and only once — during S phase
Some cells deliberately cut the cell cycle short allowing repeated S phases without completing mitosis and/or cytokinesis. This is called endoreplication.
Meiosis and the Cell Cycle
The special behavior of the chromosomes in meiosis I requires some special controls. Nonetheless, passage through the cell cycle in meiosis I (as well as meiosis II, which is essentially a mitotic division) uses many of the same players, e.g., MPF and APC. (In fact, MPF is also called maturation-promoting factor for its role in meiosis I and II of developing oocytes.
Quality Control of the Cell Cycle
The cell has several systems for interrupting the cell cycle if something goes wrong.
DNA damage checkpoints. These sense DNA damage both before the cell enters S phase (a G1 checkpoint) as well as after S phase (a G2 checkpoint). Damage to DNA before the cell enters S phase inhibits the action of Cdk2 thus stopping the progression of the cell cycle until the damage can be repaired. If the damage is so severe that it cannot be repaired, the cell self-destructs by apoptosis. Damage to DNA after S phase (the G2 checkpoint), inhibits the action of Cdk1 thus preventing the cell from proceeding from G2 to mitosis. A check on the successful replication of DNA during S phase. If replication stops at any point on the DNA, progress through the cell cycle is halted until the problem is solved.
Spindle checkpoints. Some of these that have been discovered to detect any failure of spindle fibers to attach to kinetochores and arrest the cell in metaphase until all the kinetochores are attached correctly. They detect improper alignment of the spindle itself and block cytokinesis. Furthermore, they trigger apoptosis if the damage is irreparable. All the checkpoints examined require the services of a complex of proteins. Mutations in the genes encoding some of these have been associated with cancer; that is, they are oncogenes. This should not be surprising since checkpoint failures allow the cell to continue dividing despite damage to its integrity.
Examples of checkpoints
p53
The p53 protein senses DNA damage and can halt progression of the cell cycle in G1 (by blocking the activity of Cdk2). Both copies of the p53 gene must be mutated for this to fail so mutations in p53 are recessive, and p53 qualifies as a tumor suppressor gene.
The p53 protein is also a key player in apoptosis, forcing "bad" cells to commit suicide. So if the cell has only mutant versions of the protein, it can live on — perhaps developing into a cancer. More than half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/07%3A_Cell_Division/7.02%3A_The_Cell_Cycle.txt |
This image provides a graphic illustration of the problem. It shows a bit (no more than 3%) of the single molecule of DNA released from a single human chromosome. (The chromosome was treated to remove its histones). Remembering that this is 3% of the DNA of only one of the 46 chromosomes in the human diploid cell, you can appreciate the problem faced by the cell of how to separate without error these great lengths of DNA without creating horrible tangles.
The solution to this problem is:
1. Duplicate each chromosome during the S phase of the cell cycle.
2. This produces dyads, each made up of 2 identical sister chromatids. These are held together by a ring of proteins called cohesin.
3. Condense the chromosomes into a compact form. This requires ATP and protein complexes called condensins.
4. Separate the sister chromatids and
5. Distribute these equally between the two daughter cells.
7.04: Polyploidy
Cells (and their owners) are polyploid if they contain more than two haploid (n) sets of chromosomes; that is, their chromosome number is some multiple of n greater than the 2n content of diploid cells. For example, triploid (3n) and tetraploid cell (4n) cells are polyploid.
Polyploidy in plants
Polyploidy is very common in plants, especially in angiosperms. From 30% to 70% of today's angiosperms are thought to be polyploid. Species of coffee plant with 22, 44, 66, and 88 chromosomes are known. This suggests that the ancestral condition was a plant with a haploid (n) number of 11 and a diploid (2n) number of 22, from which evolved the different polyploid descendants. In fact, the chromosome content of most plant groups suggests that the basic angiosperm genome consists of the genes on 7–11 chromosomes. Domestic wheat, with its 42 chromosomes, is probably hexaploid (6n), where n (the ancestral haploid number) was 7.
Some other examples:
Plant Probable ancestral haploid number Chromosome
number
Ploidy level
domestic oat 7 42 6n
peanut 10 40 4n
sugar cane 10 80 8n
banana 11 22, 33 2n, 3n
white potato 12 48 4n
tobacco 12 48 4n
cotton 13 52 4n
apple 17 34, 51 2n, 3n
Polyploid plants not only have larger cells but the plants themselves are often larger. This has led to the deliberate creation of polyploid varieties of such plants as watermelons, marigolds, and snapdragons.
Origin of Polyploidy
Polyploidy has occurred often in the evolution of plants. The process can begin if diploid (2n) gametes are formed. These can arise in at least two ways.
• The gametes may be formed by mitosis instead of meiosis.
• Plants, in contrast to animals, form germ cells (sperm and eggs) from somatic tissues. If the chromosome content of a precursor somatic cell has accidentally doubled (e.g., as a result of passing through S phase of the cell cycle without following up with mitosis and cytokinesis), then gametes containing 2n chromosomes are formed.
Polyploidy also occurs naturally in certain plant tissues.
• As the endosperm (3n) develops in corn (maize) kernels (Zea mays), its cells undergo successive rounds (as many as 5) of endoreplication producing nuclei that range as high as 96n.
• When rhizobia infect the roots of their legume host, they induce the infected cells to undergo endoreplication producing cells that can become 128n (from 6 rounds of endoreplication).
Polyploidy can also be induced in the plant-breeding laboratory by treating dividing cells with colchicine. This drug disrupts microtubules and thus prevents the formation of a spindle. Consequently, the duplicated chromosomes fail to separate in mitosis. Onion cells exposed to colchicine for several days may have over 1000 chromosomes inside.
Polyploidy and Speciation
When a newly-arisen tetraploid (4n) plant tries to breed with its ancestral species (a backcross), triploid offspring are formed. These are sterile because they cannot form gametes with a balanced assortment of chromosomes. However, the tetraploid plants can breed with each other. So in one generation, a new species has been formed. Polyploidy even allows the formation of new species derived from different ancestors.
In 1928, the Russian plant geneticist Karpechenko produced a new species by crossing a cabbage with a radish. Although belonging to different genera (Brassica and Raphanus respectively), both parents have a diploid number of 18. Fusion of their respective gametes (n=9) produced mostly infertile hybrids. However, a few fertile plants were formed, probably by the spontaneous doubling of the chromosome number in somatic cells that went on to form gametes (by meiosis). Thus these contained 18 chromosomes — a complete set of both cabbage (n=9) and radish (n=9) chromosomes. Fusion of these gametes produced vigorous, fully-fertile, polyploid plants with 36 chromosomes. (They had the roots of the cabbage and the leaves of the radish.)
These plants could breed with each other, but not with either the cabbage or radish ancestors, so Karpechenko had produced a new species. The process also occurs in nature. Three species in the mustard family (Brassicaceae) appear to have arisen by hybridization and polyploidy from three other ancestral species:
• B. oleracea (cabbage, broccoli, etc.) hybridized with B. nigra (black mustard) B. carinata (Abyssinian mustard).
• B. oleracea x B. rapa (turnips) B. napus (rutabaga)
• B. nigra x B. rapa B. juncea (leaf mustard)
Modern wheat and perhaps some of the other plants listed in the table above have probably evolved in a similar way.
Polyploidy in Animals
Polyploidy is much rarer in animals. It is found in some insects, fishes, amphibians, and reptiles. Until recently, no polyploid mammal was known. However, the 23 September 1999 issue of Nature reported that a polyploid (tetraploid; 4n = 102) rat has been found in Argentina. Polyploid cells are larger than diploid ones; not surprising in view of the increased amount of DNA in their nucleus. The liver cells of the Argentinian rat are larger than those of its diploid relatives, and its sperm are huge in comparison. Normal mammalian sperm heads contain some 3.3 picograms (10-12 g) of DNA; the sperm of the rat contains 9.2 pg. Although only one mammal is known to have all its cells polyploid, many mammals have polyploid cells in certain of their organs, e.g, the liver.
7.05: Endoreplication
Endoreplication is the replication of DNA during the S phase of the cell cycle without the subsequent completion of mitosis and/or cytokinesis. Endoreplication is also known as endoreduplication. Endoreplication occurs in certain types of cells in both animals and plants. There are several variations:
• replication of DNA with completion of mitosis, but no cytokinesis.
• repeated replication of DNA without forming new nuclei in telophase. This can result in:
1. Polyploidy: the replicated chromosomes retain their individual identity.
2. Polyteny: the replicated chromosomes remain in precise alignment forming "giant" chromosomes.
3. various intermediate conditions between 1 and 2
The photomicrograph shows the polytene chromosomes in a salivary gland cell of a Drosophila melanogaster larva. Such chromosomes are found in other large, active cells as well.
• Each of Drosophila's 4 pairs of chromosomes has undergone 10 rounds of DNA replication.
• The maternal and paternal homologs — as well as all their duplicates — are aligned in exact register with each other.
• So each chromosome consists of a cable containing 2048 identical strands of DNA.
• These are so large that they can be seen during interphase; even with a low-power light microscope.
Function of polyteny
The probable answer: gene amplification. Having multiple copies of genes permits a high level of gene expression; that is, abundant transcription and translation to produce the gene products. This would account for polyteny being associated with large, metabolically active cells (like salivary glands). Polytene chromosomes are subdivided into some 5,000 dense bands separated by light interbands. The bands are further subdivided into:
• dark bands of heterochromatin where the DNA is tightly compacted and there is little gene transcription;
• gray bands of euchromatin where the DNA is more loosely compacted and there is active gene transcription.
These eight photomicrographs () show the changes in the puffing pattern of equivalent segments of chromosome 3 in Drosophila melanogaster over the course of some 20 hours of normal development.
Note that during this period, when the larvae were preparing to pupate, certain puffs formed, regressed, and formed again. However, the order in which they did often differed. For example, in the larva, band 62E becomes active before 63E (c, d, and e), but when pupation begins, the reverse is true (g, h).
In general, early puffs reflect the activation of genes encoding transcription factors. These proteins then bind to the promoters of other genes, turning them on and causing a puff to appear at their loci. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/07%3A_Cell_Division/7.03%3A_Mitosis.txt |
The nuclei of human cells contain 22 autosomes and 2 sex chromosomes. In females, the sex chromosomes are the 2 X chromosomes. Males have one X chromosome and one Y chromosome. The presence of the Y chromosome is decisive for unleashing the developmental program that leads to a baby boy.
The Y Chromosome
In making sperm by meiosis, the X and Y chromosomes must separate in anaphase just as homologous autosomes do. This occurs without a problem because, like homologous autosomes, the X and Y chromosome synapse during prophase of meiosis I. There is a small region of homology shared by the X and Y chromosome and synapsis occurs at that region.
This image, shows synapsis of the X and Y chromosomes of a mouse during prophase of meiosis I. Crossing over occurs in two regions of pairing, called the pseudoautosomal regions. These are located at opposite ends of the chromosome.
The Pseudoautosomal Regions
The pseudoautosomal regions get their name because any genes located within them (so far only 9 have been found) are inherited just like any autosomal genes. Males have two copies of these genes: one in the pseudoautosomal region of their Y, the other in the corresponding portion of their X chromosome. So males can inherit an allele originally present on the X chromosome of their father and females can inherit an allele originally present on the Y chromosome of their father.
Genes outside the pseudoautosomal regions
Although 95% of the Y chromosome lies between the pseudoautosomal regions, only 27 different functional genes have been found here. Over half of this region is genetically-barren heterochromatin. Of the 27 genes found in the euchromatin, some encode proteins used by all cells. The others encode proteins that appear to function only in the testes. A key player in this latter group is SRY.
SRY
SRY (for sex-determining region Y) is a gene located on the short (p) arm just outside the pseudoautosomal region. It is the master switch that triggers the events that converts the embryo into a male. Without this gene, you get a female instead.
What is the evidence?
1. On very rare occasions aneuploid humans are born with such karyotypes as XXY, XXXY, and even XXXXY. Despite their extra X chromosomes, all these cases are male.
2. This image shows two mice with an XX karyotype (and thus they should be female). However, as you may be able to see, they have a male phenotype. This is because they are transgenic for SRY. Fertilized XX eggs were injected with DNA carrying the SRY gene.
Although these mice have testes, male sex hormones, and normal mating behavior, they are sterile.
1. Another rarity: XX humans with testicular tissue because a translocation has placed the SRY gene on one of the X chromosomes
2. Still another rarity that demonstrates the case: women with an XY karyotype who, despite their Y chromosome, are female because of a destructive mutation in SRY.
In 1996, a test based on a molecular probe for SRY was used to ensure that potential competitors for the women's Olympic events in Atlanta had no SRY gene. But because of possibilities like that in case 4, this testing is no longer used to screen female Olympic athletes.
The X Chromosome
The X chromosome carries nearly 1,000 genes but few, if any, of these have anything to do directly with sex. However, the inheritance of these genes follows special rules. These arise because:
• males have only a single X chromosome
• almost all the genes on the X have no counterpart on the Y; thus
• any gene on the X, even if recessive in females, will be expressed in males.
Genes inherited in this fashion are described as sex-linked or, more precisely, X-linked.
X-Linkage example
Hemophilia is a blood clotting disorder caused by a mutant gene encoding either
• clotting factor VIII, causing hemophilia A or
• clotting factor IX, causing hemophilia B.
Both genes are located on the X chromosome (shown here in red). With only a single X chromosome, males who inherit the defective gene (always from their mother) will be unable to produce the clotting factor and suffer from difficult-to-control episodes of bleeding. In heterozygous females, the unmutated copy of the gene will provide all the clotting factor they need. Heterozygous females are called "carriers" because although they show no symptoms, they pass the gene on to approximately half their sons, who develop the disease, and half their daughters, who also become carriers.
X Y
X XX XY
Xh XhX XhY
Women rarely suffer from hemophilia because to do so they would have to inherit a defective gene from their father as well as their mother. Until recently, few hemophiliacs ever became fathers.
X-chromosome Inactivation (XCI)
Human females inherit two copies of every gene on the X chromosome, whereas males inherit only one (with some exceptions: the 9 pseudoautosomal genes and the small number of "housekeeping" genes found on the Y). But for the hundreds of other genes on the X, are males at a disadvantage in the amount of gene product their cells produce? The answer is no, because females have only a single active X chromosome in each cell.
During interphase, chromosomes are too tenuous to be stained and seen by light microscopy. However, a dense, stainable structure, called a Barr body (after its discoverer) is seen in the interphase nuclei of female mammals. The Barr body is one of the X chromosomes. Its compact appearance reflects its inactivity. So, the cells of females have only one functioning copy of each X-linked gene — the same as males.
X-chromosome inactivation occurs early in embryonic development. In a given cell, which of a female's X chromosomes becomes inactivated and converted into a Barr body is a matter of chance (except in marsupials like the kangaroo, where it is always the father's X chromosome that is inactivated). After inactivation has occurred, all the descendants of that cell will have the same chromosome inactivated. Thus X-chromosome inactivation creates clones with differing effective gene content. An organism whose cells vary in effective gene content and hence in the expression of a trait, is called a genetic mosaic.
Mechanism of X-chromosome inactivation
Inactivation of an X chromosome requires a gene on that chromosome called XIST.
• XIST is transcribed into a long noncoding RNA.
• XIST RNA accumulates along the X chromosome containing the active XIST gene and proceeds to inactivate all (or almost all) of the hundreds of other genes on that chromosome.
• Barr bodies are inactive X chromosomes "painted" with XIST RNA.
The Sequence of Events in Mice
• During the first cell divisions of the female mouse zygote, the XIST locus on the father's X chromosome is expressed so most of his X-linked genes are silent.
• By the time the blastocyst has formed, the silencing of the paternal X chromosome still continues in the trophoblast (which will go on to form the placenta) but
• in the inner cell mass (the ICM, which will go on to form the embryo) transcription of XIST ceases on the paternal X chromosome allowing its hundreds of other genes to be expressed. The shut-down of the XIST locus is done by methylating XIST regulatory sequences. So the pluripotent stem cells of the ICM express both X chromosomes.
• However, as embryonic development proceeds, X-chromosome inactivation begins again. But this time it is entirely random. There is no predicting whether it will be the maternal X or the paternal X that is inactivated in a given cell.
Some genes on the X chromosome escape inactivation
What about those 18 genes that are found on the Y as well as the X? There should be no need for females to inactivate one copy of these to keep in balance with the situation in males. And, as it turns out, these genes escape inactivation in females. Just how they manage this is still under investigation.
X-chromosome Abnormalities
As we saw above, people are sometimes found with abnormal numbers of X chromosomes. Unlike most cases of aneuploidy, which are lethal, the phenotypic effects of aneuploidy of the X chromosome are usually not severe.
Examples:
• Females with but a single intact X chromosome (usually the one she got from her mother) in some (thus a genetic mosaic) or all of her cells show a variable constellation of phenotypic traits called Turner syndrome. For those girls that survive to birth, the phenotypic effects are generally mild because each cell has a single functioning X chromosome like those of XX females. Number of Barr bodies = zero.
• XXX, XXXX, XXXXX karyotypes: all females with mild phenotypic effects because in each cell all the extra X chromosomes are inactivated. Number of Barr bodies = number of X chromosomes minus one.
• Klinefelter's syndrome: people with XXY or XXXY karyotypes are males (because of their Y chromosome). But again, the phenotypic effects of the extra X chromosomes are mild because, just as in females, the extra Xs are inactivated and converted into Barr bodies.
Sex Determination in Other Animals
Although the male fruit fly, Drosophila melanogaster, is X-Y, the Y chromosome does not dictate its maleness but rather the absence of a second X. Furthermore, instead of females shutting down one X to balance the single X of the males — as we do — male flies double the output of their single X relative to that of females.
In birds, moths, schistosomes, and some lizards, the male has two of the same chromosome (designated ZZ), whereas the female has "heterogametic" chromosomes (designated Z and W). In chickens, a single gene on the Z chromosome (designated DMRT1), when present in a double dose (ZZ), produces males while the presence of only one copy of the gene produces females (ZW).
Environmental Sex Determination
In some cold-blooded vertebrates such as
• fishes
• reptiles (e.g. certain snakes, lizards, turtles, and all crocodiles and alligators)
• invertebrates (e.g. certain crustaceans),
sex is determined after fertilization — not by sex chromosomes deposited in the egg.
The choice is usually determined by the temperature at which early embryonic development takes place.
• In some cases (e.g. many turtles and lizards), a higher temperature during incubation favors the production of females.
• In other cases (e.g., alligators), a higher temperature favors the production of males.
Even in cases (e.g. some lizards) where there are sex chromosomes, a high temperature can convert a genotypic male (ZZ) into a female.
Hermaphrodites
Hermaphrodites have both male and female sex organs. Many species of fish are hermaphroditic.
Some start out as one sex and then, in response to stimuli in their environment, switch to the other.
Other species have both testes and ovaries at the same time (but seldom fertilize themselves). However, populations of C. elegans consist mostly of hermaphrodites and these only fertilize themselves.
Hermaphroditic fishes have no sex chromosomes. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/07%3A_Cell_Division/7.06%3A_Sex_Chromosomes.txt |
Mitosis produces two cells with the same number of chromosomes as the parent cell. Mitosis of a diploid cell (2n) produces two diploid daughter cells. If two diploid cells went on to participate in sexual reproduction, their fusion would produce a tetraploid (4n) zygote. The solution for this problem is Meiosis.
Meiosis
Meiosis is a process of cell division in eukaryotes characterized by:
• two consecutive divisions: meiosis I and meiosis II
• no DNA synthesis (no S phase) between the two divisions
• the result: 4 cells with half the number of chromosomes of the starting cell, e.g., 2nn
Fusion of two such cells produces a 2n zygote.
Meiosis in Animals
Used to produced the gametes: sperm and eggs
Meiosis in Plants
Used to produce spores. Spores are the start of the gametophyte generation which, in time, will produce gametes (by mitosis because the starting cells are already haploid).
Meiosis I
Prophase of meiosis I (prophase I) is a more elaborate process than prophase of mitosis (and usually takes much longer).
Here is a brief overview of the process. A more detailed view is provided below.
• When the chromosomes first become visible they are already doubled, each homologue having been duplicated during the preceding S phase.
• Result: pairs of homologous dyads each dyad consisting of two sister chromatids held together by a protein complex called cohesin.
• Pairing: Each pair of homologous dyads align lengthwise with each other.
• Result: a tetrad. (These structures are sometimes referred to as bivalents because at this stage you cannot distinguish the individual sister chromatids under the microscope.)
• The two homologous dyads are held together by
• one or more chiasmata (sing. = chiasma) which form between two nonsister chromatids at points where they have crossed over.
• the synaptonemal complex (SC), a complex assembly of proteins (including cohesin)
At metaphase I, microtubules of the spindle fibers attach to the
• sister kinetochores of one homologue, pulling both sister chromatids toward one pole of the cell;
• sister kinetochores of the other homologue pulling those sisters toward the opposite pole.
Result: one homologue is pulled above the metaphase plate, the other below. The chiasmata keep the homologues attached to each other, and the cohesin keeps the sister chromatids together.
At anaphase I,
• the cohesin between the chromosome arms breaks down allowing
• the chiasmata to slip apart.
• Result: the homologous dyads separate and migrate toward their respective poles.
Meiosis II
Chromosome behavior in meiosis II is like that of mitosis.
• At metaphase II, spindle fibers attach one kinetochore of the dyad to one pole, the other to the opposite pole.
• At anaphase II, the chromatids separate and (each now an independent chromosome) move to their respective poles.
Genetic Recombination
Meiosis not only preserves the genome size of sexually reproducing eukaryotes but also provides three mechanisms to diversify the genomes of the offspring.
Crossing Over
Chiasmata represent points where earlier (and unseen) nonsister chromatids had swapped sections. The process is called crossing over. It is reciprocal; the segments exchanged by each nonsister chromatid are identical (but may carry different alleles).
Each chromatid contains a single molecule of DNA. So the problem of crossing over is really a problem of swapping portions of adjacent DNA molecules. It must be done with great precision so that neither chromatid gains or loses any genes. In fact, crossing over has to be sufficiently precise that not a single nucleotide is lost or added at the crossover point if it occurs within a gene. Otherwise a frameshift would result and the resulting gene would produce a defective product or, more likely, no product at all.
In the diagram above, only a single chiasma is shown. However, multiple chiasmata are commonly found (in humans the average number of chiasmata per tetrad is just over two). In this photomicrograph, a tetrad of the grasshopper Chorthippus parallelus shows 5 chiasmata.
Random Assortment
In meiosis I, the orientation of paternal and maternal homologues at the metaphase plate is random. Therefore, although each cell produced by meiosis contains only one of each homologue, the number of possible combinations of maternal and paternal homologues is 2n, where n = the haploid number of chromosomes. In this diagram, the haploid number is 3, and 8 (23) different combinations are produced.
Random assortment of homologues in humans produces 223 (8,388,608) different combinations of chromosomes.
Furthermore, because of crossing over, none of these chromosomes is "pure" maternal or paternal. The distribution of recombinant and non-recombinant sister chromatids into the daughter cells at anaphase II is also random.
So I think it is safe to conclude that of all the billions of sperm produced by a man during his lifetime (and the hundreds of eggs that mature over the life of a woman), no two have exactly the same gene content.
Fertilization
By reducing the number of chromosomes from 2n to n,the stage is set for the union of two genomes. If the parents differ genetically, new combinations of genes can occur in their offspring.
Taking these three mechanisms together, I think that it is safe to conclude that no two human beings have ever shared an identical genome unless they had an identical sibling; that is a sibling produced from the same fertilized egg.
The behavior of chromosomes during meiosis (2nn) and fertilization (n + n2n) provide the structural basis for Mendel's rules of inheritance.
Prophase I — a detailed view
The lengthy and complex events of prophase I can be broken down into 5 stages.
Leptotene
• All the chromosomes condense.
• Pairing. Homologous dyads (pairs of sister chromatids) find each other and align themselves from end to end with the aid of an axial element (that contains cohesin). In budding yeast (and perhaps other eukaryotes) the process follows a period of trial-and-error. Any two dyads pair at their centromeres. If they are not homologs, they separate and try again.
• How the nonsisters recognize their shared regions of DNA homology is uncertain. Double-stranded breaks (DSBs) often occur in the DNA of the chromatids, and these may be necessary for the homologs to recognize each other.
Zygotene
• Synapsis. The synaptonemal complex begins to form.
• DNA strands of nonsister chromatids begin the process of recombination. How they are able to do so across the synaptonemal complex, which is over 100 nm thick, is unknown.
Pachytene
• Synapsis is now complete.
• Recombination nodules appear (at least in some organisms, including humans). They are named for the idea that they represent points where DNA recombination is occurring.
• There must be at least one for each bivalent if meiosis is to succeed. There are often more, each one presumably representing the point of a crossover.
• They contain enzymes known to be needed for DNA recombination and repair.
• The steps in recombining DNA continue to the end of pachytene.
Diplotene
• DNA recombination is complete.
• The synaptonemal complex begins to break down.
• The chromatids begin to pull apart revealing
• chiasmata. At first the chiasmata are located at the sites of the recombination nodules, but later they migrate towards the ends of the chromatids.
Diakinesis
In some organisms, the chromosomes decondense and begin to be transcribed for a time. This is followed by the chromosomes recondensing in preparation for metaphase I.
In creatures where this does not occur, the chromosomes condense further in preparation for metaphase I.
Quality Control of Meiosis
It shouldn't be surprising that things can go wrong in such a complicated process. However, cells going through meiosis have checkpoints that monitor each pair of homologues for
• proper recombination of their DNA
• correct formation of the synaptonemal complex
Any failure that is detected stops the process and usually causes the cell to self-destruct by apoptosis.
However, despite these checkpoints, errors occasionally do go uncorrected.
Errors in Meiosis
It is estimated that from 10–25% of all human fertilized eggs contain chromosome abnormalities, and these are the most common cause of pregnancy failure (35% of the cases).
These chromosome abnormalities
• arise from errors in meiosis, usually meiosis I;
• occur more often (90%) during egg formation than during sperm formation;
• become more frequent as a woman ages.
• Aneuploidy — the gain or loss of whole chromosomes — is the most common chromosome abnormality. It is caused by nondisjunction, the failure of chromosomes to correctly separate:
• homologues during meiosis I or
• sister chromatids during meiosis II
• Zygotes missing one chromosome ("monosomy") cannot develop to birth (except for females with a single X chromosome).
• Three of the same chromosome ("trisomy") is also lethal except for chromosomes 13, 18, and 21 (trisomy 21 is the cause of Down syndrome).
• Three or more X chromosomes are viable because all but one of them are inactivated. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/07%3A_Cell_Division/7.07%3A_Meiosis.txt |
• 8.1: Mendel's Monohybrid Crosses
Gregor Mendel (1822-1884) was an Austrian monk who discovered the basic rules of inheritance. From 1858 to 1866, he bred garden peas in his monastery garden and analyzed the offspring of these matings. The garden pea was good choice of experimental organism because many varieties were available that bred true for clear-cut, qualitative traits.
• 8.2: Crossing Over and Genetic Recombination in Meiosis
• 8.3: The Evidence of Creighton and McClintock
In 1932, the geneticists Harriet Creighton and Barbara McClintock provided an elegant demonstration that the recombination of genes linked on a chromosome requires the physical exchange of segments of the chromosome with its homologous partner. During their studies of linkage in corn, they developed a strain of corn that had one chromosome (number 9 of 10 pairs) with two unusual features: (1) a knob at one end of the chromosome and (2) an extra piece at the other.
• 8.4: Genetic linkage and Genetic Maps
• 8.5: Gene Mapping with Three-point Crosses
The closer the intervals examined, the more accurate the map. A three-point cross also gives the gene order immediately.
• 8.6: Quantitative Trait Loci
The rules of inheritance discovered by Mendel depended on his wisely choosing traits that varied in a clear-cut, easily distinguishable, qualitative way. But humans are not either tall or short nor are they either heavy or light. Many traits differ in a continuous, quantitative way throughout a population. This can be explained by assuming it is controlled by several pairs of genes — called quantitative trait loci (QTL) — the effects of which are added together (called polygenic inheritance).
• 8.7: Mapping the Genes of T2
• 8.8: rII Locus of T4
08: The Genetic Consequences of Meiosis
Gregor Mendel (1822-1884) was an Austrian monk who discovered the basic rules of inheritance. From 1858 to 1866, he bred garden peas in his monastery garden and analyzed the offspring of these matings. The garden pea was good choice of experimental organism because many varieties were available that bred true for clear-cut, qualitative traits like
• seed texture (round vs wrinkled)
• seed color (green vs yellow)
• flower color (white vs purple)
• tall vs dwarf growth habit
• and three others that also varied in a qualitative — rather than quantitative — way.
Furthermore, peas are normally self-pollinated because the stamens and carpels are enclosed within the petals. By removing the stamens from unripe flowers, Mendel could brush pollen from another variety on the carpels when they ripened.
The First Cross
Mendel crossed a pure-breeding round-seeded variety with a pure-breeding wrinkled-seeded one. The parents (designated the P generation) were pure-breeding because each was homozygous for the alleles at the gene locus (on chromosome 7) controlling seed texture (RR for round; rr for wrinkled).
The results
All the peas produced in the second or hybrid generation were round.
Interpretation
All the peas of this F1 generation have an Rr genotype. All the haploid sperm and eggs produced by meiosis received one chromosome 7. All the zygotes received one R allele (from the round parent) and one r allele (from the wrinkled parent). Because the round trait is dominant, the phenotype of all the seeds was round.
P gametes (round parent)
R R
P gametes
(wrinkled parent)
r Rr Rr
r Rr Rr
The Second Cross
Mendel then allowed his hybrid peas to self-pollinate.
The results
The wrinkled trait — which had disappeared in his hybrid generation — reappeared in 25% of the new crop of peas.
Interpretation
Random union of equal numbers of R and r gametes produced an F2 generation with 25% RR and 50% Rr — both with the round phenotype — and 25% rr with the wrinkled phenotype.
F1 gametes
R r
F1 gametes R RR Rr
r Rr rr
The Third Cross
Mendel then allowed some of each phenotype in the F2 generation to self-pollinate. His results:
• All the wrinkled seeds in the F2 generation produced only wrinkled seeds in the F3.
• One-third (193/565) of the round F1 seeds produced only round seeds in the F3 generation, but
• two-thirds (372/565) of them produced both types of seeds in the F3 and — once again — in a 3:1 ratio.
Interpretation
One-third of the round seeds and all of the wrinkled seeds in the F2 generation were homozygous and produced only seeds of the same phenotype. But two thirds of the round seeds in the F2 were heterozygous and their self-pollination produced both phenotypes in the ratio of a typical F1 cross.
Phenotype ratios are approximate
The union of sperm and eggs is random. So the pod in the color photo () — with its 9 smooth seeds and 3 wrinkled seeds! — represents something of a statistical fluke. As the size of the sample gets larger, however, chance deviations become minimized and the ratios approach the theoretical predictions more closely. The table shows the actual seed production by ten of Mendel's F1 plants. While his individual plants deviated widely from the expected 3:1 ratio, the group as a whole approached it quite closely.
Round Wrinkled
1. 45 12
2. 27 8
3. 24 7
4. 19 16
5. 32 11
6. 26 6
7. 88 24
8. 22 10
9. 28 6
10. 25 7
Total 336 107
Mendel's Hypothesis
To explain his results, Mendel formulated a hypothesis that included the following:
1. In the organism there is a pair of factors that controls the appearance of a given characteristic. (We call them genes.)
2. The organism inherits these factors from its parents, one from each.
3. Each is transmitted from generation to generation as a discrete, unchanging unit. (The wrinkled seeds in the F2 generation were no less wrinkled than those in the P generation although they had passed through the round-seeded F1 generation.)
4. When the gametes are formed, the factors separate and are distributed as units to each gamete. This statement is often called Mendel's rule of segregation.
5. If an organism has two unlike factors (we call them alleles) for a characteristic, one may be expressed to the total exclusion of the other (dominant vs recessive).
The Testcross: A Test of Mendel's Hypothesis
A good hypothesis meets several standards.
• It should provide an adequate explanation of the observed facts.
• If two or more hypotheses meet this standard, the simpler one is preferred.
• It should be able to predict new facts.
So if a generalization is valid, then certain specific consequences can be deduced from it. To test his hypothesis, Mendel predicted the outcome of a breeding experiment that he had not yet carried out. He crossed heterozygous round peas (Rr) with wrinkled (homozygous, rr) ones. He predicted that in this case one-half of the seeds produced would be round (Rr) and one-half wrinkled (rr)
F1 gametes
R r
P gametes r Rr rr
r Rr rr
To a casual observer in the monastery garden, the cross appeared no different from the P cross described above: round-seeded peas being crossed with wrinkled-seeded ones. But Mendel predicted that this time he would produce both round and wrinkled seeds and in a 50:50 ratio. He performed the cross and harvested 106 round peas and 101 wrinkled peas.
This kind of mating is called a testcross. It "tests" the genotype in those cases where two different genotypes (like RR and Rr) produce the same phenotype.
Mendel did not stop here.
• He went on to cross pea varieties that differed in six other qualitative traits. In every case, the results supported his hypothesis.
• He crossed peas that differed in two traits. He found that the inheritance of one trait was independent of that of the other and so framed his second rule: the rule of independent assortment.
Mendel's rules today
Little attention was paid when Mendel published his findings in 1866. Not until 1900, 34 years later and 16 years after his death, was his work brought to light. By then, three men — working independently — discovered the same principles. So the present remarkable development of genetics dates from only the start of the 20th century.
The discovery of chromosomes — and their behavior during meiosis (2n -> n) and fertilization (n + n -> 2n) — established the structural basis for Mendel's rules.
What is the status today of Mendel's rules? Although many important exceptions to them have been discovered — three examples:
• both members of many allelic pairs affect the phenotype; that is, neither is fully dominant
• several different pairs of genes — often on different chromosomes — affect a phenotype additively with none being fully dominant.
• many gene loci are not inherited independently but show linkage (because they are relatively close together on the same chromosome)
His rules still form the foundation upon which the science of genetics rests. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/08%3A_The_Genetic_Consequences_of_Meiosis/8.01%3A_Mendel%27s_Monohybrid_Crosses.txt |
Crossing over occurs between equivalent portions of two nonsister chromatids.
Each chromatid contains a single molecule of DNA. So the problem of crossing over is really a problem of swapping portions of adjacent DNA molecules.
It must be done with great precision so that neither chromatid gains or loses any genes. In fact, crossing over has to be sufficiently precise that not a single nucleotide is lost or added at the crossover point if it occurs within a gene. Otherwise a frameshift would result and the resulting gene would produce a defective product or, more likely, no product at all.
8.03: The Evidence of Creighton and McClintock
Here was an organism with a rare chromosomal aberration that made it possible to distinguish two homologs from each other under the microscope. Furthermore, this unusual chromosome carried the dominant allele for colored kernels (C) and the recessive allele for waxy endosperm (wx). Its normal-appearing mate carried the recessive allele for colorless kernels (c) and the dominant allele for normal (starchy) endosperm (Wx). Thus the plant was a dihybrid for these two linked traits and, in addition, one chromosome of the pair was visibly marked at each end.
Creighton and McClintock reasoned that this plant would produce 4 kinds of gametes: The parental kinds (Cwx and cWx) and the recombinant kinds produced by crossing over (cwx and CWx). Fertilization of these gametes by gametes containing a chromosome of normal appearance and both recessive alleles cwx (a typical testcross) should produce 4 kinds of kernels:
• colored waxy (Ccwxwx) kernels
• colorless kernels with normal endosperm (ccWxwx)
• colorless waxy (ccwxwx) and
• colored kernels with normal endosperm (CcWxwx).
1. In the first case, there should be one normal chromosome and one extra-long chromosome with the knob at the end.
2. In the second case, both chromosomes should be of normal appearance. However,
3. in the third case (colorless, waxy), where crossing over had occurred, one would hope to find evidence that a physical exchange of parts between the homologous chromosomes of the dihybrid parent had occurred. Either a chromosome of normal length, but with a knob at one end, or an extra-long chromosome with no knob should be present. Creighton and McClintock found the latter, thus indicating that the gene locus for wx was associated with (and thus near) the end of the chromosome with the extra segment. The gene locus for kernel color must then be neared the end with the knob.
4. Examination of the plants in class 4 (colored kernels and normal endosperm) revealed a chromosome of normal length but with a knob at the end.
Thus, behavior of the genes as revealed by the study of the phenotypes produced was shown to be directly related to the behavior of chromosomes as seen under the microscope. The recombination of genes occurs when homologous chromosomes exchange parts.
8.04: Genetic linkage and Genetic Maps
Background
Mendel then crossed these dihybrids. If it is inevitable that round seeds must always be yellow and wrinkled seeds must be green, then he would have expected that this would produce a typical monohybrid cross: 75% round-yellow; 25% wrinkled-green. But, in fact, his mating generated seeds that showed all possible combinations of the color and texture traits.
• 9/16 of the offspring were round-yellow
• 3/16 were round-green
• 3/16 were wrinkled-yellow, and
• 1/16 were wrinkled-green
Rule of Independent Assortment
Finding in every case that each of his seven traits was inherited independently of the others, he formed his "second rule" the Rule of Independent Assortment:
The inheritance of one pair of factors (genes) is independent of the inheritance of the other pair.
Today we know that this rule holds only if two conditions are met:
• the genes are on separate chromosomes or
• the genes are widely separated on the same chromosome.
Mendel was lucky in that every pair of genes he studied met one requirement or the other. The table shows the chromosome assignments of the seven pairs of alleles that Mendel studied. Although all of these genes showed independent assortment, several were, in fact, syntenic with three loci occurring on chromosome 4 and two on chromosome 1. However, the distance separating the syntenic loci was sufficiently great that the genes were inherited as though they were on separate chromosomes.
Trait Phenotype Alleles Chromosome
Seed form round-wrinkled R-r 7
Seed color yellow-green I-i 1
Pod color green-yellow Gp-gp 5
Pod texture smooth-wrinkled V-v 4
Flower color purple-white A-a 1
Flower location axial-terminal Fa-fa 4
Plant height tall-dwarf Le-le 4
Start with two different strains of corn (maize).
• one that is homozygous for two traits
• yellow kernels (C,C) which are filled with endosperm causing the kernels to be
• smooth (Sh,Sh).
• a second that is homozygous for
• colorless kernels (c,c) that are wrinkled because their endosperm is
• shrunken (sh,sh)
When the pollen of the first strain is dusted on the silks of the second (or vice versa), the kernels produced (F1) are all yellow and smooth. So the alleles for yellow color (C) and smoothness (Sh) are dominant over those for colorlessness (c) and shrunken endosperm (sh).
To simplify the analysis, mate the dihybrid with a homozygous recessive strain (ccshsh). Such a mating is called a test cross because it exposes the genotype of all the gametes of the strain being evaluated.
According to Mendel's second rule, the genes determining color of the endosperm should be inherited independently of the genes determining texture. The F1 should thus produce gametes in approximately equal numbers.
• CSh, as inherited from one parent.
• csh, as inherited from the other parent
• Csh, a recombinant
• cSh, the other recombinant.
In fact, the recombination frequency is 2.0%, telling us that the actual order of loci is
cshbz.
Mapping by linkage analysis is best done with loci that are relatively close together; that is, within a few centimorgans of each other. Why? Because as the distance between two loci increases, the probability of a second crossover occurring between them also increases.
But a second crossover would undo the effect of the first and restore the parental combination of alleles. These would show up as nonrecombinants. Thus as the distance between two loci increases, the percentage of recombinants that forms understates the actual distance in centimorgans that separates them. And, in fact, that has happened in this example. Using a three-point cross reveals the existence of a small number of double recombinants and tells us that the actual distance c—bz is indeed 5 cM as we would expect by summing
• c—sh = 3 cM
• sh—bz = 2 cM
and not the 4.6 cM revealed by the dihybrid cross.
A three-point cross also tells us the gene order in a single cross rather than the three we needed here.
There are other problems with preparing genetic maps of chromosomes.
• The probability of a crossover is not uniform along the entire length of the chromosome.
• Crossing over is inhibited in some regions (e.g., near the centromere).
• Some regions are "hot spots" for recombination (for reasons that are not clear). Approximately 80% of genetic recombination in humans is confined to just one-quarter of our genome.
• In humans, the frequency of recombination of loci on most chromosomes is higher in females than in males. Therefore, genetic maps of female chromosomes are longer than those for males.
A genetic map of chromosome 9 (the one that carries the C, Sh, and bz loci) of the corn plant (Zea mays) is shown above. If one maps in small intervals from one end of a chromosome to the other, the total number of centimorgans often exceeds 100 (as you can see for chromosome 9). However, even for widely-separated loci, the maximum frequency of recombinants that can form is 50%. And this is also the frequency of recombinants that we see for genes independently assorting on separate chromosomes. So we cannot tell by simply counting recombinants whether a pair of gene loci is located far apart on the same chromosome or are on different chromosomes. As we saw above, several of Mendel's independently assorting traits are controlled by genes on the same chromosome but located so far apart that they are inherited as if they were located on different chromosomes.
Genes that are present on the same chromosome are called syntenic. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/08%3A_The_Genetic_Consequences_of_Meiosis/8.02%3A_Crossing_Over_and_Genetic_Recombination_in_Meiosis.txt |
Mechanisms discussed previously show how carrying out three different dihybrid test crosses in the corn plant reveals the order of the gene loci and the distance between them (in centimorgans, cM). Here we shall see how a single test cross of a trihybrid corn plant; that is, one parent is heterozygous for three linked alleles (C,Sh, Bz, on one chromosome; c,sh,bz on the other) and the other parent is homozygous for the recessive version of all three genes (c,c,sh,sh,bz,bz) reveals the gene order and gives a more accurate measurement of the distance in cM separating the outermost loci (in this case C and Bz) than a dihybrid cross involving those loci would. Hypothetical breeding data are shown in Table 8.5.1.
Table 8.5.1: Hypothetical breeding data
Group Expressed Alleles
(Phenotype)
Crossovers Number Totals
1 CShBz None; the parentals 479 952
2 cshbz 473
3 C|shbz Single; between C and others 15 28
4 c|ShBz 13
5 CSh|bz Single, between Bz and others 9 18
6 csh|Bz 9
7 C|sh|Bz Double recombinants 1 2
8 c|Sh|bz 1
Totals 1000
Eight different phenotypes — representing the 8 possible genotypes (23 = 8) are produced. Scoring them reveals
• The percentage of recombinants between C and Sh is 3.0%: 28/1000 of single recombinants plus 2/1000 double recombinants.
• That between Sh and Bz is 2.0%: 18/1000 single recombinants plus 2/1000 double recombinants.
• That between C and Bz is 4.6%: 28 + 18 = 46/1000.
But adding the distances between C and Sh and Sh and Bz gives a map distance between C and Bz of 5.0 cM not the 4.6 cM revealed by the data (and the same number that a C,c,Bz,bz dihybrid cross would have produced). Why the discrepancy? Because the double recombinants restored the parental configuration, they were missed in the scoring. So the two rare classes of double recombinants need to be added (twice) to the data.
\[28 + 18 + 2 + 2 = 50/1000 = 5\%\]
to get the true value. So the map of this region of the chromosome is:
This exercise underscores the rule that the closer the intervals examined, the more accurate the map. A three-point cross also gives the gene order immediately. The procedure is:
1. Determine the rarest classes (here, C,sh,Bz and c,Sh,bz) because two crossovers between a pair of loci will be rarer than one.
2. In these two groups, the alleles that specify the trait that was not seen in the parents (sh and Sh) occupy the middle locus.
8.06: Quantitative Trait Loci
The rules of inheritance discovered by Mendel depended on his wisely choosing traits that varied in a clear-cut, easily distinguishable, qualitative way. But humans are not either tall or short nor are they either heavy or light. Many traits differ in a continuous, quantitative way throughout a population.
This histogram shows the distribution of heights among a group of male secondary-school seniors. As you can see, the plot resembles a bell-shaped curve. Such distributions are typical of quantitative traits. Some of the variation can be explained by differences in diet and perhaps other factors in the environment. Environment alone is not, however, sufficient to explain the full range of heights or weights.
An understanding of how genes can control quantitative traits emerged in 1908 from the work of the Swedish geneticist Nilsson-Ehle who studied quantitative traits in wheat. Using Mendel's methods, he mated pure-breeding red-kernel strains with pure-breeding white-kernel strains. The offspring were all red, but the intensity of color was much less that in the red parent. It seemed as though the effect of the red allele in the F1 generation was being modified by the presence of the white allele.
Nilsson-Ehle: Genetics of Two Crosses
When Nilsson-Ehle mated two F1 plants, he produced an F2 generation in which red-kerneled plants outnumbered white-kerneled plants 15:1. But the red kernels were not all alike. They could quite easily be sorted into four categories. One sixteenth of them were deep red, like the P type. Four sixteenths were medium dark red, six sixteenths were medium red (like the F1 generation), and four sixteenths were light red. The genetics of the two crosses is shown here. The alleles at one locus are indicated with prime marks; at the other, without.
These results could be explained by assuming that kernel color in wheat is controlled by not one, but two pairs of genes, the effects of which add up without distinct dominance. Each pair is located on a different chromosome or so far apart on the same chromosome that there is no linkage. Four alleles for red produce a deep red kernel. Four alleles for white produce a white kernel. Just one red allele out of four produces a light red kernel. Any two out of the four produce a medium red kernel. Any three of the four produce a medium dark red kernel. If one plots the numbers of the different colored offspring in the F2 generation against color intensity, one gets a graph like Figure 8.6.3.
In other wheat varieties, Nilsson-Ehle found F2 generations with a ratio of red kernels to white of 63:1. These could be explained by assuming that three pairs of alleles were involved. In these cases, six different shades of red could be detected, but the color differences were very slight. Environmental influences also caused alterations in intensity so that in practice the collection of kernels displayed a continuous range of hues all the way from deep red to white.
So the occurrence of continuous variation of a trait in a population can be explained by assuming it is controlled by several pairs of genes — called quantitative trait loci (QTL) — the effects of which are added together. This is called polygenic inheritance or the multiple-factor hypothesis. At first the study of quantitative traits was mostly confined to animal husbandry and the breeding of agricultural crops. It was based on the premise that
• When two extreme types ae mated (e.g., AABB and aabb). the offspring are intermediate in type.
• When two intermediate types are mated, most of their offspring are also intermediate, but some extreme types will be produced.
• The results of random matings in a large population will be a large range of types with the greatest number in the middle range and the fewest at the extremes.
In more recent times, the search for quantitative trait loci has turned to humans. A number of diseases, cancers for example, are thought to be caused by the additive effects of genes at different loci. Pedigree analysis has provided some insights, but the use of microarrays promises to provide more. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/08%3A_The_Genetic_Consequences_of_Meiosis/8.05%3A_Gene_Mapping_with_Three-point_Crosses.txt |
T2 and its close relative T4 are viruses that infect the bacterium E. coli. The infection ends with destruction (lysis) of the bacterial cell so these viruses are examples of bacteriophages ("bacteria eaters"). Each virus particle (virion) consists of:
• a protein head (~0.1 µm) inside of which is a single, circular molecule of double-stranded DNA containing 166,000 base pairs.
• a protein tail from which extend
• thin protein fibers
Life Cycle
The virus attaches to the E. coli cell (a). This requires a precise molecular interaction between the fibers and the cell wall of the host.
• The DNA molecule is injected into the cell (b).
• Within 1 minute, the viral DNA begins to be transcribed and translated into some of the viral proteins, and synthesis of host proteins is stopped.
• At 5 minutes, viral enzymes needed for synthesis of new viral DNA molecules are produced (c).
• At 8 minutes, some 40 different structural proteins for the viral head and tail are synthesized.
• At 13 minutes, assembly of new viral particles begins (d).
• At 25 minutes, the viral lysozyme destroys the bacterial cell wall and the viruses burst out — ready to infect new hosts (e).
• If the bacterial cells are growing in liquid culture, it turns clear.
• If the bacterial cells are growing in a "lawn" on the surface of an agar plate, then holes, called plaques, appear in the lawn.
Occasionally, new phenotypes appear such as a change in the appearance of the plaques or even a loss in the ability to infect the host.
Examples:
• h
• Some strains of E. coli, e.g. one designated B/2, gain the ability to resist infection by normal ("wild-type") T2. The mutation has caused a change in the structure of their cell wall so that the tail fibers of T2 can no longer bind to it. However, T2 can strike back. Occasional T2 mutants appear that overcome this resistance. The mutated gene, designated h (for "host range"), encodes a change in the tail fibers so they can once again bind to the cell wall of strain B/2. The normal or "wild-type" gene is designated h+ .
• When plated on a lawn containing both E. coli B and E. coli B/2,
• the mutant (h) viruses can lyze both strains of E. coli, producing clear plaques, while
• the wild-type (h+) viruses can only lyze E. coli B producing mottled or turbid plaques.
• r
• Occasional T2 mutants appear that break out of their host cell earlier than normal.
• The mutation occurs in a gene designated r (for "rapid lysis"). It reveals itself by the extra-large plaques that it forms.
• The wild-type gene, producing a normal time of lysis, is designated r+. It forms normal-size plaques.
As with so many organisms, the occurrence of mutations provides the tools to learn about such things as
• the function of the gene
• its location in the DNA molecule (mapping)
Mapping by Recombination Frequencies
As we have seen, E. coli strain B can be infected by both h+ and h strains of T2. In fact, a single bacterial cell can be infected simultaneously by both. Let us infect a liquid culture of E. coli B with two different mutant T2 viruses: h r+ and h+ r. When this is done in liquid culture, and then plated on a mixed lawn of E. coli B and B/2, four different kinds of plaques appear.
Genotype Phenotype Number of Plaques
hr+ clear, small 460
h+r turbid, large 460
h+r+ turbid, small 40
hr clear, large 40
Total = 1000
The most abundant (460 each) are those representing the parental types; that is, the phenotypes are those expected from the two infecting strains. However, small numbers (40 each) of two new phenotypes appear. These can be explained by genetic recombination having occasionally occurred between the DNA of each parental type within the bacterial cell.
Just as in higher organisms, one assumes that the frequency of recombinants is proportional to the distance between the gene loci. In this case, 80 out of 1000 plaques were recombinant, so the distance between the h and r loci is assigned a value of 8 map units or centimorgans (cM). Now coinfect E. coli B with two other strains of T2:
• hm+ and
• h+m
hm+ 470
h+m 470
h+m+ 30
hm 30
Total = 1000
Again, 4 kinds of plaques are produced: parental (470 each) and recombinant (30 each).
The smaller number of recombinants indicates that these two gene loci (h and m) are closer together (6 cM) than h and r (8 cM). But the order of the three loci could be either
• m–6–h—8—r
or
• h–6–m-2-r
To find out which is the correct order, perform a third mating using
• mr+ and
• m+r
mr+ 440
m+r 440
m+r+ 60
mr 60
Total = 1000
This makes it clear that the order is m—h—r, not h—m—r.
But why only 12 cM between the outside loci (m and r) instead of the 14 cM produced by adding the map distances found in the first two matings?
A Three-Point Cross
The answer comes from performing a mating between T2 viruses differing at all three loci:
• hmr
and
• h+m+r+
(Note: this time one parent has all mutant; the other all wild-type alleles — don't be confused!)
Group 1 hmr 435
Group 2 h+m+r+ 435
Group 3 h+mr+ 25
Group 4 hm+r 25
Group 5 hmr+ 35
Group 6 h+m+r 35
Group 7 hm+r+ 5
Group 8 h+mr 5
Total = 1000
The result: 8 different types of plaques are formed.
• parentals; that is, nonrecombinants in Groups 1 and 2;
• recombinants — all the others
Analyzing these data shows how the two-point cross between m and r understated the true distance between them.
Let's first look at single pairs of recombinants as we did before (thus ignoring the third locus).
• If we look at all the recombinants between h and r but ignore m (as in the first experiment), we find that they are contained in Groups 5, 6, 7, and 8 — giving the total of 80 that we found originally.
• If we look at recombinants between h and m but ignore r (as in the second experiment), we find that they are contained in Groups 3, 4,7, and 8 — giving the same total of 60 that we found before.
• But if we focus only on m and r (as we did in the third experiment), we find that the recombinants are contained in Groups 3, 4, 5, and 6 — giving the same total of 120 as before while the non-recombinants are not only in Groups 1 and 2 but also in Groups 7 and 8. The reason: a double-crossover occurred in these cases, restoring the parental configuration of the m and r alleles.
• Because these double crossovers were hidden in the third experiment, the map distance (12 cM) was understated. To get the true map distance, we add their number to each of the other recombinant groups (Groups 3,4,5, and 6) so 25 + 5 +25 +5 +35 + 5 + 35 + 5 = 140, and the true map distance between m and r is the 14 cM that we found by adding the map distances between h and r (8 cM) and h and m (6 cM).
The three-point cross is also useful because it gives the gene order simply by inspection:
• Find the rarest genotypes (here Groups 7 and 8)
• The gene NOT in the parental configuration (here h) is always the middle one. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/08%3A_The_Genetic_Consequences_of_Meiosis/8.07%3A_Mapping_the_Genes_of_T2.txt |
Mapping Within A Gene: the RII Locus
T2 and its close relative T4 are viruses that infect the bacterium E. coli. The infection ends with destruction (lysis) of the bacterial cell so these viruses are examples of bacteriophages ("bacteria eaters"). They have been enormously useful in genetic studies because:
• Viruses of two (or more) different genotypes can simultaneously infect a single bacterium.
• The DNA molecules of one of the infecting viruses can recombine with that of another forming recombinant molecules.
• The huge number of viruses released from a huge number of bacterial hosts enables even rare recombination events to be detected.
Another page — Bacteriophage Genetics — describes how T2 is used to map the order and relative spacing of genes on the single circular molecule of DNA that is the virus's genome. Here let us see how T4 can be used to detect mutations within a single gene and speed up the process of mapping these point mutations by the use of deletion mutants.
Detecting Mutation within a Single Gene
In Bacteriophage Genetics, we examined mutation of a gene designated r, for "rapid lysis". It turned out that actually there are three different gene locirI, rII, and rIII — mutations in any one of which produced a rapid-lysis phenotype. But, in addition, there were many mutations found in each of these. Could wild-type virus be formed by recombination between mutations within the same gene? Seymour Benzer decided to find out.
As we saw in Bacteriophage Genetics, the recombination frequency between different genes is low (on the order of 10-2). One would expect that recombination frequencies between mutations in a single gene would be far lower (10-4 or less). Fortunately Benzer could exploit a phenomenon to enable him to detect such rare events: rII mutants as well as wild-type T4 can infect and complete their life cycle in a strain of E. coli designated B. However, while rII mutants can infect a strain of E. coli designated K, they cannot complete their life cycle in strain K. Wild-type T4 can.
The procedure was to infect strain B in liquid culture with two mutants to be tested (designated here as rx and ry). After incubation, these were plated on a lawn of:
• strain B — which supports the growth of all viruses thus giving the total number of viruses liberated.
• strain K — on which only wild-type viruses can grow.
The recombination frequency between any pair of mutations is calculated as
$\text{Recombination Frequency} = \dfrac{2 \times \text{number of wild-type plaques (strain K plaques)}}{\text{total number of plaques (on strain B)}}$
You have to double the number found on strain K because you only see one-half the recombinants — the other half consists of double mutants. Using this technique, Benzer eventually found some 2000 different mutations in the rII gene. The recombination frequency between some pairs of these was as low as 0.02.
• The T4 genome has 160,000 base pairs of DNA extending over ~1,600 centimorgans (cM).
• So 1 cM ≅ 100 base pairs
• So 0.02 cM represents a pair of adjacent nucleotides.
• From these data, Benzer concluded that the
• smallest unit of mutation and
• the smallest unit of recombination
was a single base pair of DNA.
In other words, these mutations represent a change in a single base pair — we call these point mutations. Recombination between two molecules of DNA can occur at any pair of nucleotides.
Mapping Point Mutations Within A Gene
The relative order and spacing of any two point mutations in a single gene like rII can be done using the procedure describe in Bacteriophage Genetics. But with some 2000 different mutations to test, the process would be tremendously time-consuming. (Even using the procedure to be described now, Benzer spent some 10 years on the project.) Benzer was able to speed up the mapping process by taking advantage of the discovery that some of his mutants did not have point mutations but deletions instead. In contrast to the properties of T4 viruses with point mutations, T4 viruses with deletions in rII showed no recombination with other rII mutants or any other genes for that matter. Moreover, these deletions never back-mutated.
Deletion Mapping
Deletions can be mapped by the same procedure used for point mutations. Simply cross pairs of deletion mutants and see if they produce progeny that can grow on E. coli strain K. Here is a hypothetical example. Each of 6 strains of deletion mutants are crossed with each of the others.
Table 8.8.1:
Strains 1 2 3 4 5 6
1 0 0 + 0 0 0 1 and 3 do not overlap
2 0 + + 0 0 must shift 4 away from 2
3 0 + + 0 6 must extend under 3
4 0 + + right-hand end of 4 must be removed from over 6
5 0 + left-hand end of 6 must not overlap 5 but must continue to overlap 2.
∴ shorten right-hand end of 5
6 0
From the results, one can draw a map showing the order and relative size of the deletions.
With such a deletion map, one can now quickly map the location of point mutations by coinfecting each of the different deletion strains (here 1–6) with the mutant strain ("x").
There is no longer any need to count plaques; simple see whether there is growth or not.
Coinfect with strain 1 2 3 4 5 6
and mutant "x"
Results →
0 0 + + 0 +
From these results, we learn that the point mutation "x" is located on the T4 DNA within the region shown above in blue.
Complementation
As we saw above, rapid lysis (r) mutants were found that mapped to three different regions of the T4 genome: rI, rII, and rIII. This meant that those in different regions were not alleles of the same gene and more than one gene product participated in the lysis function. Even within one "locus", rII, there turned out to be two different stretches of DNA both of which were needed intact for the lysis function. This was revealed by the complementation test that Benzer used. In this test,
• E. coli strain K (which rII mutants can infect but not complete their life cycle) — growing in liquid culture — was
• coinfected with two different rII mutants (shown in the figure as "1" and "2").
Note that this procedure differs from the earlier one (recombination) in that the nonpermissive E. coli K is used for the initial infection (not strain B as before). Neither strain rII"1" nor strain rII"2" is able to grown in E. coli K. But if the lost function in rII"1" is NOT the same as the lost function in rII"2", then
• each should be able to produce the gene product missing in the other — complementation — and
• living phages will be produced. (Again, there is no need to count plaques; simply see if they are formed or not.)
Mutant strains 1 2 3 4 5
1 0 0 + 0 +
2 0 + 0 +
3 0 + 0
4 0 +
5 0
From these results, you can deduce that these 5 rII mutants fall into two different complementation groups, which Benzer designated A (containing strains 1, 2, and 4) and B (containing strains 3 and 5). Later work showed that the function of rII depended on the polypeptide products encoded by two adjacent regions (A and B) of rII (perhaps acting as a heterodimer). In terms of function, then, both A and B qualify as independent genes. In coinfections by two mutant strains,
• If either A or B is mutated on the same DNA molecule ("cis"), there is no function while
• if A is mutated in one DNA molecule and B in the other ("trans"), function is restored.
Complementation, then, is the ability of two different mutations to restore wild-type function when they are in the "trans" (on different DNA molecules), but not when they are in "cis" (on the same DNA molecule). Benzer coined the term cistron for these genetic units of function. But today, we simply modify earlier concepts of the "gene" to fit this operational definition. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/08%3A_The_Genetic_Consequences_of_Meiosis/8.08%3A_rII_Locus_of_T4.txt |
• 9.1: Regulation of Gene Expression in Bacteria
Within its tiny cell, the bacterium E. coli contains all the genetic information it needs to metabolize, grow, and reproduce. It can synthesize every organic molecule it needs from glucose and a number of inorganic ions. Many of the genes in E. coli are expressed constitutively; that is, they are always turned "on". Others, however, are active only when their products are needed by the cell, so their expression must be regulated.
• 9.2: The Tryptophan Repressor
In E. coli, the synthesis of the amino acid tryptophan from precursors available to the cell requires 5 enzymes. The genes encoding these are clustered together in a single operon with its own promoter and operator. When tryptophan is available to the cell, its presence shuts down the operon.
• 9.3: Regulation of Gene Expression in Eukaryotes
There are several methods used by eukaryotes. regulate gene expression. Including altering the rate of transcription of the gene, altering the rate at which RNA transcripts are processed, altering the stability of messenger RNA molecules and altering the efficiency with which ribosomes translate the mRNA into a polypeptide.
• 9.4: Steroid Response Elements
Steroid hormone receptors are proteins that have a binding site for a particular steroid molecule. Their response elements are DNA sequences that are bound by the complex of the steroid bound to its receptor. The response element is part of the promoter of a gene. Binding by the receptor activates or represses, as the case may be, the gene controlled by that promoter. It is through this mechanism that steroid hormones turn genes on (or off).
• 9.5: Epigenetics
Epigenetics can be defined as a change in phenotype that is heritable but does not involve a change in the nucleotide sequence in DNA; that is, a change in genotype. This definition is very broad encompassing a variety of phenomena.
• 9.6: Visualization of Transcription and Translation in Bacteria
Each polysome is attached to the DNA fiber by a complex of proteins that includes a molecule of RNA polymerase. Thus the DNA is transcribed by RNA polymerase molecules moving from top to bottom, and the growing mRNA molecules are translated by ribosomes moving in a proximal -> distal direction. IIn E. coli, then, and probably in all bacteria, the transcription of DNA into mRNA and the translation of mRNA into polypeptides (not visible here) are closely coordinated in both time and space.
• 9.7: Footprinting
Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds.
• 9.8: Chromatin Immunoprecipitation
Many DNA-binding proteins, such as transcription factors, bind to specific sequences of nucleotides in, for example, promoters and enhancers of genes. The binding of protein to DNA is done by noncovalent forces and is easily reversible. The identification of a specific site in DNA bound by a particular protein at a particular time can be discovered by the technique of chromatin immunoprecipitation.
• 9.9: Isolating Transcription Factors
Transcription factors are extraordinarily diverse, and any one factor represents only a tiny fraction of the protein molecules present in the cell. This page describes how one can isolate and purify such rare molecules.
• 9.10: Palindromes
A palindrome is a sequence of letters and/or words, that reads the same forwards and backwards. Palindromes also occur in a DNA and there are two types.
• 9.11: Cell-specific gene expression
• 9.12: Imprinted Genes
Imprinted genes are genes whose expression is determined by the parent that contributed them. Imprinted genes violate the usual rule of inheritance that both alleles in a heterozygote are equally expressed.
• 9.13: Ribozymes
Some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules). Catalytic RNA molecules are called ribozymes.
09: Regulation of Gene Expression
The Operon
Within its tiny cell, the bacterium E. coli contains all the genetic information it needs to metabolize, grow, and reproduce. It can synthesize every organic molecule it needs from glucose and a number of inorganic ions. Many of the genes in E. coli are expressed constitutively; that is, they are always turned "on". Others, however, are active only when their products are needed by the cell, so their expression must be regulated.
Two examples:
• If the amino acid tryptophan (Trp) is added to the culture, the bacteria soon stop producing the five enzymes previously needed to synthesize Trp from intermediates produced during the respiration of glucose. In this case, the presence of the products of enzyme action represses enzyme synthesis.
• Conversely, adding a new substrate to the culture medium may induce the formation of new enzymes capable of metabolizing that substrate. If we take a culture of E. coli that is feeding on glucose and transfer some of the cells to a medium contain lactose instead, a revealing sequence of events takes place.
• At first the cells are quiescent: they do not metabolize the lactose, their other metabolic activities decline, and cell division ceases.
• Soon, however, the culture begins growing rapidly again with the lactose being rapidly consumed. What has happened? During the quiescent interval, the cells began to produce three enzymes.
The three enzymes are
• a permease that transports lactose across the plasma membrane from the culture medium into the interior of the cell
• beta-galactosidase which converts lactose into the intermediate allolactose and then hydrolyzes this into glucose and galactose. Once in the presence of lactose, the quantity of beta-galactosidase in the cells rises from a tiny amount to almost 2% of the weight of the cell.
• a transacetylase whose function is still uncertain.
The lac operon
The capacity to respond to the presence of lactose was always there. The genes for the three induced enzymes are part of the genome of the cell. But until lactose was added to the culture medium, these genes were not expressed (β-galactosidase was expressed weakly — just enough to convert lactose into allolactose). The most direct way to control the expression of a gene is to regulate its rate of transcription; that is, the rate at which RNA polymerase transcribes the gene into molecules of messenger RNA (mRNA).
Gene transcription begins at a particular nucleotide shown in the figure as "+1". RNA polymerase actually binds to a site "upstream" (i.e., on the 5' side) of this site and opens the double helix so that transcription of one strand can begin. The binding site for RNA polymerase is called the promoter. In bacteria, two features of the promoter appear to be important:
• a sequence of TATAAT (or something similar) centered 10 nucleotides upstream of the +1 site and
• another sequence (TTGACA or something quite close to it) centered 35 nucleotides upstream.
The exact DNA sequence between the two regions does not seem to be important. Each of the three enzymes synthesized in response to lactose is encoded by a separate gene. The three genes are arranged in tandem on the bacterial chromosome.
In the absence of lactose, the repressor protein encoded by the I gene binds to the lac operator and prevents transcription. Binding of allolactose to the repressor causes it to leave the operator. This enables RNA polymerase to transcribe the three genes of the operon. The single mRNA molecule that results is then translated into the three proteins.
The lac repressor binds to a specific sequence of two dozen nucleotides called the operator. Most of the operator is downstream of the promoter. When the repressor is bound to the operator, RNA polymerase is unable to proceed downstream with its task of gene transcription. The lac repressor represents only a tiny fraction of the proteins in the E. coli cell.
The operon is the combination of the operator and the three protein-encoding genes associated with it.
The gene encoding the lac repressor is called the I gene. It happens to be located just upstream of the lac promoter. However, its precise location is probably not important because it achieves its effect by means of its protein product, which is free to diffuse throughout the cell. And, in fact, the genes for some repressors are not located close to the operators they control.
Although repressors are free to diffuse through the cell, how does — for example — the lac repressor find the single stretch of 24 base pairs of the operator out of the 4.6 million base pairs of DNA in the E. coli genome? It turns out the repressor is free to bind anywhere on the DNA using both
• hydrogen bonds and
• ionic (electrostatic) interactions between its positively-charged amino acids (Lys, Arg) and the negative charges on the deoxyribose-phosphate backbone of the DNA.
Once astride the DNA, the repressor can move along it until it encounters the operator sequence. Now an allosteric change in the tertiary structure of the protein allows the same amino acids to establish bonds — mostly hydrogen bonds and hydrophobic interactions — with particular bases in the operator sequence.
The lac repressor is made up of four identical polypeptides (thus a "homotetramer"). Part of the molecule has a site (or sites) that enable it to recognize and bind to the 24 base pairs of the lac operator. Another part of the repressor contains sites that bind to allolactose. When allolactose unites with the repressor, it causes a change in the shape of the molecule, so that it can no longer remain attached to the DNA sequence of the operator. Thus, when lactose is added to the culture medium, it causes the repressor to be released from the operator and RNA polymerase can now begin transcribing the 3 genes of the operon into a single molecule of messenger RNA.
Hardly does transcription begin, before ribosomes attach to the growing mRNA molecule and move down it to translate the message into the three proteins. You can see why punctuation codons — UAA, UAG, or UGA — are needed to terminate translation between the portions of the mRNA coding for each of the three enzymes. This mechanism is characteristic of bacteria, but differs in several respects from that found in eukaryotes:
• Genes in eukaryotes are not linked in operons (except for nematodes like C. elegans and tunicates like Ciona intestinalis).
• Primary transcripts in eukaryotes contain the transcript of only a single gene (with the above exceptions).
• Transcription and translation are not physically linked in eukaryotes as they are in bacteria; transcription occurs in the nucleus while translation occurs in the cytosol (with a few exceptions).
C. elegans
C. elegans differs from most eukaryotes in having a substantial fraction (15–20%) of its genes grouped in operons containing from 2 to 8 genes each. Like bacteria, all the genes in an operon are transcribed from a single promoter producing a single primary transcript (pre-mRNA). Some of the genes in these operons appear — as in bacteria — to be involved in the same biochemical function, but this may not be the case for most. C. elegans operons also differ from those in bacteria in that each pre-mRNA is processed into a separate mRNA for each gene rather than being translated as a unit.
Corepressors
As mentioned above, the synthesis of tryptophan from precursors available in the cell requires 5 enzymes. The genes encoding these are clustered together in a single operon with its own promoter and operator. In this case, however, the presence of tryptophan in the cell shuts down the operon. When Trp is present, it binds to a site on the Trp repressor and enables the Trp repressor to bind to the operator. When Trp is not present, the repressor leaves its operator, and transcription of the 5 enzyme-encoding genes begins.
The above picure shows stereo view of the tryptophan repressor (right side of each panel) bound to its operator DNA (left side). The repressor is a homodimer of two identical polypeptides (on either side of the horizontal red line). Binding to DNA occurs only when a molecule of tryptophan (red rings) is bound to each monomer of the repressor. The usefulness to the cell of this control mechanism is clear. The presence in the cell of an essential metabolite, in this case tryptophan, turns off its own manufacture and thus stops unneeded protein synthesis. As its name suggests, repressors are negative control mechanisms, shutting down operons
• in the absence of a substrate (lactose in our example) or
• the presence of an essential metabolite (tryptophan is our example).
However, some gene transcription in E. coli is under positive control.
Positive Control of Transcription: CAP
Absence of the lac repressor is essential but not sufficient for effective transcription of the lac operon. The activity of RNA polymerase also depends on the presence of another DNA-binding protein called catabolite activator protein (CAP). Like the lac repressor, CAP has two types of binding sites: One binds the nucleotide cyclic AMP and the other binds a sequence of 16 base pairs upstream of the promoter
However, CAP can bind to DNA only when cAMP is bound to CAP. so when cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase cannot begin its work, even in the absence of the repressor. So the lac operon is under both negative (the repressor) and positive (CAP) control. Why?
It turns out that it is not simply a matter of belt and suspenders. This dual system enables the cell to make choices. What, for example, should the cell do when fed both glucose and lactose? Presented with such a choice, E. coli (for reasons about which we can only speculate) chooses glucose. It makes its choice by using the interplay between these two control devices.
Although the presence of lactose removes the repressor, the presence of glucose lowers the level of cAMP in the cell and thus removes CAP.Without CAP, binding of RNA polymerase is inhibited even though there is no repressor to interfere with it if it could bind. The molecular basis for its choices is shown in the above figure.
CAP consists of two identical polypeptides (hence it is a homodimer). Toward the C-terminal, each has two regions of alpha helix with a sharp bend between them. The longer of these is called the recognition helix because it is responsible for recognizing and binding to a particular sequence of bases in DNA.
The above figure shows a model of CAP. The two monomers are identical. Each monomer recognizes a sequence of nucleotides in DNA by means of the region of alpha helix labeled F. Note that the two recognition helices are spaced 34Å apart, which is the distance that it takes the DNA molecule (on the left) to make precisely one complete turn.
The recognition helices of each polypeptide of CAP are, of course, identical. But their orientation in the dimer is such that the sequence of bases they recognize must run in the opposite direction for each recognition helix to bind properly. This arrangement of two identical sequences of base pairs running in opposite directions is called an inverted repeat.
The strategy illustrated by CAP and its binding site has turned out to be used widely. As more and more DNA-regulating proteins have been discovered, many turn out to share the traits we find in CAP:
• They usually contain two subunits. Therefore, they are dimers.
• They recognize and bind to DNA sequences with inverted repeats.
• In bacteria, recognition and binding to a particular sequence of DNA is accomplished by a segment of alpha helix. Hence these proteins are often described as helix-turn-helix proteins. The Trp repressor shown above is a member of this group.
Riboswitches
Protein repressors and corepressors are not the only way in which bacteria control gene transcription. It turns out that the regulation of the level of certain metabolites can also be controlled by riboswitches. A riboswitch is section of the 5'-untranslated region (5'-UTR) in a molecule of messenger RNA (mRNA) which has a specific binding site for the metabolite (or a close relative). Some of the metabolites that bind to riboswitches include:
• the purines adenine and guanine
• the amino acids glycine and lysine
• flavin mononucleotide (the prosthetic group of NADH dehydrogenase)
• S-adenosyl methionine that donates methyl groups to many molecules, including DNA and the cap at the 5' end of messenger RNA
• tRNAs. When these are bound to their amino acid (aminoacyl-tRNA), they bind to the riboswitch in the mRNA that encodes the enzyme (an aminoacyl-tRNA synthetase) responsible for loading the amino acid onto the tRNA. This causes transcription of the mRNA to terminate prematurely. tRNAs with no amino acid attached also bind to the riboswitch but in such a way that transcription of the mRNA continues. Its translation (in bacteria, translation begins while transcription is still going on) produces the aminoacyl-tRNA synthetase used to load the amino acid onto the tRNA. Thus these riboswitches regulate the level of aminoacyl-tRNAs producing more when needed, less when not (a kind of feedback inhibition.)
In each case, the riboswitch regulates transcription of genes involved in the metabolism of that molecule. The metabolite binds to the growing mRNA and induces an allosteric change that for some genes causes further synthesis of the mRNA to terminate before forming a functional product and for other genes, enhances completion of synthesis of the mRNA. In both cases, one result is to control the level of that metabolite.
Some riboswitches control mRNA translation rather than its transcription. It has been suggested that these regulatory mechanisms, which do not involve any protein, are a relict from an "RNA world". | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.01%3A_Regulation_of_Gene_Expression_in_Bacteria.txt |
In E. coli, the synthesis of the amino acid tryptophan from precursors available to the cell requires 5 enzymes. The genes encoding these are clustered together in a single operon with its own promoter and operator. When tryptophan is available to the cell, its presence shuts down the operon.
Mechanism
• One molecule of tryptophan binds to a site on each monomer of the Trp repressor.
• The Trp repressor, a homodimer of two of these complexes, binds to the operator of the Trp operon.
• This shuts down transcription of the 5 genes of the operon so the enzymes used in Trp synthesis are not synthesized.
This stereoscopic view () shows the tryptophan repressor (right side of each panel) bound to its operator DNA (left side). The two identical polypeptides of the repressor are shown on either side of the horizontal red line. The two tryptophan molecules are shown as red rings. Look also for the stretches of alpha helix in each monomer. You may find it easier to fuse the two images into a 3D view by holding a sheet of 8.5 x 11" (22 x 28 cm) paper vertically between your nose and the dividing line between the two images on the screen so that your left eye sees only the left image, your right eye only the right.
9.03: Regulation of Gene Expression in Eukaryotes
The latest estimates are that a human cell, a eukaryotic cell, contains some 21,000 genes. Some of these are expressed in all cells all the time. These so-called housekeeping genes are responsible for the routine metabolic functions (e.g. respiration) common to all cells. Some are expressed as a cell enters a particular pathway of differentiation. Some are expressed all the time in only those cells that have differentiated in a particular way. For example, a plasma cell expresses continuously the genes for the antibody it synthesizes. Some are expressed only as conditions around and in the cell change. For example, the arrival of a hormone may turn on (or off) certain genes in that cell.
How is gene expression regulated? There are several methods used by eukaryotes.
• Altering the rate of transcription of the gene. This is the most important and widely-used strategy.
• However, eukaryotes supplement transcriptional regulation with several other methods:
• Altering the rate at which RNA transcripts are processed while still within the nucleus.
• Altering the stability of messenger RNA (mRNA) molecules; that is, the rate at which they are degraded.
• Altering the efficiency with which ribosomes translate the mRNA into a polypeptide.
Protein-coding genes have
• exons whose sequence encodes the polypeptide
• introns that will be removed from the mRNA before it is translated
• a transcription start site
• promoters
• a basal or core promoter located within about 40 base pairs (bp) of the start site
• "upstream" promoters, which may extend over as many as 200 bp farther upstream
• enhancers
• silencers
Adjacent genes are often separated by an insulator which helps them avoid cross-talk between each other's promoters and enhancers (and/or silencers).
Transcription start site
This is where a molecule of RNA polymerase II (pol II, also known as RNAP II) binds. Pol II is a complex of 12 different proteins (shown in the figure in yellow with small colored circles superimposed on it). The start site is where transcription of the gene into RNA begins.
The core promoter
All eukaryotic genes contain a core promoter. One common example is a sequence of bases (e.g., TATAAAAAA) called the TATA box. It is bound by a large complex of some 50 different proteins, including
• Transcription Factor IID (TFIID) which is a complex of
• TATA-binding protein (TBP), which recognizes and binds to the TATA box
• 13 other protein factors which bind to TBP, each other, and (some of them) to the DNA.
• Transcription Factor IIB (TFIIB) which binds both the DNA and pol II.
Many different genes and many different types of cells share the same transcription factors — not only those that bind at the core promoter but even some of those that bind upstream. What turns on a particular gene in a particular cell is probably the unique combination of promoter sites and the transcription factors that are chosen.
An Analogy
The rows of lock boxes in a bank provide a useful analogy. To open any particular box in the room requires two keys:
• Your key, whose pattern of notches fits only the lock of the box assigned to you (= the upstream promoter), but which cannot unlock the box without
• A key carried by a bank employee that can activate the unlocking mechanism of any box (= the core promoter) but cannot by itself open any box.
Hormones Effect
These loops are stabilized by a protein designated CTCF ("CCCTC binding factor"; named for the nucleotide sequence to which it binds). The CTCF at one site on the DNA forms a dimer with the CTCF at another site on the DNA binding the two regions together. CTCF has 11 zinc fingers. They can also be stabilized by cohesin — the same protein complex that holds sister chromatids together during mitosis and meiosis.
Michael R. Botchan and his colleagues have produced visual evidence of this model of enhancer action. They created an artificial DNA molecule with
• several (4) promoter sites for Sp1 about 300 bases from one end. Sp1 is a zinc-finger transcription factor that binds to the sequence 5' GGGCGG 3' found in the promoters of many genes, especially "housekeeping" genes.
• several (5) enhancer sites about 800 bases from the other end. These are bound by an enhancer-binding protein designated E2.
• 1860 base pairs of DNA between the two.
Significance of "Looping"
The looping of chromosomes that brings enhancers close to promoters (and promoters close to other promoters) seems to be a mechanism to ensure the expression (or inhibition) of groups of genes that must perform together. The response of a cell to the arrival of a signal (e.g., a hormone) may involve turning on (or off) hundreds of different genes whose products must be produced in a coordinated way for the cell to respond appropriately. The dynamic movement of portions of the chromosome carrying the appropriate gene loci into a "transcription factory" may be a mechanism to accomplish this. If so, we are seeing the eukaryotic equivalent of the coordinated gene expression provided by operons in bacteria.
Silencers
Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control. However, when transcription factors bind to them, expression of the gene they control is repressed.
Insulators
As you can see above, enhancers can turn on promoters of genes located thousands of base pairs away. Insulators prevent an enhancer from inappropriately binding to and activating the promoter of some other gene in the same region of the chromosome..
Insulators are
• stretches of DNA (as few as 42 base pairs may do the trick)
• located between the
• enhancer(s) and promoter(s) or
• silencer(s) and promoter(s)
of adjacent genes or clusters of adjacent genes.
The enhancer for the promoter of the gene for the delta chain of the gamma/delta T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose between one or the other. There is an insulator between the alpha gene promoter and the delta gene promoter that ensures that activation of one does not spread over to the other.
All insulators discovered so far in vertebrates work only when bound by the CTCF protein. Another example: In mammals (mice, humans, pigs), only the allele for insulin-like growth factor-2 (IGF2) inherited from one's father is active; that inherited from the mother is not — a phenomenon called imprinting.
The mechanism: the mother's allele has an insulator between the IGF2 promoter and enhancer. So does the father's allele, but in his case, the insulator has been methylated. CTCF can no longer bind to the insulator, and so the enhancer is now free to turn on the father's IGF2 promoter.
Many of the commercially-important varieties of pigs have been bred to contain a gene that increases the ratio of skeletal muscle to fat. This gene has been sequenced and turns out to be an allele of IGF2, which contains a single point mutation in one of its introns. Pigs with this mutation produce higher levels of IGF2 mRNA in their skeletal muscles (but not in their liver). This tells us that:
• Mutations need not be in the protein-coding portion of a gene in order to affect the phenotype.
• Mutations in non-coding portions of a gene can affect how that gene is regulated (here, a change in muscle but not in liver). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.02%3A_The_Tryptophan_Repressor.txt |
The DNA sequence of the glucocorticoid response element is
5' AGAACAnnnTGTTCT 3'
3' TCTTGTnnnACAAGA 5'
where n represents any nucleotide. (Note the inverted repeats.) The glucocorticoid receptor, like all steroid hormone receptors, is a zinc-finger transcription factor; the zinc atoms are the four yellow spheres. Each is attached to four cysteines.
For a steroid hormone to regulate (turn on or off) gene transcription, its receptor must:
• bind to the hormone (cortisol in the case of the glucocorticoid receptor)
• bind to a second copy of itself to form a homodimer
• be in the nucleus, moving from the cytosol if necessary
• bind to its response element
• bind to other protein cofactors
This autoradiograph shows the endometrial cells from the uterus of a guinea pig 15 minutes after an injection of radioactive progesterone. The radioactivity has concentrated within the nuclei of the endometrial cells as shown by the dark grains superimposed on the images of the nuclei. The same effect is seen when radioactive estrogens are administered.
The cells of the endometrium are target cells for both progesterone and estrogens, preparing the uterus for possible pregnancy. Nontarget cells (e.g. liver cells or lymphocytes) show no accumulation of female sex hormones. Although their DNA contains the response elements, their cells do not have the protein receptors needed.
The Nuclear Receptor Superfamily
The zinc-finger proteins that serve as receptors for glucocorticoids and progesterone are members of a large family of similar proteins that serve as receptors for a variety of small, hydrophobic molecules. These include:
• other steroid hormones like the mineralocorticoid aldosterone and estrogens
• the thyroid hormone, T3
• calcitriol, the active form of vitamin D
• retinoids: vitamin A (retinol) and its relatives
• retinal
• retinoic acid (tretinoin — also available as the drug Retin-A®); and its isomer
• isotretinoin (sold as Accutane® for the treatment of acne).
• bile acids
• fatty acids. These bind members of the superfamily called peroxisome-proliferator-activated receptors (PPARs). They got their name from their initial discovery as the receptors for drugs that increase the number and size of peroxisomes in cells.
• In every case, the receptors consists of at least three functional modules or domains. From N-terminal to C-terminal, these are:
• a domain needed for the receptor to activate the promoters of the genes being controlled
• the zinc-finger domain needed for DNA binding (to the response element)
• the domain responsible for binding the particular hormone as well as the second unit of the dimer
9.05: Epigenetics
Epigenetics can be defined as a change in phenotype that is heritable but does not involve a change in the nucleotide sequence in DNA; that is, a change in genotype. This definition is very broad encompassing a variety of phenomena.
Epigenetic changes during cellular differentiation
For example, a change in phenotype of a single cell that is then passed on to its descendants qualifies as an epigenetic phenomenon. Thus it includes the various pathways of differentiation that are taken by cells during the embryonic development of an organism. Examples:
• X-inactivation — where one of the two X chromosomes in female mammals is inactivated in each cell early in development and that same chromosome remains inactivated in all the descendants of that cell.
• Imprinting — where whether a gene in a cell lineage is expressed or not depends on which parent contributed the gene.
The great stumbling block in converting differentiated cells into induced pluripotent stem cells (iPSCs) was to find ways of reversing the epigenetic changes in the differentiated cell (e.g., a skin cell) to unlock its full developmental potential. Stable changes in gene expression are brought about in two main ways:
• DNA methylation — where its cytosines are methylated. This usually represses the activity of that DNA.
• Histone modifications — where methyl, acetyl, and other groups are added to the histones in chromatin. Prominent examples:
• adding methyl groups to the #4 lysine in histone H3 ("H3K4me"). This is associated with active genes in that region of the chromatin.
• adding methyl groups to the #27 lysine in histone H3 ("HeK27me"). This is associated with gene silencing.
Some definitions:
• epigenetic "writers": enzymes that add chemical groups to histones or DNA.
• epigenetic "erasers": enzymes that remove these groups.
• epigenetic "readers": proteins that recognize specific epigenetic modifications of histones or DNA producing a change in gene expression, e.g., increasing (or decreasing) gene transcription.
9.06: Visualization of Transcription and Translation in Bacteria
The above figure is an Electron micrograph courtesy of O. L. Miller, Jr., B. A. Hamkalo, and C. A. Thomas, Jr. It shows simultaneous transcription and translation of E. coli genes. The long fiber running from top to bottom (green arrow ) is a segment of the E. coli chromosome. Extending from it are polysomes (red arrow), the size of which generally increases from top to bottom. Each polysome consists of a backbone of messenger RNA (mRNA) to which the ribosomes are attached.
Each polysome is attached to the DNA fiber by a complex of proteins that includes a molecule of RNA polymerase. Thus the DNA is transcribed by RNA polymerase molecules moving from top to bottom, and the growing mRNA molecules are translated by ribosomes moving in a proximal -> distal direction. IIn E. coli, then, and probably in all bacteria, the transcription of DNA into mRNA and the translation of mRNA into polypeptides (not visible here) are closely coordinated in both time and space.
In eukaryotes, in contrast, while all transcription takes place in the nucleus, most (but not all) translation of mRNA occurs later in the cytosol.
9.07: Footprinting
Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples:
• hormone-receptor complexes that bind to their hormone response elements
• transcription factors that bind eukaryotic operators, enhancers, and silencers
• the lac repressor that shuts down the lac operon in E. coli
• Clone a piece of DNA that contains the operator site to which the repressor binds.
• Label one end of the DNA molecules with a radioactive molecule, e.g. radioactive ATP.
• Digest the DNA with DNase I.
• DNase I cuts DNA molecules randomly (in contrast to restriction enzymes that cut where they find a particular sequence)
• Choose such gentle conditions that most molecules will be cut only once.
• The result will be a mixture of radioactive fragments of varying length, with the smallest increment in length represented by a single nucleotide.
• Separate the fragments by electrophoresis.
• Binding of the lac repressor to the sequence of 24 base pairs in the operator prevents DNase I from attacking that region of the molecule.
• When the fragments are separated by electrophoresis, those representing the lengths covered by the repressor will be missing from the autoradiogram.
• The resulting gap is the "footprint".
• The same sample of DNA (unprotected by the repressor) is subjected to normal DNA sequencing and the resulting ladder aligned with the footprint autoradiogram.
• The exact sequence of bases in the lac operator can then be read directly because they represent the rungs of the ladder missing in the footprint. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.04%3A_Steroid_Response_Elements.txt |
Many DNA-binding proteins, such as transcription factors, bind to specific sequences of nucleotides in, for example, promoters and enhancers of genes. Some examples:
• a generalized eukaryotic promoter
• multiple transcription factors bound to the Drosophila eve promoter
• the glucocorticoid receptor (protein) bound to its response element (DNA)
• the tryptophan repressor bound to its operator
The binding of protein to DNA is done by noncovalent forces and is easily reversible. In fact, as conditions in a cell change, there is a dynamic coming and going of DNA-binding proteins throughout the genome. The identification of a specific site in DNA bound by a particular protein at a particular time can be discovered by the technique of chromatin immunoprecipitation (ChIP).
The Procedure
1. Harvest your cell population at the desired time. Some examples:
• yeast cells growing on a particular nutrient;
• HeLa cells exposed to a particular cytokine;
• salivary gland cells of Drosophila at the time of pupation.
2. Treat the cells with formaldehyde (HCHO) which creates covalent bonds between the proteins and nucleotides to which they have been bound noncovalently.
3. Break open the cells releasing their contents.
4. Use ultrasound to break the DNA into fragments averaging about 500 bp long.
5. Add an antibody that specifically binds to the protein you are interested in.
6. Add beads coated with Protein A or Protein S — both proteins that bind to any antibody.
7. Centrifuge down the complexes of bead—antibody—target protein—DNA.
8. Heat the complexes to break the covalent crosslinks between the target protein and the DNA.
9. Digest the protein with a protease leaving purified DNA fragments.
10. Perform PCR on the fragments.
11. Use any of several methods to identify the amplified DNA, for example,
• Southern blotting
• DNA chip analysis ("ChIP on chip")
• clone and sequence ("ChIP-Seq")
9.09: Isolating Transcription Factors
Transcription factors are extraordinarily diverse, and any one factor represents only a tiny fraction of the protein molecules present in the cell. This page describes how one can isolate and purify such rare molecules.
Example: Isolating the lac Repressor
An E. coli cell contains only 10-20 copies of the lac repressor. This represents a ratio of only 1 molecule in 50,000 protein molecules in the cell. However, the specificity of the lac repressor for the DNA sequence of the operator provides a mechanism for fishing it out of the mixture.
Affinity Chromatography
The following procedure is carried out for an affinity chromatograph:
• Learn the sequence to which the repressor binds (by footprinting).
• Synthesize a segment of DNA containing the sequence.
• Attach this artificial molecule to beads of an inert, solid medium (the matrix).
• Pour an extract of E. coli cells over the beads.
• Only molecules specific for the DNA sequence — in this case, molecules of the lac repressor — will bind to the beads.
• After irrelevant protein molecules have passed through the column, wash the beads with a buffer that will release the lac repressor molecules so they can be studied.
9.10: Palindromes
A palindrome is a sequence of letters and/or words, that reads the same forwards and backwards. "able was I ere I saw elba" is a palindrome. Palindromes also occur in a DNA. There are two types.
Palindromes that occur on opposite strands of the same section of DNA helix
5' GGCC 3'
3' CCGG 5'
This type of palindrome serves as the target for most restriction enzymes. The graphic shows the palindromic sequences "seen" by five restriction enzymes (named in blue) commonly used in recombinant DNA work.
Inverted Repeats
In these cases, two different segments of the double helix read the same but in opposite directions.
5' AGAACAnnnTGTTCT 3'
3' TCTTGTnnnACAAGA 5'
Inverted repeats are commonly found in
• The DNA to which transcription factors bind.
The DNA sequence shown above is that of the glucocorticoid response element where n represents any nucleotide. Transcription factors are often dimers of identical proteins homodimers so it is not surprising that each member of the pair needs to "see" the same DNA sequence in the same orientation.
• The DNA of many transposons is flanked by inverted repeats such as this one:
• 5' GGCCAGTCACAATGG..~400 nt..CCATTGTGACTGGCC 3'
3' CCGGTCAGTGTTACC..~400 nt..GGTAACACTGACCGG 5'
• Inverted repeats at either end of retroviral gene sequences aid in inserting the DNA copy into the DNA of the host.
• Duplicated Genes.
The human Y chromosome contains 7 sets of genes — each set containing from 2 to 6 nearly-identical genes — oriented back-to-back or head-to-head; that is, they are inverted repeats like the portion shown here. (The dashes represent the thousands of base pairs that separate adjacent palindromes.)
CTCCCACAACCCATGGGATTTGTG... 3'
GAGGGTGTTGGGTACCCTAAACAC... 5'
This orientation and redundancy may help ensure that a deleterious mutation in one copy of the set can be repaired using the information in another copy of that set. All that is needed is to form a loop so that the two sequences line up side-by-side. Repairs can then be made (probably by the mechanism of homologous recombination). Here, for example, the single difference in the sequences can be eliminated (red for blue or vice versa). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.08%3A_Chromatin_Immunoprecipitation.txt |
An example: Make Drosophila transgenic for recombinant DNA containing:
• the Z gene for beta-galactosidase
• even-skipped (eve), (another homeobox gene).
Any cell with the transcription factors for turning on the even-skipped promoter will begin to make beta-galactosidase. Given the proper substrate, the enzyme produces a colored product.
The above photomicrograph shows 7 bands of this colored product identifying the cells that were expressing the even-skipped gene. This event was "reported" by the lacZ gene. The 7 dark stripes reveal regions that alternate with the 7 bands formed by the cells expressing fushi-tarazu.
Green fluorescent protein (GFP)
In nature, green fluorescent protein (GFP) is produced by, Aequorea victoria, the Pacific Northwest jellyfish. The protein has become of great interest to cell and molecular biologists because it can reveal gene expression in living cells.
This is done by fusing the gene for GFP to the gene whose expression you are interested in. When that gene is turned on in a cell, not only is its protein synthesized, but GFP is synthesized as well. Illuminating the cells with near-ultraviolet light causes them to fluoresce a bright green. In this way, the experimenter can see when and where the gene is expressed in the living organism.
DNA Chips
All the methods described so far are limited to monitoring the expression of one or, at most, a few genes. But as conditions change in a cell, the transcription and translation of literally hundreds of genes may be altered.
Thanks to the marriage of
• semiconductor chip technology
• automated synthesis of oligodeoxynucleotides
• automated fluorescence scanners
• computer software,
it is now possible to monitor the activity of literally thousands of genes in one kind of cell. For examples:
• mammalian cells when they are transferred from a "minimal" culture medium to one enriched in growth factors;
• the skeletal muscles of mice as they age.
The Chip
• Examine published gene sequences.
• For each gene, pick out ~20 different stretches of ~25 nucleotides that seem characteristic of that gene.
• Synthesize oligodeoxynucleotides corresponding to these.
• Also synthesize oligodeoxynucleotides for each of the above that have one nucleotide altered (usually near the middle). These will provide a control.
• Using robotic chip-making machines, spot these oligonucleotides individually in arrays, each spot receiving millions of copies that are fixed to the chip surface.
With the partial completion of the human genome project, three companies are now selling DNA chips containing from 36,000 to 50,000 pieces of DNA thought to represent different human genes.
The Assay
• Harvest your cells. Presumably they are expressing a characteristic subset of their genes; that is, transcribing them into messenger RNA (mRNA) molecules.
• Extract the RNA.
• Make complementary DNA (cDNA) by treating the RNA mixture with reverse transcriptase.
• Transcribe the cDNA back into now much-amplified RNA.
• Attach fluorescent tags to the RNA.
• Flood the chip with this mixture.
• RNAs finding their complementary sequences on the chip will bind to them. (They will bind less strongly to adjacent spots with the single-nucleotide change if the binding is truly specific.
• Illuminate the chip and automatically record the intensities of the color at each spot.
• Use a computer to analyze the pattern.
Results of monitoring genome-wide expression
• This work was reported by V. R. Iyer, et al in the 1 January 1999 issue of Science. It involved the monitoring the expression of 8613 different genes.
• Mice raised on a restricted diet did not show such dramatic shifts in gene expression as they aged. This fits well with data that mice on restricted diets age more slowly than those on rich diets. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.11%3A_Cell-specific_gene_expression.txt |
Imprinted genes are genes whose expression is determined by the parent that contributed them. Imprinted genes violate the usual rule of inheritance that both alleles in a heterozygote are equally expressed.
Examples of the usual rule:
• If a child inherits the gene for blood group A from either parent and the gene for group B from the other parent, the child's blood group will be AB.
• If a child inherits the gene encoding hemoglobin A from either parent and the gene encoding hemoglobin S from the other parent, the child's red blood cells will contain roughly equal amounts of the two types of hemoglobin.
But there are a few exceptions to this rule. A small number of genes in mammals (~80 of them at a recent count) and in angiosperms have been found to be imprinted. Because most imprinted genes are repressed, either
• the maternal (inherited from the mother) allele is expressed exclusively because the paternal (inherited from the father) allele is imprinted or
• vice versa.
The process begins during gamete formation when
• in males certain genes are imprinted in developing sperm and
• in females, others are imprinted in the developing egg.
All the cells in a resulting child will have the same set of imprinted genes from both its father and its mother EXCEPT for those cells ("germplasm") that are destined to go on to make gametes. All imprints — both maternal and paternal — are erased in them.
Examples
IGF2
— the gene encoding the insulin-like growth factor-2
In humans (and other mammals like mice and pigs) the IGF2 allele inherited from the father (paternal) is expressed; the allele inherited from the mother is not.
If both alleles should begin to be expressed in a cell, that cell may develop into a cancer.
IGF2r
— the gene encoding the cell receptor for Igf-2
In mice the IGF2r allele inherited from the mother is expressed; that from the father is not. Differential imprinting accounts for this, and the mechanism is described below.
XIST
— the gene encoding the RNA that converts one of the X chromosomes in a female cell into an inactive Barr body. This process is random in the cells of the female fetus and thus is NOT an example of imprinting. However, all the cells of her extraembryonic membranes (which form the amnion, placenta, and umbilical cord) have the father's X chromosome inactivated. Imprinting of the XIST locus accounts for this.
Mechanism of parental imprinting
The process of imprinting starts in the gametes where the allele destined to be inactive in the new embryo (either the father's or the mother's as the case may be) is "marked". The mark appears to be methylation of the DNA in the promoter(s) of the gene.
Methyl groups are added to cytosines (Cs) in the DNA. When this occurs at stretches of alternating Cs and Gs called CpG sites in a promoter, it prevents binding of transcription factors to the promoter thus shutting down expression of the gene.
Although methylation seems to be the imprinting signal, keeping the gene shut down may require the production of RNA.
Methylation — and thus inactivation — of the promoters of tumor suppressor genes is frequently found in cancer cells.
The IGF2r gene
A report in Nature (16 October 1997) by Wutz et al, reveals that:
In the mother's (maternal) copy of the gene,
• there is an upstream (left) promoter that is unmethylated and active
• binding of transcription factors to this upstream promoter enables transcription of the sense strand of the gene to produce Igf2r messenger RNA.
• There is also a downstream set of CpG sites that are methylated
In the father's (paternal) copy of the IGF2r gene (the imprinted version)
• the promoter for IGF2r transcription is methylated (and inactive),
• but the downstream promoter is unmethylated and active.
• Transcription of the antisense strand from the downstream promoter produces an antisense RNA (a long noncoding RNA) that participates in shutting his gene down.
XIST
The XIST locus on the X chromosome encodes a long noncoding RNA that shuts down all (or almost all) of the other genes on the chromosome, converting it into an inactive Barr body.
Is imprinting important?
Yes.
• Deliberate (in mice) or accidental (in humans) inheritance of two copies of a particular chromosome from one parent and none from the other parent is usually fatal (even though a complete genome is present).
• Inheritance of two copies of one of mother's genes and no copy of the father's (or vice versa) can produce serious developmental defects.
• Failure to inherit several nonimprinted genes on the father's chromosome #15 causes a human congenital disorder called Prader-Willi syndrome.
• Absence or mutation of a nonimprinted gene (UBE3A) on the mother's chromosome #15 causes Angelman syndrome.
• Failure of imprinting in somatic cells may lead to cancer.
• The cancerous cells in some cases of a malignancy called Wilms´ tumor and many cases of colon cancer have both copies of the IGF2 gene expressed (where only one, the father's, should be).
• Reduced methylation — and hence increased expression — of proto-oncogenes can lead to cancer, while
• increased methylation — and hence decreased expression — of tumor suppressor genes can also do so.
Imprinting and Parthenogenesis
Imprinting is the reason that parthenogenesis ("virgin birth") does not occur in mammals. Two complete female genomes cannot produce viable young because of the imprinted genes. For example, the embryo needs the father's Igf2 gene because the mother's copy has been imprinted and is inactive.
• An insulator — with a bound protein designated CTCF ("CCCTC binding factor") (named for a nucleotide sequence found in all insulators) — prevents her Igf2 gene from interacting with the enhancers needed to turn it ON.
• The father's copy of the gene can be turned on because methylation of his insulator prevents binding by CTCF so the enhancers can interact with the gene.
However, two healthy laboratory mice have been produced by parthenogenesis; that is, containing two female (haploid) genomes. (See Kono, T. et al., Nature, 22 April 2004.)
This was done by fusing two oocytes (thus each cell haploid):
• a normal oocyte with its imprinted (inactivated) Igf2 gene
• an immature oocyte
• harvested before imprinting occurs and
• containing a deletion of the insulator that blocks enhancer activation of the Igf2 gene. Thus the Igf2 gene from this oocyte could be expressed in the developing embryo.
Out of several hundred attempts, two resulting blastocysts not only implanted successfully in a surrogate mother but went on to be born normally. One even grew up and had babies of her own.
Imprinting in Plants
Some genes in the endosperm of angiosperms are imprinted by the addition of methyl groups. For some, both maternal copies (endosperm is 3n) are expressed (demethylated) while the male allele remains shut down. For other genes, it is the female alleles that are imprinted and thus not expressed while the male allele is functional. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.12%3A_Imprinted_Genes.txt |
Until about 20 years ago, all known enzymes were proteins. But then it was discovered that some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules). Catalytic RNA molecules are called ribozymes. Most classes of RNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA) are transcribed as precursors that are larger than the final product. These precursors often contain "head" (5') and "tail" (3') sequences and intron sequences that must be removed to make the final product. Some of the processing steps employ other RNA molecules (always associated with proteins).
Ribonuclease P
Almost all living things synthesize an enzyme — called Ribonuclease P (RNase P) — that cleaves the head (5') end of the precursors of transfer RNA (tRNA) molecules. In bacteria, ribonuclease P is a heterodimer containing a molecule of RNA and one of protein. Separated from each other, the RNA retains its ability to catalyze the cleavage step (although less efficiently than the intact dimer), but the protein alone cannot do the job.
Figure 9.13.1: Crystal structure of a bacterial ribonuclease P holoenzyme in complex with tRNA (yellow), showing metal ions involved in catalysis (pink spheres), PDB: 3Q1R. (CC BY SA 30; RNAMacGyver).
Group I Introns
Some ribosomal RNA (rRNA) genes, including those in the mitochondrial genome of certain fungi (e.g., yeast), in some chloroplast genomes and in the nuclear genome of some "lower" eukaryotes (e.g., the ciliated protozoan Tetrahymena thermophila and plasmodial slime moldPhysarum polycephalum) contain introns that must be spliced out to make the final product.
The splicing reaction is self-contained; that is, the intron — with the help of associated proteins — splices itself out of the precursor RNA. Once excision of the intron and splicing of the adjacent exons are completed, the story is over. In other words, although the action is catalyzed by the RNA, only a single molecule of substrate is involved (unlike protein enzymes that repeatedly catalyze a reaction).
However, synthetic versions of Group I introns made in the laboratory can — in vitro — act repeatedly; that is, like true enzymes. The DNA of some Group I introns includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome. All the Group I introns share a characteristic secondary structure and mode of action that distinguishes them from the next group.
Group II Introns
Some messenger RNA (mRNA) genes in the mitochondrial genome of yeast and other fungi (encoding the proteins cytochrome b and subunits of cytochrome c oxidase) and in some chloroplast genomes also contain self-splicing introns. Because their secondary structure and the details of the splicing reaction differ from the rRNA introns discussed above, these are called Group II introns. The DNA of some Group II introns also includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome.
Spliceosomes
Spliceosomes remove introns and splice the exons of most nuclear genes. They are composed of 5 kinds of small nuclear RNA (snRNA) molecules and over 100 different protein molecules. It is the RNA — not the protein — that catalyzes the splicing reactions. The molecular details of the reactions are similar to those of Group II introns, and this has led to speculation that this splicing machinery evolved from them.
Viroids
Viroids are DNA molecules that infect plant cells as conventional viruses do, but are far smaller (one has only 246 nucleotides). They are naked; that is, they are not encased in a capsi like viruses. Some viroidlike molecules get into the cell as passengers inside a conventional plant virus. These are called virusoids or viroidlike satellite RNAs.
In both cases, the molecules consists of single-stranded RNA whose ends are covalently bonded to form a circle. There are several regions where base-pairing occurs across adjacent portions of the molecule. New viroids and virusoids are synthesized by the host cell as long precursors in which the viroid structure is tandemly repeated. These repeats must be cut out and ligated to form the final product. Most virusoids and at least one viroid are self-splicing; that is, they can cut themselves out of the precursor and ligate their ends without the aid of any host enzymes. Thus they represent another class of ribozyme.
Both viroids and virusoids are responsible for a number of serious diseases of economically important plants, e.g. the coconut palm and chrysanthemums. (The problem is so severe with chrysanthemums that all growers in the U.S. now secure their stock from a few companies that raise the plants in "clean" rooms using stringent precautions to prevent infection by the viroid.) | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/09%3A_Regulation_of_Gene_Expression/9.13%3A_Ribozymes.txt |
• 10.1: Mutations - Causes and Significance
In the living cell, DNA undergoes frequent chemical change, especially when it is being replicated (in S phase of the eukaryotic cell cycle). Most of these changes are quickly repaired. Those that are not result in a mutation. Thus, mutation is a failure of DNA repair.
• 10.2: Testing for Mutagenic Chemicals in Bacteria and Mice
Ames test is a test for determining if a chemical is a mutagen. It is named for its developer, Bruce Ames.
• 10.3: Radiation and its effect on DNA
For biologists, the most significant forms of radiation are light, heat, and ionizing radiation. Ionizing radiation can penetrate cells and create ions in the cell contents. These, in turn, can cause permanent alterations in DNA (i.e., mutations). Ionizing radiation includes X rays, gamma rays, neutrons, electrons ("beta" particles), and alpha particles (helium nuclei).
• 10.4: Transposons - "jumping genes"
Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may cause mutations and increase (or decrease) the amount of DNA in the genome of the cell, and if the cell is the precursor of a gamete, in the genomes of any descendants. These mobile segments of DNA are sometimes called "jumping genes" and there are two distinct types.
10: Mutation
In the living cell, DNA undergoes frequent chemical change, especially when it is being replicated (in S phase of the eukaryotic cell cycle). Most of these changes are quickly repaired. Those that are not result in a mutation. Thus, mutation is a failure of DNA repair.
Single-base substitutions
A single base, say an A, becomes replaced by another. Single base substitutions are also called point mutations. (If one purine [A or G] or pyrimidine [C or T] is replaced by the other, the substitution is called a transition. If a purine is replaced by a pyrimidine or vice-versa, the substitution is called a transversion.)
Missense mutations
With a missense mutation, the new nucleotide alters the codon so as to produce an altered amino acid in the protein product.
Deasese: Sickle Cell anemia
The replacement of A by T at the 17th nucleotide of the gene for the beta chain of hemoglobin changes the codon GAG (for glutamic acid) to GTG (which encodes valine). Thus the 6th amino acid in the chain becomes valine instead of glutamic acid.
Nonsense mutations
With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA). Therefore, translation of the messenger RNA transcribed from this mutant gene will stop prematurely. The earlier in the gene that this occurs, the more truncated the protein product and the more likely that it will be unable to function.
Cystic Fibrosis
Here is a sampling of mutations that have been found in patients with cystic fibrosis. Each of these mutations occurs in a huge gene that encodes a protein (of 1480 amino acids) called the cystic fibrosis transmembrane conductance regulator (CFTR). The protein is responsible for transporting chloride and bicarbonate ions through the plasma membrane. The gene encompasses over 188,000 base pairs on chromosome 7 embedded in which are 27 exons encoding the protein. The numbers in the mutation column represent the number of the nucleotides affected. Defects in the protein cause the various symptoms of the disease. Unlike sickle-cell disease, then, no single mutation is responsible for all cases of cystic fibrosis. People with cystic fibrosis inherit two mutant genes, but the mutations need not be the same.
In one patient with cystic fibrosis (Patient B), the substitution of a T for a C at nucleotide 1609 converted a glutamine codon (CAG) to a STOP codon (TAG). The protein produced by this patient had only the first 493 amino acids of the normal chain of 1480 and could not function.
Silent mutations
Most amino acids are encoded by several different codons. For example, if the third base in the TCT codon for serine is changed to any one of the other three bases, serine will still be encoded. Such mutations are said to be silent because they cause no change in their product and cannot be detected without sequencing the gene (or its mRNA).
Splice-site mutations
The removal of intron sequences, as pre-mRNA is being processed to form mRNA, must be done with great precision. Nucleotide signals at the splice sites guide the enzymatic machinery. If a mutation alters one of these signals, then the intron is not removed and remains as part of the final RNA molecule. The translation of its sequence alters the sequence of the protein product.
Insertions and Deletions (Indels)
Extra base pairs may be added (insertions) or removed (deletions) from the DNA of a gene. The number can range from one to thousands. Collectively, these mutations are called indels.
Indels involving one or two base pairs (or multiples of two) can have devastating consequences to the gene because translation of the gene is "frameshifted". This figure shows how by shifting the reading frame one nucleotide to the right, the same sequence of nucleotides encodes a different sequence of amino acids. The mRNA is translated in new groups of three nucleotides and the protein specified by these new codons will be worthless. Scroll up to see two other examples (Patients C and D).
Frameshifts often create new STOP codons and thus generate nonsense mutations. Perhaps that is just as well as the protein would probably be too garbled anyway to be useful to the cell.
Indels of three nucleotides or multiples of three may be less serious because they preserve the reading frame (see the above figure).
However, a number of inherited human disorders are caused by the insertion of many copies of the same triplet of nucleotides. Huntington's disease and the fragile X syndrome are examples of such trinucleotide repeat diseases.
Disease: Fragile X Syndrome
Several disorders in humans are caused by the inheritance of genes that have undergone insertions of a string of 3 or 4 nucleotides repeated over and over. A locus on the human X chromosome contains such a stretch of nucleotides in which the triplet CGG is repeated (CGGCGGCGGCGG, etc.). The number of CGGs may be as few as 5 or as many as 50 without causing a harmful phenotype (these repeated nucleotides are in a noncoding region of the gene). Even 100 repeats usually cause no harm. However, these longer repeats have a tendency to grow longer still from one generation to the next (to as many as 4000 repeats).
This causes a constriction in the X chromosome, which makes it quite fragile. Males who inherit such a chromosome (only from their mothers, of course) show a number of harmful phenotypic effects including mental retardation. Females who inherit a fragile X (also from their mothers; males with the syndrome seldom become fathers) are only mildly affected.
The above image shows the pattern of inheritance of the fragile X syndrome in one family. The number of times that the trinucleotide CGG is repeated is given under the symbols. The gene is on the X chromosome, so women (circles) have two copies of it; men (squares) have only one. People with a gene containing 80–90 repeats are normal (light red), but this gene is unstable, and the number of repeats can increase into the hundreds in their offspring. Males who inherit such an enlarged gene suffer from the syndrome (solid red squares). (Data from C. T. Caskey, et al.).
Polyglutamine Diseases
In these disorders, the repeated trinucleotide is CAG, which adds a string of glutamines (Gln) to the encoded protein. These have been implicated in a number of central nervous system disorders including
• Huntington's disease (where the protein called huntingtin carries the extra glutamines). The abnormal protein increases the level of the p53 protein in brain cells causing their death by apoptosis.
• some cases of Parkinson's disease where the extra glutamines are in the protein ataxin-2.
Muscular Dystrophy
Some forms of muscular dystrophy that appear in adults are caused by tri- or tetranucleotide, e.g. (CTG)n and (CCTG)n, repeats where n may run into the thousands. The huge RNA transcripts that result interfere with the alternative splicing of other transcripts in the nucleus.
Amyotrophic Lateral Sclerosis (ALS)
ALS is a neurodegenerative disorder leading to dementia and muscle weakness. (ALS is often called "Lou Gehrig's disease" after the baseball player who died from it.)
The most common mutation in ALS is an expansion of the number of repeats of the hexanucleotide GGGGCC in a gene on chromosome 9 from the normal two, or at least fewer than three dozen, to hundreds or even several thousand. Translation of both the sense and the antisense strands containing these repeats (and in all 3 reading frames; there is no ATG start codon) produces polymers with long strings of gly-ala, gly-pro, gly-arg (from the sense strand) as well as pro-ala, another pro-gly, and pro-arg from the antisense strand. These proteins, especially those containing arginine (arg) form aggregates that damage brain cells.
Duplications
Duplications are a doubling of a section of the genome. During meiosis, crossing over between sister chromatids that are out of alignment can produce one chromatid with a duplicated gene and the other (not shown) with the two genes with deletions. In the case shown here, unequal crossing over created a second copy of a gene needed for the synthesis of the steroid hormone aldosterone.
However, this new gene carries inappropriate promoters at its 5' end (acquired from the 11-beta hydroxylase gene) that cause it to be expressed more strongly than the normal gene. The mutant gene is dominant: all members of one family (through four generations) who inherited at least one chromosome carrying this duplication suffered from high blood pressure and were prone to early death from stroke.
Gene duplication has also been implicated in several human neurological disorders.
Gene duplication has occurred repeatedly during the evolution of eukaryotes. Genome analysis reveals many genes with similar sequences in a single organism. Presumably these paralogous genes have arisen by repeated duplication of an ancestral gene.
Such gene duplication can be beneficial.
• Over time, the duplicates can acquire different functions.
• The proteins they encode can take on different functions; for example, if the original gene product carried out two different functions (see "pleiotropy"), each duplicated gene can now specialize at one function and do a better job at it than the parental gene.
• But even if they do not, changes in the regulatory sequences of the genes (promoters and enhancers) may cause the same protein to be expressed at different times, at different levels, and/or in different tissues.
Either situation can provide the basis for adaptive evolution.
• But even while two paralogous genes are still similar in sequence and function, their existence provides redundancy ("belt and suspenders"). This may be a major reason why knocking out genes in yeast, "knockout mice", etc. so often has such a mild effect on the phenotype. The function of the knocked out gene can be taken over by a paralog.
• After gene duplication, random loss — or inactivation — of one of these genes at a later time in
• one group of descendants
• different from the loss in another group
could provide a barrier (a "post-zygotic isolating mechanism") to the two groups interbreeding. Such a barrier could cause speciation: the evolution of two different species from a single ancestral species.
Translocations
Translocations are the transfer of a piece of one chromosome to a nonhomologous chromosome. Translocations are often reciprocal; that is, the two nonhomologues swap segments.
Translocations can alter the phenotype is several ways:
• the break may occur within a gene destroying its function
• translocated genes may come under the influence of different promoters and enhancers so that their expression is altered. The t(8;14) translocation in Burkitt's lymphoma (figure) is an example.
• the breakpoint may occur within a gene creating a hybrid gene. This may be transcribed and translated into a protein with an N-terminal of one normal cell protein coupled to the C-terminal of another. The Philadelphia chromosome found so often in the leukemic cells of patients with chronic myelogenous leukemia (CML) is the result of a translocation which produces a compound gene (bcr-abl).
Frequency of Mutations
Mutations are rare events. This is surprising. Humans inherit 3 x 109 base pairs of DNA from each parent. Just considering single-base substitutions, this means that each cell has 6 billion (6 x 109) different base pairs that can be the target of a substitution. Single-base substitutions are most apt to occur when DNA is being copied; for eukaryotes that means during S phase of the cell cycle.
No process is 100% accurate. Even the most highly skilled typist will introduce errors when copying a manuscript. So it is with DNA replication. Like a conscientious typist, the cell does proofread the accuracy of its copy. But, even so, errors slip through. It has been estimated that in humans and other mammals, uncorrected errors (= mutations) occur at the rate of about 1 in every 50 million (5 x 107) nucleotides added to the chain. (Not bad — I wish that I could type so accurately.) But with 6 x 109 base pairs in a human cell, that means that each new cell contains some 120 new mutations.
Should we be worried? The evidence is not clear. Only 1.2% of our DNA encodes the exons of our proteome, and for a long time it was thought that much of the rest was "junk" DNA. Mutations in it would most likely be harmless. And even in coding regions, the existence of synonymous codons could result in the altered (mutated) gene still encoding the same amino acid in the protein. But it now appears that as much as 80% of our DNA seems to participate in regulating which genes are expressed, and how strongly, in each of the multitude of differentiated cell types in our body as each responds to the signals (nutrients, hormones, etc.) it receives. So mutations in these regions might well have harmful, if subtle, effects.
As more vertebrate genomes are sequenced, it turns out that some of these stretches of DNA that do not encode proteins none-the-less have been remarkably conserved during vertebrate evolution. Some of these regions have accumulated even fewer mutations than protein-encoding genes have. This suggests that these sequences are extremely important to the welfare of the organism. However, other regions of the genome seem able to sustain point mutations with no detectible harm.
Recent advances have enabled the coding portions of the genome of single cells to be sequenced. Preliminary results indicate that each normal cell in an adult has accumulated ~20 somatic mutations, and that its collection of mutations differs from cell to cell. Cancer cells accumulate many more mutations (often in the hundreds).
How can we measure the frequency at which phenotype-altering mutations occur? In humans, it is not easy.
• First we must be sure that the mutation is newly-arisen. (Some populations have high frequencies of a particular mutation, not because the gene is especially susceptible, but because it has been passed down through the generations from a early "founder".
• Recessive mutations (most of them are) will not be seen except on the rare occasions that both parents contribute a mutation at the same locus to their child.
• This leaves us with estimating mutation frequencies for genes that are inherited as
• autosomal dominants
• X-linked recessives; that is, recessives on the X chromosome which will be expressed in males because they inherit only one X chromosome.
Examples
Frequency is expressed as the frequency of mutations occurring at that locus in the gametes
• Autosomal dominants
• Retinoblastoma
in the RB gene: about 8 per million (8 x 10-6)
• Osteogenesis imperfecta
in one or the other of the two genes that encode Type I collagen: about 1 per 100,000 (10-5)
• Inherited tendency to polyps (and later cancer) in the colon.
in a tumor suppressor gene (APC): ~10-5
• X-linked recessives
• Hemophilia A
~3 x 10-5 (the Factor VIII gene)
• Duchenne Muscular Dystrophy (DMD)
>8 x 10-5 (the dystrophin gene)
Why should the mutation frequency in the dystrophin gene be so much larger than most of the others? It's probably a matter of size. The dystrophin gene stretches over 2.4 x 106 base pairs of DNA. This is almost 0.1% of the entire human genome! Such a huge gene offers many possibilities for damage.
Measuring Mutation Rate
The frequency with which a given mutation is seen in a population (e.g., the mutation that causes cystic fibrosis) provides only a rough approximation of mutation rate — the rate at which fresh mutations occur — because of historical factors at work such as natural selection (positive or negative), drift, and founder effect. In addition, most methods for counting mutations require that the mutation have a visible effect on the phenotype. Thus
• many (but not all) mutations in noncoding DNA
• mutations that produce
• synonymous codons (encode the same amino acid)
• or, sometimes, new codons that encode a chemically-similar amino acid
• mutations which disrupt a gene whose functions are redundant; that is, can be compensated for by other genes
will not be seen. But now these problems have been largely solved. The story is told in a report by D. R. Denver, et al. in the 5 August 2004 issue of Nature.
C. elegans
The Procedure
• Their organism = C. elegans
• Its advantages
• compact genome
• hermaphroditic — it fertilizes its own eggs and any new germline mutation will soon be either lost or appear on both homologous chromosomes.
• rapid generation time (4 days)
• They created 198 different experimental lines of worms.
• They grew them under optimum conditions to minimize any effects of natural selection.
• Only one offspring was kept at each new generation.
• Each line was maintained for several hundred generations.
• At the end of this time, random stretches of DNA
• derived from multiple locations on each of the six C. elegans chromosomes and
• totalling an average of ~21 thousand base pairs for each line
were sequenced from each of the 198 lines and the sequences compared with the same loci in natural populations of C. elegans.
Results
Examining the DNA sequences from their experimental animals (a total of over 4 million base pairs!), and comparing them with the controls, turned up a total of 30 mutations.
• 17 of these were insertions or deletions ("indels')
• 7 in exons — all but 2 of which produced frameshifts and a premature STOP codon.
• 10 in introns or between genes
• 13 of these were single base substitutions ("point" mutations)
• 3 in exons : one "silent" producing a synonymous codon; two that changed the encoded amino acid.
• 10 in introns or between genes
Calculating Mutation Rate
From these results I have pooled their data to calculate an approximate rate at which spontaneous mutations occur throughout the genome.
Mutation Rate = # of mutations observed [30] ÷ (# of experimental lines [198]) x (average # of generations [339]) x (average # of base pairs sequenced [~21,000])
yielding a rate of 2.1 x 10-8 mutations per base pair per generation.
The total C. elegans genome contains some 108 base pairs so this tells us that two new germline mutations occur somewhere in each of C. elegans's two haploid genomes in each generation.
A similar analysis for Drosophila (whose genome is about the same size as that of C. elegans) showed a similar mutation rate: ~10-8 mutations per base pair per generation. As for the green plant Arabidopsis thaliana, its spontaneous mutation rate is slightly lower: ~7 x 10-9 mutations per base pair per generation.
In the 30 April 2010 issue of Science, Roach, J. C., et al., reported that the rate for humans is in the same range: ~1.1 x 10-8 mutations per base pair in the haploid genome. With a diploid genome of 6 x 109 base pairs, that works out to some 70 new mutations in each child. They derived these numbers from comparing the complete genome sequence of two children and their parents.
In the 20 July 2012 issue of Cell, Wang, J., et al. reported the results of sequencing 8 individual sperm cells from a 40-year-old man. They found a mutation rate ranging from 2.0 x 10-8 to 3.8 x 10-8.
Should we be worried about such spontaneous mutation rates? Probably not too much. With our high proportion of noncoding DNA, many mutations will occur in regions that will have no effect on our phenotype. Evidence: out of a total of 251 mutations found in the 8 sperm cells, only 3 were missense mutations altering a gene product. However, even in noncoding DNA, point mutations may affect the expression of genes, so perhaps as many as 10% of the point mutations a child inherits may have harmful, if subtle, effects.
Males Contribute More Mutations Than Females
If most mutations occur during S phase of cell division, then males should be more at risk. This is because only two dozen (24) or so mitotic divisions occur from the fertilized egg that starts a little girl's embryonic development and the setting aside of her future eggs (which is done long before she is even born). Furthermore, the sperm of a 30-year old man, in contrast, are the descendants of at least 400 mitotic divisions since the fertilized egg that formed him.
So, fathers are more likely than mothers to transmit newly-formed mutations to their children. The sperm of a 25-year-old man might carry some 45 new mutations. This number rises at a rate of about 1 per year, so the sperm of a 40-year-old man may transmit some 60 new mutations to his children (about 20 of these in coding regions). No matter what the age of the mother, she transmits only about 15 new mutations to her offspring. (But chromosomal aberrations, like aneuploidy, are more apt to arise in eggs than in sperm, and the incidence of these increases with maternal age.) These data explain why the children of aged fathers suffer more genetic disorders than those of young fathers.
Somatic vs. Germline Mutations
The significance of mutations is profoundly influenced by the distinction between germline and soma. Mutations that occur in a somatic cell, in the bone marrow or liver for example, may
• damage the cell
• make the cell cancerous
• kill the cell
Whatever the effect, the ultimate fate of that somatic mutation is to disappear when the cell in which it occurred, or its owner, dies.
Germline mutations, in contrast, will be found in every cell descended from the zygote to which that mutant gamete contributed. If an adult is successfully produced, every one of its cells will contain the mutation. Included among these will be the next generation of gametes, so if the owner is able to become a parent, that mutation will pass down to yet another generation. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/10%3A_Mutation/10.01%3A_Mutations_-_Causes_and_Significance.txt |
Ames test
Big Blue mice are transgenic for a segment of DNA that contains the DNA of bacteriophage lambda, a virus that infects E. coli, and which serves here as the vector for 3 genetic elements from the lac operon of E. coli:
• the lacI gene
• the operator of the operon
• the beta-galactosidase (lacZ) gene
The Assay
The transgenic mice are given repeated doses of the suspected carcinogen for a week or two. If the chemical is mutagenic, it will cause random mutations throughout the genome of each mouse cell. If a mutation occurs in either the lacI gene (which encodes the lac repressor) or the operator,
the gene (lacZ) for beta-galactosidase will no longer be repressed. To detect this,
• The DNA is extracted from the tissues of the treated mouse.
• The vector is isolated and used to make functional bacteriophages.
• E. coli cells are mixed with the bacteriophage and spread on a solid culture medium.
• The bacteriophages infect and destroy ("lyze") the E. coli cells.
• This causes clear circular zones, called plaques, to appear in a "lawn" of bacteria.
• Before they die, cells that have been infected by bacteriophages carrying a mutated lacI or operator will produce beta-galactosidase.
• This reacts with a substrate in the culture medium turning it blue.
• Bacteriophages with unmutated genes produce colorless plaques because no beta-galactosidase is synthesized.
• Count both colorless and blue plaques.
• The number of blue plaques divided by the total number of plaques gives the mutation frequency.
This photograph shows one mutant (blue) plaque on a lawn of E. coli containing many non-mutant (clear) plaques.
10.03: Radiation and its effect on DNA
For biologists, the most significant forms of radiation are light, heat, and ionizing radiation. Ionizing radiation can penetrate cells and create ions in the cell contents. These, in turn, can cause permanent alterations in DNA (i.e., mutations). Ionizing radiation includes X rays, gamma rays, neutrons, electrons ("beta" particles), and alpha particles (helium nuclei).
Units of measurement
• rad: The rad represents a certain dose of energy absorbed by 1 gram of tissue. It is a unit of concentration. So if we could uniformly expose the entire body to radiation, the number of rads received would be the same whether we were speaking of a single cell, an organ (e.g., an ovary) or the entire body (just as the concentration of salt in sea water is the same whether we consider a cupful or an entire ocean).
• rem: Some forms of radiation are more efficient than others transferring their energy to the cell. To have a level playing field, it is convenient to multiply the dose in rads by a quality factor (Q) for each type of radiation. The resulting unit is the rem ("roentgen-equivalent man"). Thus, rem = rad x Q. X rays and gamma rays have a Q about 1, so the absorbed dose in rads is the same number in rems. Neutrons have a Q of about 5 and alpha particles have a Q of about 20. An absorbed dose of, say, 1 rad of these is equivalent to 5 rem and 20 rem respectively.
• The sievert (Sv) and gray (Gy): Despite the years of high-quality research reported in rems and millirems (mrem, 10-3 rem), the International Commission on Radiation Units and Measurements wants us to give up the rad in favor of the gray (Gy), a unit 100 times larger. Similarly, the rem is to be replaced by the sievert (Sv), again so that 100 rem = 1 Sv. So I will try to express all radiation doses in a single unit, the millisievert (mSv).
Table 10.2.1: An assortment of typical radiation doses (in mSv)
Used to destroy the bone marrow in preparation for a marrow transplant (given over several days) 10,000
Approximate lethal dose ("LD50") if no treatment and given to the entire body in a short period 4,500
Causes radiation sickness (when absorbed in a short period) >1,000
When delivered in a single dose, increases the risk of developing cancer by 1% 100
Increase in lifetime dose to most heavily exposed people living near Chernobyl 430
Annual dose (excluding natural background) permitted for U.S. radiation workers 50
Average annual dose (excluding natural background) for medical X-ray technicians 3.2
Maximum permissible annual dose (excluding natural background and medical exposure) to general public 1.7
Average annual dose of natural background radiation, worldwide 2.4
Natural background, Boston, MA, USA (per year)(excluding radon) 1.02
Natural background, Denver, CO, USA (per year)(excluding radon) 1.8
Additional annual dose if you live in a brick rather than a wood house 0.07
Annual dose in some houses in Ramsar, Iran >130
Average dose to person living within 10 miles of Three-Mile Island (TMI) caused by the accident of 28 March 1979 0.01
Most heavily exposed person (a fisherman) near TMI <1.0
Approximate dose received by a person spending 1 year at the fence surrounding a nuclear power station 0.001–0.006
Average dose to each person in the U. S. population from nuclear power plants (per year) 0.00002
Received by the brain during a set of dental x rays 0.005
Received by the colon during a barium enema 15
Received by the lungs during a chest x ray 0.01—0.15
Screening mammogram 0.5
Total dose received by the people living near the Fukushima Daiichi Nuclear Power Station in Japan during the first year after the reactors were damaged by a devastating tsunami. 12–25
Dose from a typical set of full-body computed tomography (CT) scans 20
Cardiac stress test using radioactive thallium 36
Typical dose received by the abdomen during a CT scan to diagnose appendicitis 10
Typical PET scan 14.0
Airline passenger crossing the U.S. 0.04
Flight crew flying regularly between New York and Tokyo (per year) 9
Hourly dose to skin holding piece of the original "Fiesta Ware" (a brand of pottery) 2–3
Annual dose to each person in the U. S. population from fallout (former weapons testing plus Chernobyl) 0.0006
Estimated average annual radiation exposure from various sources (in millisieverts) of an inhabitant of the United States (total = 5.86 mSv). Individual exposures, especially to radon and medical sources, vary widely from these average values. The use of medical imaging in the United States (some 67 million CT scans were performed here in 2006) has increased greatly in recent years. As for radon, only the lungs are exposed as the alpha particles emitted by radon cannot penetrate other tissues. (Data from the National Council on Radiation Protection and Measurements, Bethesda, MD.)
Background Radiation
About 27% of our annual exposure to radiation is from background radiation (Figure 10.3.1) and originate from three primary sources:
1. Cosmic radiation (0.27 mSv). The value increases with altitude, so the dose for people in Denver, Colorado is about 0.50 mSv.
2. Rocks and soil (0.28 mSv). This value varies with the geology of a region: people in Louisiana get as little as 0.15 mSv/yr; people on the Colorado plateau (incl. Denver!) get 1.4 mSv/yr.
3. From within the body (0.4 mSv). Most of this comes from potassium-40. About 0.02% of the potassium in nature is in the form of the radioactive isotope 40K. Living tissue cannot discriminate between radioactive and nonradioactive versions, so the same 0.02% of the total potassium in the body (about 1.7 g in a 70-kg person) is radioactive. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/10%3A_Mutation/10.02%3A_Testing_for_Mutagenic_Chemicals_in_Bacteria_and_Mice.txt |
Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may cause mutations and increase (or decrease) the amount of DNA in the genome of the cell, and if the cell is the precursor of a gamete, in the genomes of any descendants. These mobile segments of DNA are sometimes called "jumping genes" and there are two distinct types. Class II transposons consist of DNA that moves directly from place to place. Class I transposons are retrotransposons that first transcribe the DNA into RNA and then use reverse transcriptase to make a DNA copy of the RNA to insert in a new location.
Class II Transposons
Class II transposons move by a "cut and paste" process: the transposon is cut out of its location (like command/control-X on your computer) and inserted into a new location (command/control-V). This process requires an enzyme — a transposase — that is encoded within some of these transposons.
Fig.10.4.1 Transposons
Transposase binds to both ends of the transposon, which consist of inverted repeats; that is, identical sequences reading in opposite directions. They also bind to a sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site; others can insert the transposon anywhere in the genome.
The DNA at the target site is cut in an offset manner (like the "sticky ends" produced by some restriction enzymes). After the transposon is ligated to the host DNA, the gaps are filled in by Watson-Crick base pairing. This creates identical direct repeats at each end of the transposon. Often transposons lose their gene for transposase. However, as long as somewhere in the cell there is a transposon that can synthesize the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location.
Miniature Inverted-repeat Transposable Elements (MITEs)
The recent completion of the genome sequence of rice and C. elegans has revealed that their genomes contain thousands of copies of a recurring motif consisting of almost identical sequences of about 400 base pairs flanked by characteristic inverted repeats of about 15 base pairs such as
5' GGCCAGTCACAATGG..~400 nt..CCATTGTGACTGGCC 3'
3' CCGGTCAGTGTTACC..~400 nt..GGTAACACTGACCGG 5'
MITEs are too small to encode any protein. Just how they are copied and moved to new locations is still uncertain. Probably larger transposons that do encode the necessary enzyme and recognize the same inverted repeats are responsible. There are over 100,000 MITEs in the rice genome (representing some 6% of the total genome). Some of the mutations found in certain strains of rice are caused by the insertion of a MITE in the gene. MITEs have also been found in the genomes of humans, Xenopus, and apples.
Transposons in Maize
The first transposons were discovered in the 1940s by Barbara McClintock who worked with maize (Zea mays, called "corn" in the U.S.). She found that they were responsible for a variety of types of gene mutations, usually insertions and deletions (indels) and translocations. Some of the mutations (c, bz) used as examples of how gene loci are mapped on the chromosome were caused by transposons. In developing somatic tissues like corn kernels, a mutation (e.g., c) that alters color will be passed on to all the descendant cells. This produces the variegated pattern which is so prized in "Indian corn". (Photo courtesy of Whalls Farms.) It took about 40 years for other scientists to fully appreciate the significance of Barbara McClintock's discoveries. She was finally awarded a Nobel Prize in 1983.
Transposons in Drosophila
P elements are Class II transposons found in Drosophila. They do little harm because expression of their transposase gene is usually repressed. However, when male flies with P elements mate with female flies lacking them, the transposase becomes active in the germline producing so many mutations that their offspring are sterile. In nature this is no longer a problem. P elements seem to have first appeared in Drosophila melanogaster about 50 years ago. Since then, they have spread through every population of the species. Today flies lacking P elements can only be found in old strains maintained in the laboratory. P elements have provided valuable tools for Drosophila geneticists. Transgenic flies containing any desired gene can be produced by injecting the early embryo with an engineered P element containing that gene. Other transposons are being studied for their ability to create transgenic insects of agricultural and public health importance.
Transposons in bacteria
Some transposons in bacteria carry — in addition to the gene for transposase — genes for one or more (usually more) proteins imparting resistance to antibiotics. When such a transposon is incorporated in a plasmid, it can leave the host cell and move to another. This is the way that the alarming phenomenon of multidrug antibiotic resistance spreads so rapidly. Transposition in these cases occurs by a "copy and paste" mechanism. This requires an additional enzyme — a resolvase — that is also encoded in the transposon itself. The original transposon remains at the original site while its copy is inserted at a new site.
Retrotransposons
Retrotransposons also move by a "copy and paste" mechanism but in contrast to the transposons described above, the copy is made of RNA, not DNA. The RNA copies are then transcribed back into DNA — using a reverse transcriptase — and these are inserted into new locations in the genome. Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each. Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening stretch of DNA arrived there by retrotransposition. Some 50% of the entire human genome consists of retrotransposons.
LINEs (Long interspersed elements)
The human genome contains over one million LINEs (representing 19% of the genome). The most abundant of these belong to a family called LINE-1 (L1). These L1 elements are DNA sequences that range in length from a few hundred to as many as 9,000 base pairs. Only about 50 L1 elements are functional "genes"; that is, can be transcribed and translated. The functional L1 elements are about 6,500 bp in length and encode three proteins, including an endonuclease that cuts DNA and a reverse transcriptase that makes a DNA copy of an RNA transcript.
L1 activity
L1 activity proceeds as follows:
1. RNA polymerase II transcribes the L1 DNA into RNA.
2. The RNA is translated by ribosomes in the cytoplasm into the proteins.
3. The proteins and RNA join together and reenter the nucleus.
4. The endonuclease cuts a strand of "target" DNA, often in the intron of a gene.
5. The reverse transcriptase copies the L1 RNA into L1 DNA which is inserted into the target DNA forming a new L1 element there.
Through this copy-paste mechanism, the number of LINEs can increase in the genome. The diversity of LINEs between individual human genomes make them useful markers for DNA "fingerprinting". Variation occurs in the length of L1 elements: Transcription of an active L1 element sometimes continues downstream into additional DNA producing a longer transposed element. Reverse transcription of L1 RNA often concludes prematurely and produces a shortened transposed element.
While L1 elements are not functional, they may play a role in regulating the efficiency of transcription of the gene in which they reside. Occasionally, L1 activity makes and inserts a copy of a cellular mRNA (thus a natural cDNA). Lacking introns as well as the necessary control elements like promoters, these genes are not expressed. They represent one category of pseudogene.
SINEs (Short interspersed elements)
SINEs are short DNA sequences (100–400 base pairs) that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III; that is, molecules of tRNA, 5S rRNA, and some other small nuclear RNAs. The most abundant SINEs are the Alu elements. There are over one million copies in the human genome (representing 9% of our total DNA). Alu elements consist of a sequence averaging 260 base pairs that contains a site that is recognized by the restriction enzyme AluI. They appear to be reverse transcripts of 7S RNA, part of the signal recognition particle. Most SINEs do not encode any functional molecules and depend on the machinery of active L1 elements to be transposed; that is, copied and pasted in new locations.
HIV-1
HIV-1 — the cause of AIDS — and other human retroviruses (e.g., HTLV-1, the human T-cell leukemia/lymphoma virus) behave like retrotransposons. The RNA genome of HIV-1 contains a gene for reverse transcriptase and one for integrase. The integrase serves the same function as the transposases of DNA transposons. The DNA copies can be inserted anywhere in the genome. Molecules of both enzymes are incorporated in the virus particle.
Transposons and Mutations
Transposons are mutagens and can cause mutations in several ways. If a transposon inserts itself into a functional gene, it will probably damage it. Insertion into exons, introns, and even into DNA flanking the genes (which may contain promoters and enhancers) can destroy or alter the gene's activity. Faulty repair of the gap left at the old site (in cut and paste transposition) can lead to mutation there. The presence of a string of identical repeated sequences presents a problem for precise pairing during meiosis. How is the third, say, of a string of five Alu sequences on the "invading strand" of one chromatid going to ensure that it pairs with the third sequence in the other strand? If it accidentally pairs with one of the other Alu sequences, the result will be an unequal crossover — one of the commonest causes of duplications.
Note
The insertion of a retrotransposon in the DNA flanking a gene for pigment synthesis is thought to have produced white grapes from a black-skinned ancestor. Later, the loss of that retrotransposon produced the red-skinned grape varieties cultivated today.
SINEs (mostly Alu sequences) and LINEs cause only a small percentage of human mutations. (There may even be a mechanism by which they avoid inserting themselves into functional genes.) However, they have been found to be the cause of the mutations responsible for some cases of human genetic diseases, including:
• Hemophilia A (Factor VIII gene) and Hemophilia B [Factor IX gene]
• X-linked severe combined immunodeficiency (SCID) [gene for part of the IL-2 receptor]
• porphyria
• predisposition to colon polyps and cancer [APC gene]
• Duchenne muscular dystrophy [dystrophin gene]
What good are transposons?
Transposons have been called "junk" DNA and "selfish" DNA. They are "selfish" because their only function seems to make more copies of themselves and "junk" because there is no obvious benefit to their host. Because of the sequence similarities of all the LINEs and SINEs, they also make up a large portion of the "repetitive DNA" of the cell. Retrotransposons cannot be so selfish that they reduce the survival of their host. And it now appears that many, at least, confer some benefit. The ENCODE project found that some 75% of our repetitive DNA occurs within, or overlaps with, sequences, like enhancers, that regulate gene expression.
Some other possibilities:
• Retrotransposons often carry some additional sequences at their 3' end as they insert into a new location. Perhaps these occasionally create new combinations of exons, promoters, and enhancers that benefit the host.
Example:
• Thousands of our Alu elements occur in the introns of genes.
• Some of these contain sequences that when transcribed into the primary transcript are recognized by the spliceosome.
• These can then be spliced into the mature mRNA creating a new exon, which will be transcribed into a new protein product.
• Alternative splicing can provide not only the new mRNA (and thus protein) but also the old.
• In this way, nature can try out new proteins without the risk of abandoning the tried-and-true old one.
• L1 elements inserted into the introns of functional genes reduce the transcription of those genes without harming the gene product — the longer the L1 element, the lower the level of gene expression. Some 79% of our genes contain L1 elements, and perhaps they are a mechanism for establishing the baseline level of gene activity.
• Telomerase, the enzyme essential for maintaining chromosome length, is closely related to the reverse transcriptase of LINEs and may have evolved from it.
• RAG-1 and RAG-2. The proteins encoded by these genes are needed to assemble the repertoire of antibodies and T-cells receptors (TCRs) used by the adaptive immune system. The mechanism resembles that of the cut and paste method of Class II transposons , and the RAG genes may have evolved from them. If so, the event occurred some 450 million years ago when the jawed vertebrates evolved from jawless ancestors. Only jawed vertebrates have the RAG-1 and RAG-2 genes.
• In Drosophila, the insertion of transposons into genes has been linked to the development of resistance to DDT and organophosphate insecticides.
Transposons and the C-value Paradox
The genome of Arabidopsis thaliana contains ~1.2 x 108 base pairs (bp) of DNA. About 14% of this consists of transposons; the rest functional genes (25,498 of them). The maize (corn) genome contains 20 times more DNA (2.4 x 109 bp) but surely has no need for 20 times as many genes. In fact, 60% of the corn genome is made up of transposons (the figure for humans is 42%). So it seems likely that the lack of an association between size of genome and number of functional genes — the C-value paradox — is caused by the amount of transposon DNA accumulated in the genome. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/10%3A_Mutation/10.04%3A_Transposons_-_jumping_genes.txt |
• 11.1: Recombinant DNA and Gene Cloning
Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.
• 11.2: Polymerase Chain Reaction
The polymerase chain reaction is a technique for quickly "cloning" a particular piece of DNA in the test tube (rather than in living cells like E. coli). Thanks to this procedure, one can make virtually unlimited copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules.
• 11.3: Gene Therapy - Methods and Prospects
Many human diseases are caused by defective genes that are caused by a defect at a single gene locus. (The inheritance is recessive so both the maternal and paternal copies of the gene must be defective.) Is there any hope of introducing functioning genes into these patients to correct their disorder? Probably. Other diseases also have a genetic basis, but it appears that several genes must act in concert to produce the disease phenotype.
• 11.4: Recent Advances in Gene Therapy
Reaching the goal of effective gene therapies for human diseases has been a difficult one.
• 11.5: Transgenic Animals
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to the gene itself, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host.
• 11.6: Transgenic Plants
Progress is being made on several fronts to introduce new traits into plants using recombinant DNA technology. The genetic manipulation of plants has been going on since the dawn of agriculture, but until recently this has required the slow and tedious process of cross-breeding varieties. Genetic engineering promises to speed the process and broaden the scope of what can be done.
• 11.7: Restriction Fragment Length Polymorphisms
Restriction Fragment Length Polymorphisms (RFLPs) have provided valuable information in many areas of biology, including screening human DNA for the presence of potentially deleterious genes ("Case 1") and providing evidence to establish the innocence of, or a probability of the guilt of, a crime suspect by DNA "fingerprinting" ("Case 3")
• 11.8: Gel Blotting
Gel blotting is a technique for visualizing a particular subset of macromolecules — proteins, or fragments of DNA or RNA — initially present in a complex mixture.
• 11.9: Genetic Screening for Phenylketonuria
Phenylketonuria is one of the commonest inherited disorders — occurring in approximately 1 in 10,000 babies born in the U. S. It occurs in babies who inherit two mutant genes for the enzyme phenylalanine hydroxylase (PAH — "1" in the figure on the left). This enzyme normally starts the process of breaking down molecules of the amino acid phenylalanine that are in excess of the body's needs for protein synthesis.
• 11.10: Antisense RNA
Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
• 11.11: Antisense Oligodeoxynucleotides and their Therapeutic Potential
Antisense oligonucleotides are synthetic polymers. The monomers are chemically-modified deoxynucleotides like those in DNA or ribonucleotides like those in RNA. There are usually only 15–20 of them, hence "oligo". Their sequence (3′ → 5′) is antisense; that is, complementary to the sense sequence of a molecule of mRNA.
• 11.12: Forward and Reverse genetics
Since Mendel's time, most genetics has involved observing an interesting phenotype tracking down the gene responsible for it. So this "forward" genetics proceeds from phenotype -> genotype. But now with a knowledge of the DNA sequence of a gene of unknown function, one can use methods for suppressing that particular gene ("knockdown"), and then observe the effect on the phenotype. So this "reverse" genetics proceeds from genotype -> phenotype.
• 11.13: Metagenomics
All the genomes listed on my page Genome Sizes describe the complete genome of a single species. For bacteria and archaeons, this means that the organism was grown in pure culture to provide the DNA for sequencing. But it is now clear that the microbial world contains vast numbers of both groups that have never been grown in the laboratory and thus have escaped study. Soil, water, and the contents of our large intestine are examples of habitats that teem with unknown microorganisms.
Thumbnail: A DNA microarray. (CC BY-SA 3.0; Guillaume Paumier).
11: Genomics
Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.
Making Recombinant DNA (rDNA) - An Overview
• Treat DNA from both sources with the same restriction endonuclease (BamHI in this case).
• BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'
• The ends of the cut have an overhanging piece of single-stranded DNA.
• These are called "sticky ends" because they are able to base pair with any DNA molecule containing the complementary sticky end.
• In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed.
• A DNA ligase covalently links the two into a molecule of recombinant DNA.
To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo.
Cloning in vivo can be done in
• unicellular microbes like E. coli
• unicellular eukaryotes like yeast and
• in mammalian cells grown in tissue culture.
In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But here let us examine an example of cloning using E. coli as the host and a plasmid as the vector.
Plasmids
Plasmids are small (a few thousand base pairs), usually carry only one or a few genes, are circular and have a single origin of replication. Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at high levels. In nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one or more antibiotics. Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes.
In the above figure, the tangle is a portion of a single DNA molecule containing over 4.6 million base pairs encoding approximately 4,300 genes. The small circlets are plasmids.
Examples of Plasmids
pAMP
• 4539 base pairs
• a single replication origin
• a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of penicillin)
• a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5'that, as we saw above, is cut by the restriction enzyme BamHI
• a single occurrence of the sequence
5' AAGCTT 3'
3' TTCGAA 5'that is cut by the restriction enzyme HindIII
Treatment of pAMP with a mixture of BamHI and HindIII produces:
• a fragment of 3755 base pairs carrying both the ampr gene and the replication origin
• a fragment of 784 base pairs
• both fragments have sticky ends
pKAN
• 4207 base pairs
• a single replication origin
• a gene (kanr) conferring resistance to the antibiotic kanamycin.
• a single site cut by BamHI
• a single site cut by HindIII
Treatment of pKAN with a mixture of BamHI and HindIII produces:
• a fragment of 2332 base pairs
• a fragment of 1875 base pairs with the kanr gene (but no origin of replication)
• both fragments have sticky ends
These fragments can be visualized by subjecting the digestion mixtures to electrophoresis in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates toward the positive electrode (anode) when a direct current is applied. The smaller the fragment, the farther it migrates in the gel.
Ligation Possibilities
If you remove the two restriction enzymes and provide the conditions for DNA ligase to do its work, the pieces of these plasmids can rejoin (thanks to the complementarity of their sticky ends).
Mixing the pKAN and pAMP fragments provides several (at least 10) possibilities of rejoined molecules. Some of these will not produce functional plasmids (molecules with two or with no replication origin cannot function).
Fig.11.1.4 Recombinant Plasmid
One interesting possibility is the joining of
• the 3755-bp pAMP fragment (with ampr and a replication origin) with the
• 1875-bp pKAN fragment (with kanr)
Sealed with DNA ligase, these molecules are functioning plasmids that are capable of conferring resistance to both ampicillin and kanamycin. They are molecules of recombinant DNA.
Because the replication origin, which enables the molecule to function as a plasmid, was contributed by pAMP, pAMP is called the vector.
Transforming E. coli
Treatment of E. coli with the mixture of religated molecules will produce some colonies that are able to grow in the presence of both ampicillin and kanamycin.
• A suspension of E. coli is treated with the mixture of religated DNA molecules.
• The suspension is spread on the surface of agar containing both ampicillin and kanamycin.
• The next day, a few cells — resistant to both antibiotics — will have grown into visible colonies containing billions of transformed cells.
• Each colony represents a clone of transformed cells.
However, E. coli can be simultaneously transformed by more than one plasmid, so we must demonstrate that the transformed cells have acquired the recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant colonies (clones) tells the story.
• Plasmid DNA from cells that acquired their resistance from a recombinant plasmid only show only the 3755-bp and 1875-bp bands (Clone 1, lane 3).
• Clone 2 (Lane 4) was simultaneous transformed by religated pAMP and pKAN. (We cannot tell if it took up the recombinant molecule as well.)
• Clone 3 (Lane 5) was transformed by the recombinant molecule as well as by an intact pKAN.
Cloning other Genes
The recombinant vector described above could itself be a useful tool for cloning other genes. Let us assume that within its kanamycin resistance gene (kanr) there is a single occurrence of the sequence
5' GAATTC 3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.
If we treat any other sample of DNA, e.g., from human cells, with EcoRI, fragments with the same sticky ends will be formed. Mixed with EcoRI-treated plasmid and DNA ligase, a small number of the human molecules will become incorporated into the plasmid which can then be used to transform E. coli.
But how to detect those clones of E. coli that have been transformed by a plasmid carrying a piece of human DNA?
The key is that the EcoRI site is within the kanr gene, so when a piece of human DNA is inserted there, the gene's function is destroyed.
All E. coli cells transformed by the vector, whether it carries human DNA or not, can grow in the presence of ampicillin. But E. coli cells transformed by a plasmid carrying human DNA will be unable to grow in the presence of kanamycin. So,
• Spread a suspension of treated E. coli on agar containing ampicillin only
• grow overnight
• with a sterile toothpick transfer a small amount of each colony to an identified spot on agar containing kanamycin
• (do the same with another ampicillin plate)
• Incubate overnight
All those clones that continue to grow on ampicillin but fail to grow on kanamycin (here, clones 2, 5, and 8) have been transformed with a piece of human DNA.
Some recombinant DNA products being used in human therapy
Using procedures like this, many human genes have been cloned in E. coli or in yeast. This has made it possible — for the first time — to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with a human gene are being used to manufacture more than 100 products for human therapy. Some examples:
• insulin for diabetics
• factor VIII for males suffering from hemophilia A
• factor IX for hemophilia B
• human growth hormone (HGH)
• erythropoietin (EPO) for treating anemia
• several types of interferons
• several interleukins
• granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant
• granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil production (e.g., after chemotherapy) and for mobilizing hematopoietic stem cells from the bone marrow into the blood.
• tissue plasminogen activator (TPA) for dissolving blood clots
• adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID)
• parathyroid hormone
• many monoclonal antibodies
• hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
• C1 inhibitor (C1INH) used to treat hereditary angioedema | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.01%3A_Recombinant_DNA_and_Gene_Cloning.txt |
The polymerase chain reaction is a technique for quickly "cloning" a particular piece of DNA in the test tube (rather than in living cells like E. coli). Thanks to this procedure, one can make virtually unlimited copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules.
Procedure
To perform PCR, you must know at least a portion of the sequence of the DNA molecule that you wish to replicate. You must then synthesize primers: short oligonucleotides (containing about two dozen nucleotides) that are precisely complementary to the sequence at the 3' end of each strand of the DNA you wish to amplify. The DNA sample is heated to separate its strands and mixed with the primers. If the primers find their complementary sequences in the DNA, they bind to them; synthesis begins (as always 5' -> 3') using the original strand as the template.
The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), and a DNA polymerase. It helps to use a DNA polymerase that is not denatured by the high temperature needed to separate the DNA strands. Polymerization continues until each newly-synthesized strand has proceeded far enough to contain the site recognized by the other primer. Now you have two DNA molecules identical to the original molecule. You take these two molecules, heat them to separate their strands, and repeat the process. Each cycle doubles the number of DNA molecules.
Using automated equipment, each cycle of replication can be completed in less than 5 minutes. After 30 cycles, what began as a single molecule of DNA has been amplified into more than a billion copies ($2^{30} = 1.02 \times 10^9$).
With PCR, it is routinely possible to amplify enough DNA from a single hair follicle for DNA typing. Some workers have successfully amplified DNA from a single sperm cell. The PCR technique has even made it possible to analyze DNA from microscope slides of tissue preserved years before. However, the great sensitivity of PCR makes contamination by extraneous DNA a constant problem.
11.03: Gene Therapy - Methods and Prospects
Many human diseases are caused by defective genes. A few common examples are tabulate in Table \(1\); all of these diseases are caused by a defect at a single gene locus. (The inheritance is recessive so both the maternal and paternal copies of the gene must be defective.) Is there any hope of introducing functioning genes into these patients to correct their disorder? Probably. Other diseases also have a genetic basis, but it appears that several genes must act in concert to produce the disease phenotype. The prospects of gene therapy in these cases seems far more remote.
Table \(1\): Human Diseases cause by defective gene
Disease Genetic defect
hemophilia A absence of clotting factor VIII
cystic fibrosis defective chloride channel protein
muscular dystrophy defective muscle protein (dystrophin)
sickle-cell disease defective beta globin
hemophilia B absence of clotting factor IX
severe combined immunodeficiency (SCID) any one of several genes fail to make a protein essential for T and B cell function
Severe Combined Immunodeficiency (SCID)
SCID is a disease in which the patient has neither cell-mediated immune responses nor is able to make antibodies. It is a disease of young children because, until recently, the absence of an immune system left them prey to infections that ultimately killed them. About 25% of the cases of SCID are the result of the child being homozygous for a defective gene encoding the enzyme adenosine deaminase (ADA). The normal catabolism of purines is deficient, and this is particularly toxic for T cells and B cells.
Treatment Options for SCID include:
1. Raise the child in a strictly germfree environment: all food, water, and air to be sterilized. David, the "bubble boy" from Houston, survived this way until he was 12 years old.
2. Give the child a transplant of bone marrow from a normal, histocompatible, donor. Ideally, this would give the child a continuous source of ADA+ T and B cells. However, even though the child cannot reject the transplant (the child has no immune system), T cells in the transplant (unless the donor was an identical twin) can attack the cells of the child producing graft-versus-host disease. Moreover, the donor cells may be infected with a virus which could overwhelm the recipient before his or her immune system was restored. (David received a bone marrow transplant from his sister, but she, like many people, had been infected earlier with the Epstein-Barr virus (the cause of "mono")). The virus was still present in the cells she donated, and killed her brother.
3. Give injections of ADA (the enzyme is currently extracted from cows). When conjugated with polyethylene glycol (PEG) to delay its breakdown in the blood, ADA-PEG injections have kept SCID patients reasonably healthy. But just like the insulin injections of a diabetic, they must be repeated at frequent intervals. So,
4. Giving the patient functioning ADA genes - gene therapy
Gene Therapy Requirements
The gene must be identified and cloned. This has been done for the ADA gene. It must be inserted in cells that can take up long-term residence in the patient. So far, this means removing the patient's own cells, treating them in tissue culture, and then returning them to the patient. It must be inserted in the DNA so that it will be expressed adequately; that is, transcribed and translated with sufficient efficiency that worthwhile amounts of the enzyme are produced. All these requirements seem to have been met for SCID therapy using a retrovirus as the gene vector. Retroviruses have several advantages for introducing genes into human cells.
• Their envelope protein enables the virus to infect human cells.
• RNA copies of the human ADA gene can be incorporated into the retroviral genome using a packaging cell.
Packaging cells are treated so they express an RNA copy of the human ADA gene along with a packaging signal (P) needed for the assembly of fresh virus particles. They also needs inverted repeats ("R") at each end to aid insertion of the DNA copies into the DNA of the target cell. They need an RNA copy of the retroviral gag, pol, and env genes but with no packaging signal (so these genes cannot be incorporated in fresh viral particles).
Treated with these two genomes, the packaging cell produces a crop of retroviruses with:
• the envelope protein needed to infect the human target cells
• an RNA copy of the human ADA gene, complete with R sequences at each end
• reverse transcriptase, needed to make a DNA copy of the ADA gene that can be inserted into the DNA of the target cell
• none of the genes (gag, pol, env) that would enable the virus to replicate in its new host.
Once the virus has infected the target cells, this RNA is reverse transcribed into DNA and inserted into the chromosomal DNA of the host.
Target Cells: T cells
The first attempts at gene therapy for SCID children (in 1990), used their own T cells (produced following ADA-PEG therapy) as the target cells. The T cells were:
• placed in tissue culture
• stimulated to proliferate (by treating them with the lymphokine, Interleukin 2 (IL-2)
• infected with the retroviral vector
• returned, in a series of treatments, to the child
The children developed improved immune function but the injections had to be repeated because T cells live for only 6–12 months in the blood. Moreover, the children also continued to receive ADA-PEG so the actual benefit of the gene therapy was unclear
Target Cells: Stem cells
Blood ("hematopoietic") stem cells:
• produce (by mitosis) all the types of blood cells, including T and B lymphocytes
• produce (by mitosis) more stem cells, thus ensuring an inexhaustible supply
In June of 2002, a team of Italian and Israeli doctors reported on two young SCID patients that were treated with their own blood stem cells that had been transformed in vitro with a retroviral vector carrying the ADA gene. After a year, both children had fully-functioning immune systems (T, B, and NK cells) and were able to live normal lives without any need for treatment with ADA-PEG or immune globulin (IG). The doctors attribute their success to first destroying some of the bone marrow cells of their patients to "make room" for the transformed cells. Nine years later (August 2011) these two patients are still thriving and have been joined by 28 other successfully-treated children most of whom no longer need to take ADA-PEG.
Gene Therapy for X-linked SCID
Gene therapy has also succeeded for 20 baby boys who suffered from another form of severe combined immunodeficiency called X-linked SCID because it is caused by a mutated X-linked gene encoding a subunit — called γc (gamma-c) — of the receptor for several interleukins, including interleukin-7 (IL-7). IL-7 is essential for converting blood stem cells into the progenitors of T cells. Boys with X-linked SCID can make normal B cells, but because B cells need T-helper cells to function, these boys could make neither cell-mediated nor antibody-mediated immune responses and had to live in a sterile bubble before their treatment.
Their doctors
• isolated blood stem cells from the bone marrow of each infant
• treated the cells with a retroviral vector containing the normal gene for the γc interleukin receptor subunit
• returned the treated cells to each donor
The results: Now after as long as 11 years, 19 of these boys
• are able to live normal lives at home instead of inside a sterile "bubble"
• have normal (with some exceptions*) numbers of T cells of both the CD4 and CD8 subsets
• have responded to several childhood immunizations, including diphtheria, tetanus and polio by producing both T cells and antibodies specific for these agents
• Antibody production is sufficiently good that most of the boys have no need for periodic infusions of immune globulin (IG)
Five of the little boys developed leukemia (one has died):
• in one case caused by a proliferating clone of γδ T cells in which the vector has inserted itself in a gene (on chromosome 11) implicated in some cases of acute lymphoblastic leukemia (ALL)
• in a second case, the leukemia was of αβ T cells
Gene Therapy for β-thalassemia
β-thalassemia is an inherited disease. The most severe cases result from mutations in both copies of the gene encoding the beta chain of hemoglobin. Many causative mutations have been identified, and most lead to a failure to make any beta chains. The resulting hemoglobin functions poorly and the person requires frequent blood transfusions. In 2010, Cavazzana-Calvo (and many colleagues) report a single case of successful gene therapy for this disorder; their patient was an 18-year old male. Their procedure involve harvesting blood stem cells from the patient and exposing him to a retroviral vector that contained
• a human gene for beta-hemoglobin complete with its promoter, enhancer, and other control elements;
• alterations to the vector to make it safe.
• After sufficient chemotherapy to "make room" for them, the patient was injected with these cells.
The result: Almost three years later, the patient is well and no longer requires periodic blood transfusions. One-third of his hemoglobin is now manufactured by the red-cell precursors descended from the gene-altered stem cells. A similar procedure was used on several babies born with an inherited lysosomal storage disease or Wiskott-Aldrich syndrome (another type of immune deficiency). Up to two years after treatment with a retroviral vector containing the intact gene, these babies shown any signs of their disorders (reported in the 23 August 2013 issue of Science).
Adenovirus Vectors
Adenoviruses are human pathogens responsible for some cases of the human "cold". Modified versions of two strains are currently being used as vectors in gene therapy trials. Advantages of adenovirusus: Unlike retroviral vectors, they do not integrate into the host genome and thus should not be able to disrupt host genes (It was such disruption that caused some X-linked SCID patients treated with a retroviral vector to develop leukemia) and they can infect nondividing cells with high efficiency. Disadvantages are that they elicit a powerful immune response, both by T cells and by B cells (antibodies) so repeated doses soon lose their effectiveness. Moreoer, many people already have antibodies against the virus from earlier "colds", and these can inactivate the vector at the outset. A recent trial of an HIV vaccine using an adenovirus as the vector was halted when it was found not only not to be effective but, in people with preexisting high levels of anti-adenovirus antibodies, may have even increased their susceptibility to HIV. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.02%3A_Polymerase_Chain_Reaction.txt |
Reaching the goal of effective gene therapies for human diseases has been a difficult one. Some of the problems that remain to be solved include:
• how to avoid an immune response in the patient, which can interfere with gene therapy in two ways:
• The vector provokes inflammation.
• The vector elicits antibodies that destroy the vector when it is administered again.
Both of these are serious problems with adenovirus vectors.
• how to get the gene into non-dividing cells like liver, muscle, and neurons;
• how to get the gene to be replicated (in dividing cells) and expressed indefinitely but
• minimize the risk that it inserts near a proto-oncogene which it could activate producing a cancer. (This occurred in several little boys treated with a retroviral vector based on the murine leukemia virus.
• how to get the gene to be expressed as needed; that is, how to bring the gene under normal physiological controls so that its product is produced where, when, and in the amounts needed.
Adeno-Associated Virus (AAV) — A possible solution?
Adeno-associated virus gets its name because it is often found in cells that are simultaneously infected with adenovirus. However, by itself it seems to be harmless. Unlike adenovirus, AAV
• does not stimulate inflammation in the host;
• can enter non-dividing cells;
• integrates successfully into one spot in the genome of its host — on chromosome 19 in humans.
• (However, AAV vectors do elicit a strong immune response so they can be used only once.)
As for the problem of getting the transgene to be expressed appropriately, that may be solved by using two AAV vectors simultaneously:
• one carrying the desired gene (e.g., for factor VIII or adenosine deaminase, or in the case illustrated here, erythropoietin) into the cells of the host;
• the other carrying genes for the components of the transcription factors needed to turn that gene on.
Regulated production of erythropoietin (EPO)
Vector 1
This piece of DNA contained (among other things):
• the DNA of adeno-associated virus (AAV)
• a gene encoding a protein containing two domains:
• a portion of the molecule ("p65") that is needed to activate gene transcription but that by itself cannot bind to DNA
• a portion ("FRB") that binds the drug rapamycin.
• a gene encoding another protein with two domains:
• a portion of molecule ("ZFHD1") that binds specifically to the DNA sequence in the promoter of the erythropoietin gene but that by itself cannot activate transcription of the gene;
• a portion ("FKBP12") that also binds to rapamycin.
• promoters (not shown) that allow continuous expression (transcription and translation) of the two genes. But note that, by themselves, the two gene products are inactive
Vector 2
This piece of DNA contained (among other things):
• the DNA of adeno-associated virus (AAV)
• 12 identical promoters (green boxes) of the erythropoietin gene
• the gene for erythropoietin (EPO) itself
The Experiment
The experimental animals were injected (in their skeletal muscles) with many copies of both vectors. Skeletal muscle was chosen because muscle fibers are multinucleate. Once across the plasma membrane, there are many nuclei which the vectors can enter and hence many opportunities to integrate into the DNA of the host.
Later the animals were injected with rapamycin. This small molecule is an immunosuppressant and is currently being tested in transplant recipients to help them avoid rejection of the transplant. It was used here because of its ability to simultaneously bind to the FRB and FKBP12 domains of the two gene products of vector 1. The resulting trimer is an active transcription factor for the erythropoietin gene.
The Results
In mice
Injections of the two vectors had — by themselves — no effect on the production of EPO nor on the number of red blood cells (hematocrit), but every time these animals were given an injection of rapamycin, they quickly began to produce EPO (with levels increasing as much as 100 fold) and the number of red blood cells rose (hematocrits increasing from 42% to 60%). The amount of EPO produced was directly related to the amount of rapamycin given. Even after 5 months, a single injection of rapamycin produced a sharp rise in the level of EPO in the blood.
In monkeys
The results were similar to those in mice, but the effect wore off after 4 months. So here is a system where a gene introduced into an animal can then be switched on by giving the animal a small molecule. (In humans, rapamycin can be given by mouth as a pill.) and can have its output regulated by the amount of the small molecule administered.
Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats
Researchers in Seoul, Korea reported in the 23 November 2000 issue of Nature that they have used an AAV-type vector to cure
• mice with inherited IDDM (the animal equivalent of Type 1 diabetes mellitus in humans)
• rats with IDDM induced by chemical destruction of their insulin-secreting beta cells
Both groups of animals were injected (in their hepatic portal vein) with billions of copies of a complex vector containing:
• AAV
• the complementary DNA (cDNA) encoding a synthetic version of insulin
• a promoter that is active only in liver cells and is turned on by the presence of glucose
• the DNA encoding a signal sequence (so that the insulin can be secreted)
• an enhancer to elevate expression of this artificial gene
The results:
Both groups of animals gained control over their blood sugar level and kept this control for over 8 months. When given glucose, they proceeded to synthesize the synthetic insulin which then brought their blood glucose back down to normal levels.
Curing hemophilia B in mice
Researchers at the Salk Institute reported (in the 30 March 1999 issue of the Proceedings of the National Academy of Sciences) work with mice
• whose genes for clotting factor IX had been "knocked out" and
• thus were subject to uncontrolled bleeding like human patients with hemophilia B.
These mice were injected (also in the hepatic portal vein) with DNA containing
• AAV
• cDNA for factor IX (the dog gene)
• liver-specific promoter and enhancer sequences
The mice proceeded to make factor IX and were no longer susceptible to uncontrolled bleeding.
In later work, injection of embryonic stem cells with functioning factor IX genes into the liver of mice without the genes cured them.
Treating ALS
ALS (amyotrophic lateral sclerosis) is a human disease in which motor neurons degenerate. (It is often called "Lou Gehrig's disease" after the baseball player who died from it.)
A similar disease can be created in transgenic mice carrying mutant human genes (for superoxide dismutase) associated with ALS.
Researchers at the Salk Institute have slowed up the progression of the disease in these mice by injecting their skeletal muscles with an AAV vector containing the gene for insulin-like growth factor 1 (IGF-1). The vector
• invaded the muscle cells
• moved into the motor neurons attached to them and
• through their axons up to the cell bodies
The results:
Destruction of motor neurons was reduced, and the mice lived longer than they otherwise would have.
The Outlook
It's a big jump from mice to humans, but these results indicate that the principle of gene therapy for single-gene disorders is valid.
And some early trials in humans look promising.
• An intravenous injection of an AAV vector containing the cDNA of factor IX has produced functional levels of factor IX in several men with hemophilia B.
• On August 18, 2003, physicians in New York injected 3.5 x 109 copies of an AAV vector carrying a gene for the synthesis of GABA into the brain of a patient with Parkinson's disease. He was the first of a phase I clinical trial of this procedure. By 2007, several more Parkinson's patients had been treated with these injections with no harmful side effects and some improvement in their symptoms.
• Several patients with an inherited lack of a functional gene needed to synthesize 11-cis-retinal — and thus destined to be blind — have had a useful level of vision temporarily restored in one eye injected with an AAV vector containing the gene (the other eye was the untreated control). Probably the fact that
• the vector was injected directly into the eye and so not diluted throughout the body as an intravenous injection would be;
• retinal cells rarely divide so the vector would not be lost. (The vector used had the genes needed for integration into the host cell's DNA removed so it could not be duplicated in S phase and, in dividing cells, would eventually disappear.)
• the interior of the eye is an immunologically privileged site
contributed to this remarkable success.
• Several children suffering from X-linked severe combined immunodeficiency have had their immune systems restored after retroviral gene therapy.
• A few patients with hemophilia A have shown modest improvement when injected with their own cells that had earlier been harvested and transformed in vitro with a plasmid containing the factor VIII gene.
• Several gene therapy agents — using adenoviral vectors — are in clinical trials and have shown some promise.
Among these:
• a recombinant adenovirus encoding p53, a tumor-suppressor protein missing in many cancers
• a recombinant adenovirus that destroys cells lacking the p53 protein (as many cancer cells do)
People with the rare disorder lipoprotein lipase deficiency are unable to process the globules (chylomicrons) of fat and protein that appear in the blood after a fat-containing meal because they lack functional copies of the gene encoding lipoprotein lipase. Intramuscular injection of an AAV vector containing the functional gene provides sufficient improvement, with apparent safety, that in October 2012, this agent (Glybera®) received approval for use in the European Union. It is the first gene therapy to receive such approval. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.04%3A_Recent_Advances_in_Gene_Therapy.txt |
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to the gene itself, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host. Transgenic sheep and goats have been produced that express foreign proteins in their milk. Transgenic chickens are now able to synthesize human proteins in the "white" of their eggs. These animals should eventually prove to be valuable sources of proteins for human therapy.
Note
In July 2000, researchers from the team that produced Dolly reported success in producing transgenic lambs in which the transgene had been inserted at a specific site in the genome and functioned well.
Transgenic mice have provided the tools for exploring many biological questions.
Example
Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor
• can be infected by polio virus and even
• develop paralysis and other pathological changes characteristic of the disease in humans.
Two methods of producing transgenic mice are widely used:
• transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA
• injecting the desired gene into the pronucleus of a fertilized mouse egg
Fig.11.4.1 Methods to produce Transgenic mice
The Embryonic Stem Cell Method - Method 1
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.
1. Make your DNA
Using recombinant DNA methods, build molecules of DNA containing
• the gene you desire (e.g., the insulin gene)
• vector DNA to enable the molecules to be inserted into host DNA molecules
• promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in culture
Expose the cultured cells to the DNA so that some will incorporate it.
3. Select for successfully transformed cells
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer
• Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
• Transfer the embryos into her uterus.
• Hope that they implant successfully and develop into healthy pups (no more than one-third will).
6. Test her offspring
• Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
• Mate two heterozygous mice and screen their offspring for the 1 in 4 that will be homozygous for the transgene.
• Mating these will found the transgenic strain.
The Pronucleus Method - Method 2
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs
• Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
• Inject the male pronucleus with your DNA.
• When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
Example
This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a normal littermate (left). The giant mouse developed from a fertilized egg transformed with a recombinant DNA molecule containing:
• the gene for human growth hormone
• a strong mouse gene promoter
The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice.
Random vs. Targeted Gene Insertion
The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a particular gene, it is possible to design vectors that replace that gene. The replacement gene can be one that
• restores function in a mutant animal or
• knocks out the function of a particular locus.
In either case, targeted gene insertion requires
• the desired gene
• neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells
• tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog ganciclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene
Step 1
Treat culture of ES cells with preparation of vector DNA.
Results:
• Most cells fail to take up the vector; these cells will be killed if exposed to G418.
• In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but killed by gancyclovir.
• In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the vector find the homologous sequences in the host genome, and the region between these homologous sequences replaces the equivalent region in the host DNA.
Step 2
Culture the mixture of cells in medium containing both G418 and ganciclovir.
• The cells (the majority) that failed to take up the vector are killed by G418.
• The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tk gene).
• This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).
Step 3
Inject these into the inner cell mass of mouse blastocysts.
Knockout Mice: What do they teach us?
If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out"). Knockout mice are valuable tools for discovering the function(s) of genes for which mutant strains were not previously available. Two generalizations have emerged from examining knockout mice:
• Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
• Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.
Tissue-Specific Knockout Mice
While "housekeeping" genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g. the arrival of a hormone).
To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied. Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.
The Cre/loxP System
One of the bacteriophages that infects E. coli, called P1, produces an enzyme — designated Cre — that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between the two loxP sites is removed, and the remaining DNA ligated together again (so the enzyme is a recombinase). Using "Method 1" above, mice can be made transgenic for
• the gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s) of that type of cell;
• a "target" gene, the one whose function is to be studied, flanked by loxP sequences.
In the adult animal,
• those cells that
• receive signals (e.g., the arrival of a hormone or cytokine)
• to turn on production of the transcription factors needed
• to activate the promoters of the genes whose products are needed by that particular kind of cell
will also turn on transcription of the Cre gene. Its protein will then remove the "target" gene under study.
• All other cells will lack the transcription factors needed to bind to the Cre promoter (and/or any enhancers) so the target gene remains intact.
The result: a mouse with a particular gene knocked out in only certain cells.
Knock-in Mice
The Cre/loxP system can also be used to
• remove DNA sequences that block gene transcription. The "target" gene can then be turned on in certain cells or at certain times as the experimenter wishes.
• replace one of the mouse's own genes with a new gene that the investigator wishes to study.
Such transgenic mice are called "knock-in" mice.
Transgenic Sheep and Goats
Until recently, the transgenes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.
This is how it was done.
Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA:
1. 2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence in humans causes the inherited disease osteogenesis imperfecta.)
This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily accessible in the chromatin.
2. A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector.
3. The human gene encoding alpha1-antitrypsin.
Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or absence produces the disease Alpha1-Antitrypsin Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to the liver).
4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.
5. Binding sites for ribosomes for efficient translation of the beta-lactoglobulin mRNAs.
Successfully-transformed cells were then
• Fused with enucleated sheep eggs and implanted in the uterus of a ewe (female sheep)
• Several embryos survived until their birth, and two young lambs lived over a year.
• When treated with hormones, these two lambs secreted milk containing large amounts of alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the transgene).
On June 18, 2003, the company doing this work abandoned it because of the great expense of building a facility for purifying the protein from sheep's milk. Purification is important because even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins that remain.
However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the milk of transgenic goats.
Transgenic Chickens
Chickens grow faster than sheep and goats and large numbers can be grown in close quarters. They also synthesize several grams of protein in the "white" of their eggs.Two methods have succeeded in producing chickens carrying and expressing foreign genes.
• Infecting embryos with a viral vector carrying
• the human gene for a therapeutic protein
• promoter sequences that will respond to the signals for making proteins (e.g. lysozyme) in egg white
• Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.
Preliminary results from both methods indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay.
Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.
Transgenic Pigs
Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans.
Transgenic Primates
In the 28 May 2009 issue of Nature, Japanese scientists reported success in creating transgenic marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the germline and passed on to the animal's offspring. The hope is that these transgenic animals will provide the best model yet for studying human disease and possible therapies. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.05%3A_Transgenic_Animals.txt |
Progress is being made on several fronts to introduce new traits into plants using recombinant DNA technology. The genetic manipulation of plants has been going on since the dawn of agriculture, but until recently this has required the slow and tedious process of cross-breeding varieties. Genetic engineering promises to speed the process and broaden the scope of what can be done.
There are several methods for introducing genes into plants, including infecting plant cells with plasmids as vectors carrying the desired gene and physically shooting microscopic pellets containing the gene directly into the cell. In contrast to animals, there is no real distinction between somatic cells and germline cells. Somatic tissues of plants (e.g., root cells grown in culture) can be transformed in the laboratory with the desired gene and can grow into mature plants with flowers. If all goes well, the transgene will be incorporated into the pollen and eggs and passed on to the next generation. In this respect, it is easier to produce transgenic plants than transgenic animals.
Achievements
Improved Nutritional Quality
Milled rice is the staple food for a large fraction of the world's human population. Milling rice removes the husk and any beta-carotene it contained. Beta-carotene is a precursor to vitamin A, so it is not surprising that vitamin A deficiency is widespread, especially in the countries of Southeast Asia. The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In January 2000, a group of European researchers reported that they had succeeded in incorporating three transgenes into rice that enabled the plants to manufacture beta-carotene in their endosperm.
Insect Resistance
Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.
Disease Resistance
Genes that provide resistance against plant viruses have been successfully introduced into such crop plants as tobacco, tomatoes, and potatoes.
Example \(1\)
Tomato plants infected with tobacco mosaic virus (which attacks tomato plants as well as tobacco). The plants in the back row carry an introduced gene conferring resistance to the virus. The resistant plants produced three times as much fruit as the sensitive plants and the same as control plants.
Herbicide Resistance
Questions have been raised about the safety — both to humans and to the environment — of some of the broad-leaved weed killers like 2,4-D. Alternatives are available, but they may damage the crop as well as the weeds growing in it. However, genes for resistance to some of the newer herbicides have been introduced into some crop plants and enable them to thrive even when exposed to the weed killer.
Example \(2\)
Effect of the herbicide bromoxynil on tobacco plants transformed with a bacterial gene whose product breaks down bromoxynil (top row) and control plants (bottom row). "Spray blank" plants were treated with the same spray mixture as the others except the bromoxynil was left out. (Courtesy of Calgene, Davis, CA.)
Salt Tolerance
A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to grow most important crops. However, researchers at the University of California Davis campus have created transgenic tomatoes that grow well in saline soils. The transgene was a highly-expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf cells. There was no sodium buildup in the fruit.
"Terminator" Genes
This term is used (by opponents of the practice) for transgenes introduced into crop plants to make them produce sterile seeds (and thus force the farmer to buy fresh seeds for the following season rather than saving seeds from the current crop). The process involves introducing three transgenes into the plant:
• A gene encoding a toxin which is lethal to developing seeds but not to mature seeds or the plant. This gene is normally inactive because of a stretch of DNA inserted between it and its promoter.
• A gene encoding a recombinase — an enzyme that can remove the spacer in the toxin gene thus allowing to be expressed.
• A repressor gene whose protein product binds to the promoter of the recombinase thus keeping it inactive.
How they work
When the seeds are soaked (before their sale) in a solution of tetracycline
• Synthesis of the repressor is blocked.
• The recombinase gene becomes active.
• The spacer is removed from the toxin gene and it can now be turned on.
Because the toxin does not harm the growing plant — only its developing seeds — the crop can be grown normally except that its seeds are sterile. The use of terminator genes has created much controversy. Farmers — especially those in developing countries — want to be able to save some seed from their crop to plant the next season. However, Seed companies want to be able to keep selling seeds.
Transgenes Encoding Antisense RNA
Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense. When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked. This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or duplex RNA is quickly degraded by ribonucleases in the cell. With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Biopharmaceuticals
The genes for proteins to be used in human (and animal) medicine can be inserted into plants and expressed by them.
Advantages:
• Glycoproteins can be made (bacteria like E. coli cannot do this).
• Virtually unlimited amounts can be grown in the field rather than in expensive fermentation tanks.
• It avoids the danger from using mammalian cells and tissue culture medium that might be contaminated with infectious agents.
• Purification is often easier.
Corn is the most popular plant for these purposes, but tobacco, tomatoes, potatoes, rice and carrot cells grown in tissue culture are also being used.
Some of the proteins that have been produced by transgenic crop plants:
• human growth hormone with the gene inserted into the chloroplast DNA of tobacco plants
• humanized antibodies against such infectious agents as
• HIV
• respiratory syncytial virus (RSV)
• sperm (a possible contraceptive)
• herpes simplex virus, HSV, the cause of "cold sores"
• Ebola virus, the cause of the often-fatal Ebola hemorrhagic fever
• protein antigens to be used in vaccines
• An example: patient-specific antilymphoma (a cancer) vaccines. B-cell lymphomas are clones of malignant B cells expressing on their surface a unique antibody molecule. Making tobacco plants transgenic for the RNA of the variable (unique) regions of this antibody enables them to produce the corresponding protein. This can then be incorporated into a vaccine in the hopes (early trials look promising) of boosting the patient's immune system — especially the cell-mediated branch — to combat the cancer.
• other useful proteins like lysozyme and trypsin
• However, as of April 2012, the only protein to receive approval for human use is glucocerebrosidase, an enzyme lacking in Gaucher's disease. It is synthesized by transgenic carrot cells grown in tissue culture.
Controversies
The introduction of transgenic plants into agriculture has been vigorously opposed by some. There are a number of issues that worry the opponents. One of them is the potential risk of transgenes in commercial crops endangering native or nontarget species.
Examples:
• A gene for herbicide resistance in, e.g. maize (corn), escaping into a weed species could make control of the weed far more difficult.
• The gene for Bt toxin expressed in pollen might endanger pollinators like honeybees.
To date, field studies on Bt cotton and maize show that the numbers of some nontarget insects are reduced somewhat but not as much as in fields treated with insecticides.
Another worry is the inadvertent mixing of transgenic crops with nontransgenic food crops. Although this has occurred periodically, there is absolutely no evidence of a threat to human health. Despite the controversies, farmers around the world are embracing transgenic crops. Currently in the United States over 80% of the corn, soybeans, and cotton grown are genetically modified (GM) — principally to provide resistance to the herbicide glyphosate ("Roundup Ready®") thus making it practical to spray the crop with glyphosate to kill weeds without harming the crop and resistance to insect attack (by expressing the toxin of Bacillus thuringiensis). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.06%3A_Transgenic_Plants.txt |
Restriction enzymes cut DNA at precise points producing a collection of DNA fragments of precisely defined length. These can be separated by electrophoresis, with the smaller fragments migrating farther than the larger fragments. One or more of the fragments can be visualized with a "probe" — a molecule of single-stranded DNA that is complementary to a run of nucleotides in one or more of the restriction fragments and is radioactive (or fluorescent). If probes encounter a complementary sequence of nucleotides in a test sample of DNA, they bind to it by Watson-Crick base pairing and thus identify it. Polymorphisms are inherited differences found among the individuals in a population.
Restriction Fragment Length Polymorphisms (RFLPs) have provided valuable information in many areas of biology, including screening human DNA for the presence of potentially deleterious genes ("Case 1") and providing evidence to establish the innocence of, or a probability of the guilt of, a crime suspect by DNA "fingerprinting" ("Case 3").
Case 1: Screening for the sickle-cell gene
Sickle-cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (betaS) of the hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this position. The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6. This converts a GAG codon (for Glu) to a GTG codon for Val and abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized and cut by one of the restriction enzymes.
When the normal gene (betaA) is digested with the enzyme and the fragments separated by electrophoresis, the probe binds to a short fragment (between the red arrows). However, the enzyme cannot cut the sickle-cell gene at this site, so the probe attaches to a much larger fragment (between the blue arrows).
Figure \(1\) shows the pedigree of a family whose only son has sickle-cell disease. Both his father and mother were heterozygous (semifilled box and circle respectively) as they had to be to produce an afflicted child (solid box). The electrophoresis patterns for each member of the family are placed directly beneath them. Note that the two homozygous children (1 and 3) have only a single band, but these are more intense because there is twice as much DNA in them. In this example, a change of a single nucleotide produced the RFLP. This is a very common cause of RFLPs and now such polymorphisms are often referred to as single nucleotide polymorphisms or SNPs. (However, not all RFLPs arise from SNPs.
How can these tools be used?
By testing the DNA of prospective parents, their genotype can be determined and their odds of producing an afflicted child can be determined. In the case of sickle-cell disease, if both parents are heterozygous for the genes, there is a 1 in 4 chance that they will produce a child with the disease. Amniocentesis and chorionic villus sampling make it possible to apply the same techniques to the DNA of a fetus early in pregnancy. The parents can learn whether the unborn child will be free of the disease or not. They may choose to have an abortion rather than bring an afflicted child into the world.
Three problems:
• The mutations that cause most human genetic diseases are more varied than the single mutation associated with sickle-cell disease. Over a thousand different mutations in the cystic fibrosis gene can cause the disease. A probe for one will probably fail to identify a second. A mixture of probes, one for each of the more common mutations, can be used. But there remains the problem of "false negatives": people who are falsely told they do not carry a mutant gene.
• There are many diseases which result from several mutant genes working together to produce the disease phenotype.
• There are still genetic diseases for which no gene has yet been discovered. Until the gene can be located, cloned, and sequenced, no probe can be made to detect it directly. However, it is sometimes possible to find a genetic "marker" that can serve as a surrogate for the gene itself. Let's see how.
Case 2: Screening for a RFLP "marker"
If a particular RFLP is usually associated with a particular genetic disease, then the presence or absence of that RFLP can be used to counsel people about their risk of developing or transmitting the disease. The assumption is that the gene they are really interested in is located so close to the RFLP that the presence of the RFLP can serve as a surrogate for the disease gene itself. But people wanting to be tested cannot simply walk in off the street. Because of crossing over, a particular RFLP might be associated with the mutant gene in some people, with its healthy allele in others. Thus it is essential to examine not only the patient but as many members of the patient's family as possible.
The most useful probes for such analysis bind to a unique sequence of DNA; that is, a sequence occurring at only one place in the genome. Often this DNA is of unknown, if any, function. This can actually be helpful as this DNA has been freer to mutate without harm to the owner. The probe will hybridize (bind to) different lengths of digested DNA in different people depending on where the enzyme cutting sites are that each person has inherited. Thus a large variety of alleles (polymorphisms) may be present in the population. Some people will be homozygous and reveal a single band; others (e.g., all the family members shown below) will be heterozygous with each allele producing its band.
The pedigree in Figure \(2\) shows the inheritance of a RFLP marker through three generations in a single family. A total of 8 alleles (numbered to the left of the blots) are present in the family. The RFLPs of each member of the family are placed directly below his (squares) or her (circles) symbol and RFLP numbers.
If, for example, everyone who inherited RFLP 2 also has a certain inherited disorder, and no one lacking RFLP 2 has the disorder, we deduce that the gene for the disease is closely linked to this RFLP. If the parents decide to have another child, prenatal testing could reveal whether that child was apt to come down with the disease. But note, that crossing over during gamete formation could have moved the RFLP to the healthy allele. So the greater the distance between the RFLP and the gene locus, the lower the probability of an accurate diagnosis.
Case 3: DNA "typing"
Each human cell contains 6 x 109 base pairs of DNA. Some of this represents protein-encoding genes (e.g., for the beta chain of hemoglobin) that are identical in a large proportion of people. But long stretches of DNA do not encode for anything and are free to mutate extensively. It seems certain that if we could read the entire sequence of DNA in each human, we would never find two that were identical (unless the samples were from identical siblings; i.e., derived from a single zygote).
So each person's DNA is as unique as a fingerprint. This truth has not escaped the law enforcement and legal professions. Analysis of DNA, called DNA typing, is widely used to identify rapists and other criminals, determine paternity; that is, who the father of the child really is, determine whether a hopeful immigrant is, as he or she claims, really a close relative of already established residents.
Example \(1\): Rape Suspect
Figure \(3\) shows the test results in a rape case. Two probes were used: one revealing the bands at the top, the other those at the bottom.
DNA was tested from
• semen removed from the vagina of the rape victim (EVIDENCE #2);
• a semen stain left on the victim's clothing (EVIDENCE #1);
• the DNA of the victim herself (VICTIM) to be sure that the DNA didn't come from her cells;
• DNA from two suspects (SUSPECT #1, SUSPECT #2);
• a set of DNA fragments of known and decreasing length (MARKER). They provide a built-in ruler for measuring the exact distance that each fragment travels.
• the cells of a previously-tested person to be sure the probes are performing properly (CONTROL).
One the basis of this test, suspect #2 can clearly be ruled out. None of his bands matches the bands found in the semen.
Is suspect #1 guilty?
We can never be certain. The best we can do is to estimate the probability that another person, picked at random, could provide the same DNA fingerprint. As a conservative estimate, a given allele (band) might be found in 25% of the people tested. The probability of a random match of two alleles is (0.25)2 or 1 in 16. The probability that 6 alleles match, as in this case, is (0.25)6 or 1 in 4096. However, the suspect was not picked at random, so you may feel that the evidence of guilt is strong.
The more probes you use, the more confident you can be that you have gotten the right man. If, for example, a set of probes revealed 14 bands in a suspect's DNA identical to those in the semen sample, the probability that you have the wrong man drops to less than 1 in 268 million (0.25)14 = 1/268,435,456, which is almost as great as the entire population, males and females, in the United States.
Starting in 1999, law enforcement agencies in both Great Britain and the United States began switching to a new version of RFLP analysis using shorter sequences called STRs ("Short Tandem Repeats"). STRs are repeated sequences of a few (usually four) nucleotides, e.g., TCATTCATTCATTCAT. They often occur in the untranslated parts of known genes (whose sequence can be used for the PCR primers). The exact number of repeats (6, 7, 8, 9, etc.) varies in different people (and, often, in the gene on each chromosome; that is, people are often heterozygous for the marker).
When 13 STR loci — scattered over different chromosomes — are examined, the chance that two people picked at random have the same pattern is less than 1 in 1 trillion. The U.S. Federal Bureau of Investigation (FBI) wants to increase the number of loci examined to 20 further eliminating the possibility of false positives. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.07%3A_Restriction_Fragment_Length_Polymorphisms.txt |
Gel blotting is a technique for visualizing a particular subset of macromolecules — proteins, or fragments of DNA or RNA — initially present in a complex mixture. The steps:
1. Separate the molecules by electrophoresis. This is done in a gel which allows the molecules to migrate under the influence of the electric field.
2. "Blot" them with a nitrocellulose filter. For unknown reasons, the molecules stick tightly to the filter and will retain their relative positions when flooded with fluid at the next step.
3. Bathe the filter with a solution containing a "probe": a molecule that will combine specifically with the target molecules; that is, the one(s) you are looking for and carries a mean of visualization, e.g. a radioactive or fluorescent marker.
Southern Blot
The diagram illustrates the procedure for detecting DNA fragments containing a particular sequence. DNA is extracted from the cell and is partially digested by a restriction endonuclease. The resulting DNA fragments are separated by electrophoresis and then denatured to form single-stranded molecules (ssDNA). Without altering their positions, the separated bands of ssDNA are transferred to a nitrocellulose filter and exposed to radiolabeled cDNA or RNA. If the probe detects complementary DNA sequences, it will bind to them. The presence of the probe in a particular band is revealed by autoradiography.
This procedure was developed by E. M. Southern and the finished product is called a "Southern blot". The same basic procedure can also be used to separate and visualize RNA molecules and protein molecules. As a humorous extension of the term "Southern blot", these have been dubbed "Northern" and "Western" blots, respectively (Table \(1\)).
Table \(1\): Summary or Common Gel Blots
Type of Blot Molecules separated by electrophoresis Probe
Southern ssDNA cDNA or RNA
Northern denatured RNA RNA or cDNA
Western Protein Antibodies
11.09: Genetic Screening for Phenylketonuria
Phenylketonuria is one of the commonest inherited disorders — occurring in approximately 1 in 10,000 babies born in the U. S. It occurs in babies who inherit two mutant genes for the enzyme phenylalanine hydroxylase (PAH — "1" in the figure on the left). This enzyme normally starts the process of breaking down molecules of the amino acid phenylalanine that are in excess of the body's needs for protein synthesis.
The complete pathway is shown in the above figure. Phenylalanine that is in excess of the body's needs for protein synthesis is broken down as shown here and used in cellular respiration and to synthesize melanin as needed. Carbon atoms are shown in color, nitrogen atoms in black, and hydrogen atoms as short dashes.
Because we inherit two copies of the gene for the enzyme, both must be defective to produce the disease. A laboratory test that measures how quickly an injection of phenylalanine is removed from the blood can distinguish a person who has one PKU gene from a person who has none, but the person with one is perfectly healthy because the unmutated allele produces enough of the enzyme. However, these heterozygous individuals are "carriers" of the disease.
The Phenylalanine Tolerance Test
A short time after administering a measured amount of phenylalanine to the subject, the concentration of phenylalanine in the blood plasma is measured. The level is usually substantially higher in people who carry one PKU gene (even though they show no signs of disease) than in individuals who are homozygous for the unmutated gene. Both parents must be heterozygous (i.e., must be "carriers" of the trait) to produce a child with PKU. The chance of their doing so is 1 in 4.
Inability to remove excess phenylalanine from the blood during infancy and early childhood produces a variety of problems including mental retardation. Fortunately, a simple test (needing only a drop of blood) done shortly after birth can identify the genetic defect and, with close attention to the amount of phenylalanine in their diet, the children can develop normally.
Alcaptonuria is another inherited disease involving this pathway. It results from the inheritance of two mutant genes for the enzyme ("4") that converts homogentisic acid to maleylacetoacetic acid. When step 4 is blocked, homogentisic acid accumulates in the blood. The kidney excretes this excess in the urine, and oxidation of homogentisic acid by the air turns the urine black. Diseases like PKU and alcaptonuria are called "inborn errors of metabolism" because they are inherited and each is characterized by a distinct metabolic defect.
Genetic Screening
In the United States, approximately 1 person in 50 has inherited a PKU allele. This means that some 5 million people in the U.S. are "carriers". Should they be tested before they decide to become parents? By testing the DNA of prospective parents, their genotype can be determined and their odds of producing an afflicted child calculated. In the case of PKU, if both parents are heterozygous for the gene, there is a 1 in 4 chance that they will produce a child with the disease.
Problems:
• Scores of different mutations in the PAH gene can cause the disease. A probe for one will probably fail to identify a second. A mixture of probes, one for each of the more common mutations, can be used. But there remains the problem of "false negatives": people who are falsely told they do not carry a mutant gene.
• With an effective treatment for PKU available, should heterozygous parents forego having children?
• Do they want their health insurance company to know their status?
Genetic Screening Example
• Top: schematic of a portion of the gene encoding the enzyme phenylalanine hydroxylase (PAH) showing the sites cut by the restriction enzyme HindIII ("H") and the region to which the radioactive probe binds a mutant version of the gene with a deletion that destroys its function. The deletion eliminates the HindIII site in Exon 2, lengthening the DNA fragment to which the probe binds from 3.3 to 4.2 thousand base pairs (kb) (and thus revealing a RFLP).
• Middle: The daughter (solid circle) with PKU inherited one PKU allele from each of her parents (half-filled symbols). Her brother (open square) beat the odds (0.5) of inheriting at least one PKU allele and thus of being a carrier. If he had been a carrier, would he have wanted to know?
• Bottom: The blot shows the fragments to which the probe binds directly beneath each individual in the pedigree. It reveals only the 3.3 kb fragment for the brother, only the 4.2 kb fragment for the sister, and both for each parent.
The particular mutation in this family is only one of many mutant PAH alleles that cause PKU, and testing with this particular enzyme and probe would not necessarily detect the others.
Phenylalanine hydroxylase (PAH) is made in the liver
The evidence:
• A child with PKU was cured when he received a transplanted liver (needed for reasons unrelated to his PKU). (Described by Vajro, P., et. al., in the New England Journal of Medicine 329:363, 29 July 1993.)
• Hepatic Nuclear Factor 1 (HNF1) is a transcription factor that is strongly expressed in the cells of the liver (called hepatocytes). In these cells, it binds to and activates the promoters of many genes expressed in the liver. "Knockout" mice were created from embryonic stem (ES) cells carrying two mutant PAH genes.Among other problems, these mice produced no PAH and had severe PKU. (Described by Pontoglio, M., et. al., in Cell 84:575, 23 February 1996.)
Dominant or recessive
The disease PKU is clearly inherited as a recessive trait. Only if one inherits a mutant allele from each parent will one develop the disease. However, heterozygous people are easily distinguished from homozygotes by the phenylalanine tolerance test. So using the test as the criterion, the PKU allele shows partial dominance. So, the relationship between genotype and phenotype is not always straightforward. What is the criterion of phenotype in this case? the disease? the results of the tolerance test? | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.08%3A_Gel_Blotting.txt |
Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called "sense" because it results in a gene product (protein). Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
However, RNA can form duplexes just as DNA does. All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.
Example
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.
When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked. This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or because the duplex RNA is quickly degraded by ribonucleases in the cell. With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Examples
The Flavr Savr tomato
Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer. Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe. However, these tomatoes often became damaged during shipment and handling and have been taken off the market.
Transgenic Tobacco
Fig.11.9.3 tobacco flower
Flower of a tobacco plant carrying a transgene whose transcript is antisense to one of the mRNAs needed for normal flower pigmentation.
Transgenic Flower
Flower of another transgenic plant that failed to have its normal pigmentation altered.
Making transgenic plants
There are several methods for introducing genes into plants, including
• infecting plant cells with plasmid vectors carrying the desired gene
• shooting microscopic pellets containing the gene directly into the cell
In contrast to animals, there is no real distinction between somatic cells and germline cells. Somatic tissues of plants, e.g., root cells grown in culture,
• can be transformed in the laboratory with the desired gene
• grown into mature plants with flowers.
If all goes well, the transgene will be incorporated into the pollen and eggs and passed on to the next generation.
In this respect, it is easier to produce transgenic plants than transgenic animals.
Antisense RNA also occurs naturally
Do cells contain genes that are naturally translated into antisense RNA molecules capable of blocking the translation of other genes in the cell? The answer is yes, and these seem to represent another method of regulating gene expression. In both mice and humans, the gene for the insulin-like growth factor 2 receptor (Igf2r) that is inherited from the father synthesizes an antisense RNA that appears to block synthesis of the mRNA for Igf2r. An inherited difference in the expression of a gene depending on whether it is inherited from the mother or the father is called genomic or parental imprinting.
RNA interference (RNAi)
In testing the effects of antisense RNA, one should use sense RNA of the same coding region as a control. Surprisingly, preparations of sense RNA often turn out to be as effective an inhibitor as antisense RNA.
Why? It seems that the preparations of sense RNA often are contaminated with hybrids: sense and antisense strands that form a double helix of double-stranded RNA (dsRNA). Double-stranded RNA corresponding to a particular gene is a powerful suppressant of that gene. In fact, the suppressive effect of antisense RNA probably also depends on its ability to form dsRNA (using the corresponding mRNA as a template).
The ability of dsRNA to suppress the expression of a gene corresponding to its own sequence is called RNA interference (RNAi). It is also called post-transcriptional gene silencing or PTGS.
Mechanism of RNAi
The only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded RNA. If the cell finds molecules of double-stranded RNA (dsRNA), it uses an enzyme called Dicer to cut them into fragments containing ~21 base pairs (~2 turns of a double helix). The two strands of each fragment then separate — releasing the antisense strand. With the aid of a protein, it binds to a complementary sense sequence on a molecule of mRNA. If the base-pairing is exact, the mRNA is destroyed. Because of their action, these fragments of RNA have been named "small (or short) interfering RNA" (siRNA). The complex of siRNA and protein is called the "RNA-induced silencing complex" (RISC).
siRNAs can also interfere with transcription
There is growing evidence that siRNAs can also inhibit the transcription of genes
• perhaps by binding to complementary sequences on DNA or
• perhaps by binding to the nascent RNA transcript as it is being formed.
In fission yeast, at least, the siRNA is complexed with one molecule of each of three different proteins. The entire complex is called the RITS complex ("RNA-induced initiation of transcriptional gene silencing")
How these siRNAs — synthesized in the cytosol — gain access to the DNA in the nucleus is unknown.
Synthetic siRNA molecules that bind to gene promoters can — in the laboratory — repress transcription of that gene. The repression is mediated by methylation of the DNA in the promoter and, perhaps, methylation of histones in the vicinity.
There is a strain of rice (LGC-1) that produces abnormally low levels of proteins called glutelins. It turns out that of several glutelin genes found in rice
• two closely-similar glutelin genes are located back to back on the same chromosome.
• In LGC-1, a deletion has occurred between the two genes which removes the signal that would normally stop transcription after the first gene.
• Thus RNA polymerase II transcribes right past the first gene and on into the second.
• The result is a messenger RNA with almost-identical sequences running in opposite directions.
• This causes the mRNA to fold up into a molecule of double-stranded RNA (dsRNA).
• A Dicer-like enzyme cuts up the dsRNA into small interfering RNAs (siRNAs) that suppress further transcription of those genes as well as other glutelin genes.
Why RNAi?
RNAi has been found to operate in such diverse organisms as plants, fungi, and animals such as Drosophila melanogaster, Caenorhabditis elegans, and even mice and the zebrafish. Such a universal cell response must have an important function. What could it be?
Some possibilities:
• Some viruses of both plants and animals have a genome of dsRNA. And many other viruses of both plants and animals have an RNA genome that in the host cell is briefly converted into dsRNA. So RNAi may be a weapon to counter infections by these viruses by destroying their mRNAs and thus blocking the synthesis of essential viral proteins.
• Transposons may be transcribed into RNA molecules with regions that are double-stranded. RNAi could then destroy these.
• RNA interference may be the unexpected dividend of another basic process of controlling gene expression.
RNAi as a tool
In any case, the discovery of RNAi adds a promising tool to the toolbox of molecular biologists. Introducing dsRNA corresponding to a particular gene will knock out the cell's own expression of that gene. (Feeding C. elegans on E. coli manufacturing the dsRNA will even do the trick.)
Heroic Example
In the 24 March 2005 issue of Nature, Sönnichsen et al reported that they have injected dsRNAs corresponding to 20,326 of C. elegans's genes (98% of the total!) and monitored the effect of each on embryonic development from the completion of meiosis (following fertilization) through the second mitotic division that produces the 4-cell embryo.
They found that at least 661 different genes altered some process during this period:
• about half of them involved in cell division and
• half in general cell metabolism.
(Another thousand genes produced phenotypic effects that were seen at later stages of development.)
Because RNAi can be done in particular tissues at a chosen time, it often provides an advantage over conventional gene "knockouts" where the missing gene is carried in the germline and thus whose absence may kill the embryo before it can be studied.
Another Example: screening genes for their effect on drug sensitivity
• Distribute your cells in thousands of wells and add — from a "library" of thousands of siRNAs representing the entire genome — siRNA molecules targeting the expression of one gene to each well
• Add the drug to all the wells
• See which wells have cells that respond
Some other promising applications of RNAi
In mammalian cells
In mammalian cells, introducing dsRNA fragments only reduces gene expression temporarily. However, mammalian cells can be infected with a DNA vector that encodes an RNA molecule of 50–80 nucleotides called a "small hairpin RNA" (shRNA) containing a sequence corresponding to the gene that one wishes to suppress. As the shRNA is synthesized, dicer converts it into a typical siRNA molecule. Because the cell can continuously synthesize shRNA, the interference is long-lasting. In fact, with vectors that become integrated in the host genome, RNAi can be passed on to the descendants.
In plants
The 19 June 2003 issue of Nature reported on coffee plants that were engineered to express a transgene that makes siRNA that interferes — by RNAi — with the expression of a gene needed to make caffeine. So perhaps "decaf" coffee will one day no longer require the chemical removal of caffeine from coffee beans.
Monsanto is developing a transgenic corn (maize) that expresses a dsRNA corresponding to the sequence of an essential gene in the western corn rootworm, a devastating pest of the crop. After ingesting this dsRNA, the insect's own cells process it into an siRNA that targets the gene's mRNA for destruction and kills the worm in a few days.
Amplification of RNAi
In C. elegans, plants, and Neurospora, the introduction of a few molecules of dsRNA has a potent and long-lasting effect. In plants, the gene silencing spreads to adjacent cells (through plasmodesmata) and even to other parts of the plant (through the phloem). RNAi within a cell can continue after mitosis in the progeny of that cell. Triggering of RNAi in C. elegans can even pass through the germline into its descendants.
Such amplification of an initial trigger signal suggests a catalytic effect. It turns out that these organisms have RNA-dependent RNA polymerases (RdRPs) that uses the mRNA targeted by the initial antisense siRNA as a template for the synthesis of more siRNAs. Synthesis of these "secondary" siRNAs even occurs in adjacent regions of the mRNA. So not only can these secondary siRNAs target additional areas of the original mRNA, but they are potentially able to silence mRNAs of other genes that may carry the same sequence of nucleotides.
This phenomenon, called "transitive RNAi",
• may complicate the interpretation of gene suppression experiments as the expression of other genes may be suppressed in addition to the target gene;
• raises a warning flag for the use of RNAi to suppress single genes in human therapy (although RdRPs and amplification have not been observed in mammalian cells).
RNAi in human therapy
Because its target is so specific, the possibility of using RNAi to shut down the expression of a single gene has created great excitement that a new class of therapeutic agents is on the horizon. Many clinical trials are underway exploring the use of siRNA molecules in the treatment of a wide variety of diseases. To date, the most promising results have been using RNAi to target an inherited disease in which the liver secretes a mutant form of transthyretin leading to the accumulation of amyloid deposits in neurons and elsewhere.
MicroRNAs (miRNAs)
In C. elegans, successful development through its larval stages and on to the adult requires the presence of at least two "microRNAs" ("miRNAs") — single-stranded RNA molecules containing about 22 nucleotides and thus about the same size as siRNAs.
These small single-stranded transcripts are generated by the cleavage of larger precursors using the C. elegans version of Dicer.
They act by either destroying or inhibiting translation of several messenger RNAs in the worm (usually by binding to a region of complementary sequence in the 3' untranslated region [3'-UTR] of the mRNA).
The microRNAs (miRNAs) in C. elegans (which were first called "small temporal RNAs") turn out to be representatives of a large class of RNAs that are encoded by the organism's own genes.
• The initial product of gene transcription is a large molecule called pri-miRNA.
• While still within the nucleus an enzyme called Drosher cuts the pri-miRNA into a shorter molecule (~70 nucleotides) called pre-miRNA.
• The pre-miRNA is exported into the cytosol where it is cleaved (by Dicer in animals) into the miRNA.
MicroRNAs
• are found in all animals (humans generate some 1000 miRNAs) and plants but not in fungi.
• contain 19–25 nucleotides;
• are encoded in the genome
• some by stand-alone genes (that may encode several miRNAs)
• some by portions of an intron of the gene whose mRNA they will regulate.
• may be expressed in
• only certain cell types and
• at only certain times in the differentiation of a particular cell type.
While direct evidence of the function of many of these newly-discovered gene products remains to be discovered, they regulate gene expression by regulating messenger RNA (mRNA), either
• destroying the mRNA when the sequences match exactly (the usual situation in plants) or
• repressing its translation when the sequences are only a partial match. In this latter case, it probably requires several miRNAs to bind simultaneously in the 3'-UTR.
MicroRNAs have two traits ideally suited for this:
• Being so small, they can be rapidly transcribed from their genes.
• They do not need to be translated into a protein product to act (in contrast, e.g., to transcription factors).
MicroRNAs regulate (repress) expression of genes in mammals as well. Genome analysis has revealed thousands of human genes whose transcripts (mRNAs) contain sequences to which one or more of our miRNAs might bind. Probably each miRNA can bind to as many as 200 different mRNA targets while each mRNA has binding sites for multiple miRNAs. Such a system provides many opportunities for coordinated mRNA translation.
A study reported in Nature (Lim, et al., 433: 769, 17 Feb 2005) used DNA chip analysis to show that when a particular miRNA was expressed in HeLa cells,
• a miRNA normally expressed in the brain repressed mRNA production by 174 different genes while
• a miRNA normally expressed in cardiac and skeletal muscle repressed mRNA production by 96 genes — all but 8 of them different from the those repressed by the brain miRNA.
As work proceeds rapidly in this field, the pattern that begins to emerge is that:
• Many genes — especially those involved in such housekeeping activities (e.g., cellular respiration) common to all cells — do not have 3'-UTRs that can be blocked by any of the miRNAs encoded in the genome.
• The genes that must be expressed in a particular type of differentiated cell and/or at a particular time in the life of that cell
• do not express any of the miRNA genes that could block their expression but
• do express miRNA genes that block the expression of other genes for specialized functions that would not be appropriate in that cell at that time.
• Rather than being simple switches that turn gene expression on or off, miRNAs seem to exert a more subtle effect — raising or lowering the level of gene expression (much as protein transcription factors do).
Thus repression of gene expression by miRNAs appears to be a mechanism to ensure regulated and coordinated gene expression as cells differentiate along particular paths. For example, when zygote genes begin to be turned on in the zebrafish blastula, one of them encodes a miRNA that triggers the destruction of the maternal mRNAs that have been running things up to then.
So miRNAs may play as important role as transcription factors in regulating and coordinating the expression of multiple genes in a particular type of cell at particular times.
Therapeutic miRNAs?
The ease with which miRNAs can be introduced into cells and their widespread effects on gene expression have given rise to hopes that they might be useful in controlling genetic disorders, e.g., cancer.
To date, some laboratory studies have been quite promising.
• A miRNA that blocks the expression of G1 and S-phase cyclins — and thus stops the cell cycle in its tracks — protects mice from liver cancer.
• a miRNA that inhibits genes needed for metastasis suppresses the metastasis of treated human breast cancer cells.
Summary
In addition to protein transcription factors, eukaryotes use small RNA molecules to regulate gene expression — almost always by repressing it — so the phenomenon is called RNA silencing.
There are two sources of small RNA molecules:
• small interfering RNAs (siRNAs)
• Plant cells make these from the double-stranded RNA (dsRNA) of invading viruses.
• Scientists and pharmaceutical companies make these as agents to turn off the expression of specific genes (called RNA interference or RNAi).
• micro RNAs (miRNAs)
• These are encoded in the genomes of all plants and animals.
• Both siRNAs and miRNAs are processed in the same way in the cytosol of the cell.
• Both are generated by Dicer.
• Both are incorporated into an RNA-induced silencing complex (RISC).
• If the nucleotide sequence of the small RNA exactly matches that of the mRNA, the mRNA is cut and destroyed.
• If there is only a partial match (usually in its 3' UTR), translation (i.e., protein synthesis) is repressed. Both of these activities take place in the cytosol — perhaps in P bodies.
• However, for some small RNAs, the RISC complex enters the nucleus and turns off transcription of the corresponding gene(s) by
• binding to the unwound DNA sequence (or perhaps the RNA transcript as it is being formed)
• converting euchromatin to heterochromation
• methylating of lysine-9 histone H3 in the nucleosomes around the gene(s)
Aside from their use as laboratory — and perhaps therapeutic — tools, small RNAs are clearly essential to the organisms that make them.
Some examples:
• Plants and animals use them to defend themselves against viruses.
• Example: When human cells are infected by hepatitis C virus (HCV), they produce miRNAs that interfere with gene expression by this RNA virus and thus its ability to replicate.
• Some herpesviruses use them to keep their host cell alive long enough to complete viral replication (by blunting a host immune response against the infected cell and preventing its premature death by apoptosis).
• Of the 46 miRNAs expressed in the Drosophila embryo, 25 have been shown to be essential to normal development.
• Correct embryonic development in other animals (e.g., C. elegans, zebrafish, mice) also requires them.
• They protect against the danger of mutations caused by transposons moving around in the genome.
• They are also needed to regulate the size of the pool of at least some types of stem cells.
• Transgenic mice with a single miRNA gene knocked out develop severe immunodeficiency affecting dendritic cells, helper T cells, and B cells.
• Reduced, or no, expression of certain miRNAs are characteristic of several different cancers in humans. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.10%3A_Antisense_RNA.txt |
Antisense oligonucleotides are synthetic polymers. The monomers are chemically-modified deoxynucleotides like those in DNA or ribonucleotides like those in RNA. There are usually only 15–20 of them, hence "oligo". Their sequence (3′ → 5′) is antisense; that is, complementary to the sense sequence of a molecule of mRNA.
Antisense oligonucleotides are synthesized in the hope that they can be used as therapeutic agents — blocking disease processes by altering the synthesis of a particular protein. This would be achieved by the binding of the antisense oligonucleotide to the mRNA from which that protein is normally synthesized. Binding of the two may
• physically block the ability of ribosomes to move along the messenger RNA preventing synthesis of the protein;
• hasten the rate at which the mRNA is degraded within the cytosol;
• prevent splicing errors that would otherwise produce a defective protein.
To be useful in human therapy, antisense oligonucleotides must be able to enter the target cells; avoid digestion by nucleases; and not cause dangerous side-effects. To achieve these goals, antisense oligonucleotides are generally chemically modified to resist digestion by nucleases and attached to a targeting device such as the ligand for the type of receptors found on desired target cells or antibodies directed against molecules on the surface of the desired target cells.
Antisense Oligonucleotides Uses
A variety of antisense oligonucleotides are being tested as possible weapons against:
• Hepatitis C virus (HCV). Successful infection of the liver by this virus requires that the liver produce a particular microRNA (miRNA-122). Injections of HCV-infected humans with an ODN ("miravirsen") complementary to miRNA-122 suppresses the virus.
• HIV-1, the most frequent cause of AIDS in the United States
• Ebola virus, the cause of the often-fatal Ebola hemorrhagic fever
• human cytomegalovirus (HCMV); which frequently causes serious complications in AIDS patients
• asthma; inhalation of an antisense oligonucleotide targeting the mRNA of GATA3 (a transcription factor that promotes Th2 responses) provides relief to patients with allergic asthma.
• certain cancers, e.g., chronic myelogenous leukemia (CML)
• certain types of inflammation caused by cell-mediated immune reactions
• Duchenne muscular dystrophy (DMD)
• familial hypercholesterolemia — targets the mRNA for apolipoprotein B-100. On 31 January 2013, the antisense ODN mipomersen (Kynamro®) received regulatory approval for use in humans with familial hypercholesterolemia.
11.12: Forward and Reverse genetics
The Zebrafish
The zebrafish, Danio rerio, has become another popular "model" organism with which to study fundamental biological questions. It is a small (1–1.5 inches)(2.5–3.8 cm) freshwater fish that grows easily in aquaria (it is available at many pet stores). Some of its advantages for biologists:
• It breeds early and often (daily).
• It is a vertebrate, like us, and thus can provide clues to human biology that invertebrates like Drosophila and Caenorhabditis elegans may not.
• Its embryos, like those of most fishes, develop outside the body where they can be easily observed (unlike mice).
• Its embryos are transparent so defects in development can be seen easily.
• Individual cells in the embryo can be labeled with a fluorescent dye and their fate followed.
• Embryonic development is quick (they hatch in two days).
• They can absorb small molecules, such as mutagens, from the aquarium water.
• Individual cells — or clusters of cells — can be transplanted to other locations in the embryo (as Mangold did with newt embryos).
• They can be forced to develop by parthenogenesis to produce at will homozygous animals with either:
• a male-derived or
• female-derived genome.
• They can be cloned from somatic cells.
• They can be made transgenic (like mice and Drosophila)
• Its genome (1.4 x 109 base pairs) has been sequenced revealing 26,606 protein-coding genes.
Forward Genetics
Since Mendel's time, most genetics has involved observing an interesting phenotype and tracking down the gene responsible for it. So this "forward" genetics proceeds from phenotype -> genotype. Some examples in these pages:
• Mendel's work
• RFLP analysis of large families
• the one gene - one enzyme theory
These methods have been called "forward" genetics to distinguish them from a more recent approach, which has become an urgent priority with the successes of genome sequencing.
Reverse Genetics
Rapid methods of DNA sequencing has generated a vast amount of data. Thousands of suspected genes have been revealed (e.g., finding open reading frames — ORFs), but the function of many of them is still unknown. But now with a knowledge of the DNA sequence of a gene of unknown function, one can use methods for suppressing that particular gene ("knockdown"), and then observe the effect on the phenotype.
So this "reverse" genetics proceeds from genotype -> phenotype. Reverse genetics has been applied successfully to plants; mice; C. elegans; and can also be used with the zebrafish. For example, the function of a mysterious gene sequence in Danio can be studied by
• synthesizing a short antisense oligonucleotide complementary to a section of the gene.
• The oligonucleotide is chemically-modified to make it more stable than a fragment of RNA.
• Binding to its complementary sequence on the messenger RNA (mRNA) produced by transcription of the animal's gene, blocks ("knocks down") gene expression by preventing translation or disrupting normal splicing of the mRNA.
Because we share so many similar gene sequences (orthologous genes) with Danio, if one can discover the function of the gene in Danio, then we have a better idea of the role of its ortholog in humans. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.11%3A_Antisense_Oligodeoxynucleotides_and_their_Therapeutic_Potential.txt |
All the genomes listed on my page Genome Sizes describe the complete genome of a single species. For bacteria and archaeons, this means that the organism was grown in pure culture to provide the DNA for sequencing. But it is now clear that the microbial world contains vast numbers of both groups that have never been grown in the laboratory and thus have escaped study. Soil, water, and the contents of our large intestine are examples of habitats that teem with unknown microorganisms.
Thanks to the recent development of sequencing machines capable of rapidly (and inexpensively) sequencing huge amounts of DNA, it is now practical to sequence the DNA extracted from complex microbial ecosystems like that found in a soil sample. Several different approaches are used, but all depend on a first step of extracting the microbial DNA from the sample (and separating it from the far more complex DNA of any eukaryotes that may be present).
Assessing Microbial Diversity
The DNA encoding the small subunit (16S) of the ribosomes of both bacteria and archaeons contain some highly conserved regions; that is, regions of identical or almost identical sequence. Using primers that target these regions, one can then produce enough material by the polymerase chain reaction PCR to sequence the entire 16S rRNA gene.
Comparing the various sequences to a database of sequences from known organisms, one can estimate how many different types of microbes are present. Because of the substantial genetic diversity found between "strains" of a single species (e.g., E. coli K-12 and E.coli O157:H7), closely-related (> 97% identity) 16S rDNA sequences are assigned to a single "phylotype" because we cannot be sure whether they belong to separate species or to two strains of the same species. In either case, the collection of 16S rDNA sequences can be arranged to form a phylogenetic tree to show the patterns of relatedness.
Cataloging the Genes in a Microbial Ecosystem
Analyzing the 16S rDNA genes in a sample tells us who is there, but, of course, is not a complete genome and tells us nothing about the other genes present in the various members of the population. This information can be gained by "shotgun" sequencing of the environmental DNA sample.
The Steps:
• Break the DNA in short fragments.
• Insert these into a vector, e.g. a plasmid capable of growing in E. coli K-12.
• Expose E. coli cells to this random mix and grow the individual bacterial cells into colonies.
• The result: a library containing millions of random DNA fragments from the original sample.
• Isolate the plasmids and sequence them. Sequence "reads" average around 100 nucleotides — far shorter than a gene but often enough to move on to the next step.
• Use a powerful computer to attempt to assemble the fragments into a linear sequence of DNA. The computer looks for identical stretches of nucleotides in different fragments and uses the overlap to assemble them into a "contig".
• Look (have the computer look) for open reading frames (ORFs) of protein-encoding genes.
• Compare the ORFs with those of known microbes already in databases to see if a function can be deduced.
The sheer diversity of organisms in most microbial ecosystems makes it virtually impossible to find enough contigs to assemble a complete genome for any one organism like those listed in Genome Sizes. What you get instead is a window into the many kinds of genes present in one inhabitant or another of that ecosystem. For example, you may discover genes that encode proteins able to degrade environmental pollutants or genes able to synthesize a new antibiotic.
Finding New Functions in Microbial Populations
Another way of exploiting metagenomics is to look for new functions in the host (e.g. E. coli) if it can express the new gene with which it was transformed. For example, screening the library of E. coli clones for the ability to resist an antibiotic can reveal genes involved in antibiotic resistance — a worrisome development in recent years.
Some Applications of Metagenomics
The Sargasso Sea
Metagenomic analysis of the DNA extracted from sea water in the Sargasso Sea revealed the presence of over a thousand different 16S rDNA genes (and thus approximately that number of different species) and over a million protein-encoding genes.
The Human Colon
0.3 g fecal samples from two healthy humans produced 78 million base pairs of sequence. Each subject produced some 25 thousand open reading frames (ORFs) of which about half could be recognized as already-known bacterial or archaeal genes. Included were genes encoding enzymes for the synthesis of vitamins (e.g., vitamin B1), amino acids, and enzymes for the digestion of complex polysaccharides in our diet which would otherwise be indigestible. Perhaps as much as 10% of the energy we extract from our food is made available to us by the activity of these microorganisms.
Acid Mine Drainage
Metagenomic analysis of the acidic water (pH ~0.5) flowing from an abandoned metal mine in California revealed a much simpler ecosystem than those described above: only 3 species of bacteria and 2 of archaea. With such limited diversity, it was possible to assemble almost-complete genomes for two of these organisms.
A South African Gold Mine
Simpler still was the ecosystem found in water 2.8 km (1.7 miles) down in a gold mine. Only one organism turned up: an autotrophic bacterium capable of extracting energy from inorganic substances in its environment and synthesizing all the molecules needed for its life from them. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/11%3A_Genomics/11.13%3A_Metagenomics.txt |
A cancer is an uncontrolled proliferation of cells. In some the rate is fast; in others, slow; but in all cancers the cells never stop dividing. This distinguishes cancers — malignant tumors — from benign growths like moles where their cells eventually stop dividing (usually). Even more important, benign growths differ from malignant ones in not producing metastases; that is, they do not seed new growths elsewhere in the body.
• 12.1: Cancer in General
A cancer is an uncontrolled proliferation of cells. In some the rate is fast; in others, slow; but in all cancers the cells never stop dividing. This distinguishes cancers — malignant tumors — from benign growths like moles where their cells eventually stop dividing (usually). Even more important, benign growths differ from malignant ones in not producing metastases; that is, they do not seed new growths elsewhere in the body.
• 12.2: Cancer Cells in Culture
Both normal cells and cancer cells can be cultured in vitro in the laboratory. However, they behave quite differently. Normal cells pass through a limited number of cell divisions (70 is about the limit for cells harvested from young animals) before they decline in vigor and die. This is called replicative senescence. It may be caused by their inability to synthesize telomerase. Cancer cells may be immortal; that is, proliferate indefinitely in culture.
• 12.3: Oncogenes
An oncogene is a gene that when mutated or expressed at abnormally-high levels contributes to converting a normal cell into a cancer cell. Cancer cells are cells that are engaged in uncontrolled mitosis. Normal cells growing in culture will not divide unless they are stimulated by one or more growth factors present in the culture medium (e.g, Epidermal Growth Factor (EGF)).
• 12.4: Tumor Suppressor Genes
Some genes suppress tumor formation. Their protein product inhibits mitosis. When mutated, the mutant allele behaves as a recessive; that is, as long as the cell contains one normal allele, tumor suppression continues. (Oncogenes, by contrast, behave as dominants; one mutant, or overly-active, allele can predispose the cell to tumor formation).
• 12.5: BCL-2
BCL-2 is a human proto-oncogene located on chromosome 18. Its product is an integral membrane protein (called Bcl-2) located in the membranes of the endoplasmic reticulum (ER), nuclear envelope, and in the outer membranes of mitochondria. The gene was discovered as the translocated locus in a B-cell leukemia. This translocation is also found in some B-cell lymphomas.
• 12.6: Burkitt's Lymphoma
Burkitt's lymphoma is a solid tumor of B lymphocytes, the lymphocytes that the immune system uses to make antibodies. The genes for making antibodies are located on chromosomes 14 (the heavy [H] chains), 2 (kappa light chains), and 22 (lambda light chains). These genes are expressed only in B lymphocytes because only B cells have the necessary transcription factors for the promoters and enhancers needed to turn these antibody genes "on".
• 12.7: Chronic Myelogenous Leukemia (CML)
Leukemia is an uncontrolled proliferation of one kind of white blood cell (or leukocyte). Like all cancers (probably), all the leukemic cells are descended from a single cell that lost the ability to maintain normal control over the cell cycle. There are a number of types of leukemia, as you would expect from the number of types of white blood cells (5) and the number of stages they pass through as they mature. One of the most common is chronic myelogenous leukemia or CML.
• 12.8: Fighting Cancer with Inhibitors of Angiogenesis
Once a nest of cancer cells reaches a certain size (1–2 mm in diameter), it must develop a blood supply in order to grow larger. Diffusion is no longer adequate to supply the cells with oxygen and nutrients and to take away wastes. Cancer cells (probably like all tissues) secrete substances that promote the formation of new blood vessels — a process called angiogenesis. Over a dozen substances have been identified that promote angiogenesis.
• 12.9: Immunotherapy of Cancer
Most cancer patients are treated with some combination of surgery, radiation, and chemotherapy. Radiation and chemotherapy have the disadvantage of destroying healthy as well as malignant cells and thus can cause severe side-effects. One long-held dream is that the specificity of immune mechanisms could be harnessed against tumor cells. This might use the patient's own immune system or the transfer of antibodies or T cells from an outside source.
• 12.10: Cancer- The Causes and Prevention of Cancer
The effort to eliminate synthetic pesticides because of unsubstantiated fears about residues in food will make fruits and vegetables more expensive, decrease consumption, and thus increase cancer rates. The levels of synthetic pesticide residues are trivial in comparison to natural chemicals, and thus their potential for cancer causation is extremely low.
• 12.11: Estimating Cancer Risks
We live surrounded by radiation and by chemicals that cause mutations in test organisms (like bacteria, yeast, and mice) and cause an increase in the rate of cancers in experimental animals (rats and mice). Is there any safe dose for humans of these agents (which include oxygen!) The question is exceedingly difficult to answer and, I believe, at low doses, unanswerable.
• 12.12: The LD50 test
The LD50 is a standardized measure for expressing and comparing the toxicity of chemicals. The LD50 is the dose that kills half (50%) of the animals tested (LD = "lethal dose"). The animals are usually rats or mice, although rabbits, guinea pigs, hamsters, and so on are sometimes used.
• 12.13: Dioxin
Name given members of a family of closely-related chemicals. The term dioxin is often used for one of these: 2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD. This substance was present as a contaminant in the herbicide agent orange, which was so widely used during the Vietnam war. When ingested or injected, TCDD is extremely poisonous to laboratory animals. At sub-lethal concentrations, it causes cancer and birth defects in them.
• 12.14: Magnetic Fields and Cancer
"There is no convincing evidence that high-voltage power lines are a health hazard or a cause of cancer...18 years of research have produced considerable paranoia, but little insight and no prevention. It is time to stop wasting our research resources. We should redirect them to research that will be able to discover the true biologic causes of the leukemic clones that threaten the lives of children."
Thumbnail: This is a photograph of a basal cell carcinoma on the back taken by me. Basal cell carcinoma is the most common skin cancer. (Public Domain; John Hendrix).
12: Cancer
A cancer is an uncontrolled proliferation of cells. In some the rate is fast; in others, slow; but in all cancers the cells never stop dividing. This distinguishes cancers — malignant tumors — from benign growths like moles where their cells eventually stop dividing (usually). Even more important, benign growths differ from malignant ones in not producing metastases; that is, they do not seed new growths elsewhere in the body.
Cancers are clones. No matter how many trillions of cells are present in the cancer, they are all descended from a single ancestral cell. Evidence: Although normal tissues of a woman are a mosaic of cells in which one X chromosome or the other has been inactivated, all her tumor cells — even if from multiple sites — have the same X chromosome inactivated.
Cancers begin as a primary tumor. Most (maybe all) solid tumors shed cells into the lymph and blood. Most of these lack the potential to develop into tumors. However, some of the shed cells are able to take up residence and establish secondary tumors — metastases — in other locations of the body. These metastases, not the primary tumor, are what usually kills the patient.
Cancer cells are usually less differentiated than the normal cells of the tissue where they arose. Many people feel that this reflects a process of dedifferentiation, but I doubt it. Rather, evidence is accumulating that cancers arise in precursor cells — stem cells or "progenitor cells" — of the tissue: cells that are dividing by mitosis producing daughter cells that are not yet fully differentiated.
A cancer is an uncontrolled proliferation of cells.
Cancer is a Genetic Disease
What probably happens is:
• A single cell — perhaps an adult stem cell or progenitor cell — in a tissue suffers a mutation (red line) in a gene involved in the cell cycle, e.g., an oncogene or tumor suppressor gene.
• This results in giving that cell a slight growth advantage over other dividing cells in the tissue.
• As that cell develops into a clone, some if its descendants suffer another mutation (red line) in another cell-cycle gene.
• This further deregulates the cell cycle of that cell and its descendants.
• As the rate of mitosis in that clone increases, the chances of further DNA damage increases.
• Eventually, so many mutations have occurred that the growth of that clone becomes completely unregulated.
• The result: full-blown cancer. (Genetic analysis reveals an average of 63 mutations in pancreatic cancers; almost as many in one type of adult brain cancer, but only 11 somatic mutations in a case of brain cancer in a child.)
• Sequencing samples from several areas in a primary tumor, as well as from some of its metastases, reveals a different collection of mutations from sample to sample. This finding is reinforced by the sequencing of the genome of individual cells from a single tumor each of which shows a unique pattern of shared and unique mutations. (The ability to sequence the genome of a single cell reveals that even normal cells in an adult have accumulated a suite of somatic mutations that differs from cell to cell. However, the rate of somatic mutations in these normal cells is only a fourth of that in cancer cells.)
So even though all the malignant cells in a cancer are descended from a single original cell — and thus are members of a single clone — they are no longer genetically-identical. As the tumor develops, its various cells develop a variety of additional mutations, and these give rise to "subclones" of varying degrees of malignancy with varying
• propensity to metastasize;
• susceptibility to treatment by anticancer drugs;
• propensity to relapse after apparently-successful therapy.
These findings should stimulate a reexamination of the use of chemotherapy.
• While chemotherapy may wipe out dominant subclones in a tumor, there is evidence that is also exerts a selective pressure for the expansion of more malignant, previously-minor, subclones.
• Most chemotherapeutic agents damage DNA so while killing off some cells, they will raise the mutation rate in any surviving cells perhaps encouraging the outgrowth of even more malignant subclones.
Evidence: In a group of patients with chronic lymphocytic leukemia, those receiving chemotherapy survived for shorter periods than those that did not.
Cancer Stem Cells
Stem cells are cells that divide by mitosis to form either two stem cells, thus increasing the size of the stem cell "pool", or one daughter that goes on to differentiate, and one daughter that retains its stem-cell properties. There is growing evidence that most of the cells in leukemias, breast, brain, skin, ovarian, and colon cancers are not able to proliferate out-of-control (and to metastasize). Only those members of the clone that retain their stem-cell-like properties (~2.5% of the cells in a tumor of the colon) can do so.
There is a certain logic to this. Most terminally-differentiated cells have limited potential to divide by mitosis and, seldom passing through S phase of the cell cycle, are limited in their ability to accumulate the new mutations that predispose to becoming cancerous. Furthermore, they often have short life spans — being eliminated by apoptosis (e.g., lymphocytes) or being shed from the tissue (e.g., epithelial cells of the colon). The adult stem cell pool, in contrast, is long-lived, and its members have many opportunities to acquire new mutations as they produce differentiating daughters as well as daughters that maintain the stem cell pool.
Colon cancer
• Begins with the development of polyps in the epithelium of the colon. Polyps are benign growths.
• As time passes, the polyps may get bigger.
• At some point, nests of malignant cells may appear within the polyps
• If the polyp is not removed, some of these malignant cells will escape from the primary tumor and metastasize throughout the body.
Examination of the cells at the earliest, polyp, stage, reveals that they contain one or two mutations associated with cancer. Frequently these include
• the deletion of a healthy copy of the APC (adenomatous polyposis coli) gene on chromosome 5 leaving behind a mutant copy of this tumor suppressor gene
Two results:
1. One of the functions of the APC gene product is to destroy the transcription factor β-catenin thus preventing it from turning on genes that cause the cell to divide. With no, or a defective, APC protein, the normal brakes on cell division are lifted.
2. Another function of the APC protein is to help attach the microtubules of the mitotic spindle to the kinetochores of the chromosomes. With no, or a defective, APC product available, chromosomes are lost from the spindle producing aneuploid progeny.
• a mutant oncogene (often RAS).
• deletion and/or mutation of the tumor suppressor gene p53
The graph also explains why cancer has become such a common cause of death during the twentieth century. It probably has very little to do with exposure to the chemicals of modern living and everything to do with the increased longevity that has been such a remarkable feature of the 20th century. A population whose members increasingly survive accidents and infectious disease is a population increasingly condemned to death from such "organic" diseases as cancer.
Causes of Cancer
Cancers are caused by
• anything that damages DNA; that is anything that is mutagenic
• radiation that can penetrate to the nucleus and interact with DNA
• chemicals that can penetrate to the nucleus and damage DNA. Chemicals that cause cancer are called carcinogens.
• anything that stimulates the rate of mitosis. This is because a cell is most susceptible to mutations when it is replicating its DNA during the S phase of the cell cycle.
• certain hormones (e.g., hormones that stimulate mitosis in tissues like the breast and the prostate gland)
• chronic tissue injury (which increases mitosis in the stem cells needed to repair the damage)
• agents that cause inflammation (which generates DNA-damaging oxidizing agents in the cell)
• certain other chemicals; some the products of technology
• certain viruses
(Considering that from conception to death, an estimated 1016 mitotic cell divisions occur in humans, it is remarkable that cancer is not more common than it is.)
Viruses and Cancer
Many viruses have been studied that reliably cause cancer when laboratory animals are infected with them. What about humans? The evidence obviously is indirect but some likely culprits are:
• two papilloma viruses that can cause cancer of the cervix and other regions of the genitals (male as well as female).
• the hepatitis B and hepatitis C viruses, which infect the liver and are closely associated with liver cancer (probably because of the chronic inflammation they produce)
• some herpes viruses such as the Epstein-Barr virus (implicated in Burkitt's lymphoma) and KSHV that is associated with Kaposi's sarcoma (a malignancy frequently seen in the late stages of AIDS)
• two human T-cell lymphotropic viruses, HTLV-1 and HTLV-2
But note that the viral infection only contributes to the development of cancer.
• Many people are infected by these viruses and do not develop cancer.
• When cancers do arise in infected people, they still follow our rule of clonality. Many cells have been infected, but only one (usually) develops into a tumor.
So again it appears that only if an infected cell is unlucky enough to suffer several other types of damage will it develop into a tumor.Nevertheless, widespread vaccination against these viruses should not only prevent disease but lower the incidence of the cancers associated with them. A vaccine against hepatitis B is available as are two vaccines (Gardasil® and Cervarix®) against the most dangerous papilloma viruses.
Are Cancers Contagious?
The short answer is NO.
The reason: Cancer cells, like all cells in the body, express histocompatibility molecules on their surface. So like any organ or tissue transplant between two people (other than identical twins), they are allografts and are recognized and destroyed by the recipient's immune system.
However, there are some exceptions.
1. Although tumors are not transmissible, viruses are. So any of the viruses described in the previous section can be spread from person to person and predispose them to the relevant cancers.
2. There have been a number of cases where, unbeknownst to the surgeon, an organ (e.g., a kidney) from a donor with melanoma has allowed the growth of the same melanoma in the recipient. Transplant recipients must have their immune system suppressed if the transplant is not to be rejected, but their immunosuppression also prevents their immune system from attacking the melanoma cells. Stopping immune suppression cures the recipient (but also causes loss of the kidney).
3. Canine transmissible venereal tumor (CTVT). This tumor spreads from dog to dog during copulation. Although the MHC alleles on the tumor cells are only weakly expressed, they do eventually cause the tumor to be rejected.
4. Devil facial tumor disease (DFTD). The carnivorous Tasmanian devil is a marsupial living in Tasmania, Australia. The population is threatened by a facial cancer that is spread through bites. The population is highly inbred, thus closely-related genetically, and the MHC alleles on the tumor are only weakly expressed. So it may be these factors that allow the tumor to grow unchecked.
5. The soft-shell clam, Mya arenaria, along the North Atlantic coast of North America is being devastated by a leukemia that spreads from animal to animal perhaps as these filter feeders ingest sea water in which leukemic cells have been shed. These mollusks are invertebrates and lack powerful tissue rejection molecules like the MHC of vertebrates.
6. There are extremely rare cases where a pregnant woman with cancer (a leukemia or melanoma) has transmitted the cancer across the placenta to her fetus (whose immune system has yet to develop).
The Hallmarks of Cancer
In the year 2000 Douglas Hanahan and Robert Weinberg published a paper — The Hallmarks of Cancer — outlining 6 characteristics that are acquired as a cell progresses toward becoming a full-blown cancer. In the 4 March 2011 issue of Cell, they add 4 other features.
1. Uncontrolled proliferation.
2. Evasion of growth suppressors. Among the many mutations found in cancers, one or more inactivate tumor suppressor genes.
3. Resistance to apoptosis (programmed cell death).
4. Develop replicative immortality; i.e., avoid the normal process of cell senescence.
5. Induce angiogenesis; that is, promote the development of a blood supply.
6. Invasion and metastasis — the ability of tumor cells to invade underlying tissue and then to be carried to other parts of the body where secondary tumors develop (metastasis). During this process, the normal adhesion of cells to each other and to the underlying extracellular matrix (ECM) are disrupted.
7. Genomic instability. Cancer cells develop chromosomal aberrations and many (hundreds) of mutations. Most of the latter are "passenger" mutations, but as many as 10 may be "drivers" of the cancerous transformation.
8. Inflammation. Tumors are invaded by cells of the immune system, which promote inflammation. One effect of inflammation is the production of reactive oxygen species (ROS). These damage DNA and other molecules.
9. Changed energy metabolism. Even if well-supplied with oxygen, cancer cells get most of their ATP from glycolysis not cellular respiration.
10. Evade the immune system. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.01%3A_Cancer_in_General.txt |
The photographs (courtesy of G. Steven Martin) show mouse fibroblasts (connective tissue cells) growing in culture. The cells in the top photo show contact inhibition. Those below do not. The cells below are said to be transformed. These cells (called 3T3 cells) were not derived from a mouse cancer but were produced by laboratory treatment of normal cells. Radiation, certain chemicals, and certain viruses are capable of transforming cells. Although transformed cells are not derived from cancers, they can often develop into malignant tumors when injected into an appropriate test animal (like a nude mouse).
Normal cells are exceedingly fussy about the nutrients that must be supplied to them in their tissue culture medium.
Cancer cells (and transformed cells) can usually grow on much simpler culture medium.
Normal cells ordinarily have the normal set of chromosomes of the species; that is, have a normal karyotype.
Cancer cells almost always have an abnormal karyotype with
• abnormal numbers of chromosomes (polyploid or aneuploid)
• chromosomes with abnormal structure:
• translocations
• deletions
• duplications
• inversions
12.03: Oncogenes
An oncogene is a gene that when mutated or expressed at abnormally-high levels contributes to converting a normal cell into a cancer cell. Cancer cells are cells that are engaged in uncontrolled mitosis.
The signals for normal mitosis
Normal cells growing in culture will not divide unless they are stimulated by one or more growth factors present in the culture medium (e.g, Epidermal Growth Factor (EGF)). The growth factor binds to its receptor, an integral membrane protein embedded in the plasma membrane with its ligand-binding site exposed at the surface of the cell. Examples:
• the Epidermal Growth Factor Receptor (EGFR). The gene encoding it, EGFR, is also known as HER1.
• another growth factor receptor is encoded by the gene ERBB2 (also known as HER2.)
• Binding of a growth factor to its receptor triggers a cascade of signaling events within the cytosol. Many of these involve
• kinases — enzymes that attach phosphate groups to other proteins. Examples: the proteins encoded by SRC, RAF, ABL, and the fusion protein encoded by BCR/ABL found in chronic myelogenous leukemia (CML).
• or molecules that turn on kinases. Example: RAS. RAS molecules reside on the inner surface of the plasma membrane where they serve to link receptor activation to "downstream" kinases like RAF.
• In most cases, phosphorylation activates the protein and eventually transfers the signal into the nucleus.
Here phosphorylation activates transcription factors that bind to promoters and enhancers in DNA, turning on their associated genes. Examples: AP-1, a heterodimer of the proteins encoded by jun and fos. Some of the genes turned on by these transcription factors encode other transcription factors (e.g., myc).
Some of the genes turned on by these downstream transcription factors encode cyclins that prepare the cell to undergo mitosis. Genes that participate in any one of the steps above can become oncogenes if they become mutated so that their product becomes constitutively active (that is, active all the time even in the absence of a positive signal) or they produce their product in excess. Possible causes include if their promoter and/or enhancer has become mutated (e.g., the oncomouse: a transgenic mouse that has both copies of its myc gene under the influence of extra-powerful promoters) or loss (e.g., by a translocation) of the 3'-UTR of their mRNA so that a microRNA (miRNA) that normally represses translation can no longer do so.
All these oncogenes act as dominants; if the cell has one normal gene (called a proto-oncogene) and one mutated gene (the oncogene) at a pair of loci, the abnormal product takes control. No single oncogene can, by itself, cause cancer. It can, however, increase the rate of mitosis of the cell in which it finds itself. Dividing cells are at increased risk of acquiring mutations, so a clone of actively dividing cells can yield subclones of cells with a second, third, etc. oncogene. When a clone loses all control over its mitosis, it is well on its way to developing into a cancer.
This graph (based on the work of E. Sinn et al, Cell 49:465,1987) shows the synergistic effect of two oncogenes. The fraction (%) of transgenic mice without tumors is shown as a function of age.
Other types of potential cancer-promoting genes
• Genes that inhibit apoptosis: The suicide of damaged cells — apoptosis — provides an important mechanism for ridding the body of cells that could go on to form a cancer. It is not surprising then that inhibiting apoptosis can promote the formation of a cancer. Example: Bcl-2. The product of this gene inhibits apoptosis. Overexpression of the gene is a hallmark of B-cell cancers.
• Genes involved in repairing DNA or stopping mitosis if they fail: Mutations arise from an unrepaired error in DNA. So any gene whose product participates in DNA repair probably can also behave as an oncogene when mutated. For example: ATM. ATM (="ataxia telangiectasia mutated") gets its name from a human disease of that name, whose patients — among other things — are at increased risk of cancer. The ATM protein is also involved in detecting DNA damage and interrupting the cell cycle when damage is found. It is estimated that fully 1% of the ~21,000 genes in the human genome are proto-oncogenes.
• Tumor-Suppressor Genes: The products of some genes inhibit mitosis. These genes are called tumor suppressor genes. In contrast to oncogenes, these behave as recessives — both alleles must be defective to lose their braking effect on mitosis.
Contributors and Attributions
John W. Kimball. This content is distributed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license and made possible by funding from The Saylor Foundation. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.02%3A_Cancer_Cells_in_Culture.txt |
Some genes suppress tumor formation.
• Their protein product inhibits mitosis.
• When mutated, the mutant allele behaves as a recessive; that is, as long as the cell contains one normal allele, tumor suppression continues. (Oncogenes, by contrast, behave as dominants; one mutant, or overly-active, allele can predispose the cell to tumor formation).
RB - the retinoblastoma gene
Retinoblastoma is a cancerous tumor of the retina. It occurs in two forms:
• Familial retinoblastoma: Multiple tumors in the retinas of both eyes occurring in the first weeks of infancy.
• Sporadic retinoblastoma: A single tumor appears in one eye sometime in early childhood before the retina is fully developed and mitosis in it ceases.
Familial retinoblastoma
Familial retinoblastoma occurs when a baby inherits from one of its parents a chromosome (number 13) that has its RB locus deleted (or otherwise mutated). The normal Rb protein controls the cell cycle. It integrates the signals reaching the cell to determine whether it is safe for the cell to complete the passage from G1 of the cell cycle to mitosis.
Mechanism
The unphosphorylated Rb protein prevents cells from entering S phase of the cell cycle. It does this by binding to transcription factors called E2F. This prevents the E2Fs from binding to the promoters of such proto-oncogenes as c-myc and c-fos. Transcription of c-myc and c-fos is needed for mitosis so blocking the transcription factors needed to turn on these genes prevents cell division. However, if conditions are adequate for the cell to successfully complete mitosis, the Rb protein becomes phosphorylated, releases the E2Fs, and the cell can proceed through the cell cycle.
The Rb protein also plays a role in mitosis itself: it is needed for proper chromosome condensation starting in prophase, as well as their proper attachment to the spindle. Failure of Rb function during mitosis can lead to aneuploidy and chromosome breakage.
A random mutation of the remaining RB locus in any retinal cell — which are nondividing cells and should not enter the cell cycle — completely removes the inhibition provided by the Rb protein, and the affected cell grows into a tumor. So, in this form of the disease, a germline mutation plus a somatic mutation of the second allele leads to the disease.
Sporadic retinoblastoma
In this disease, both inherited RB genes are normal and a single cell must be so unlucky as to suffer a somatic mutation (often a deletion) in both in order to develop into a tumor. Such a double hit is an exceedingly improbable event, and so only rarely will such a tumor occur. (In both forms of the disease, the patient's life can be saved if the tumor(s) is detected soon enough and the affected eye(s) removed.)
p53
The product of the tumor suppressor gene p53 is a protein of 53 kilodaltons (hence the name). (You will find that the human gene is variously designated as P53, TP53 ["tumor protein 53"], and TRP53 ["transformation-related protein 53"])
The p53 protein prevents a cell from completing the cell cycle if its DNA is damaged or the cell has suffered other types of damage.
When
• the damage is minor, p53 halts the cell cycle — hence cell division — until the damage is repaired.
• the damage is major and cannot be repaired, p53 triggers the cell to commit suicide by apoptosis.
These functions make p53 a key player in protecting us against cancer; that is, it is an important tumor suppressor gene. More than half of all human lung, ovarian, and colorectal cancers harbor p53 mutations and have no functioning p53 protein.
Mice have been cured of cancer by treating them with a peptide that turns on production of the p53 protein in the tumor cells. However, there may be a tradeoff involved: excess production of the p53 protein leads to accelerated aging in mice.
Elephants are very long-lived but seldom develop cancers. It turns out that their cells contain 40 copies of the p53 gene (TP53) compared with the two that we and other mammals have.
p16INK4a
The product of the tumor suppressor gene INK4a is a protein of 16 kilodaltons (hence the name).
Like p53, it blocks progression through the cell cycle — in this case by inhibiting the action of the cyclin-dependent kinase Cdk4.
As an animal ages, its cells produce increasing amounts of p16INK4a. This is probably a good thing in that it reduces the risk of the cell entering uncontrolled mitosis, i.e., becoming a cancer. However, again like p53, there is a tradeoff. As levels of p16INK4a rise in adult stem cells and progenitor cells, their ability to reproduce and thus replace lost or damaged tissue diminishes.
p16INK4a is not simply a reflection of an aging cell but is actively involved in the process.
• Mice expressing higher-than-normal levels of p16INK4a show earlier replicative senescence while
• mice in which p16INK4a activity is blocked continue to repair damaged tissue efficiently but run a higher risk of getting cancer.
• In mice, eliminating senescent cells (they are high in p16INK4a) prevents (in young mice) and partially reverses (in older mice) some of the signs of aging such as cataracts, and loss of adipose tissue and skeletal muscle mass.
In humans, deletions and other mutations of p16INK4a are found in a variety of cancers.
Loss Of Heterozygosity (LOH)
Because tumor suppressor genes are recessive, cells that contain one normal and one mutated gene — that is, are heterozygous — still behave normally. (Exception: one X-linked tumor suppressor gene [WTX] has been found. In males, having only one X chromosome, a damaging point-mutation in WTX or its deletion is all that is needed to eliminate tumor-suppression. Females are also at risk if the mutation or deletion occurs on the X chromosome that is not inactivated.)
However, there are several mechanisms which can cause a cell to lose its normal gene and thus be predisposed to develop into a tumor. These may result in a "loss of heterozygosity" or "LOH".
Mechanisms of LOH:
1. Deletion of
• the normal allele;
• the chromosome arm containing the normal allele;
• the entire chromosome containing the normal allele (resulting in aneuploidy).
2. In females, X-inactivation of the X chromosome carrying the normal allele.
3. Loss of the chromosome containing the normal allele followed by duplication of the chromosome containing the mutated allele.
4. Mitotic recombination. The study of tumor suppressor genes revealed (for the first time) that crossing over — with genetic recombination — occasionally occurs in mitosis (as it always does in meiosis).
In #3 and #4, the resulting cell now carries two copies of the "bad" gene. This is called "reduction to homozygosity".
LOH can work both ways.
When LOH occurs by mitotic recombination (process #4 above), one daughter cell becomes homozygous for the mutant allele but the other becomes homozygous for the normal ("wild-type") allele. This is of no help when tumor-suppressor genes are involved but in other situations it can be.
A rare skin disease of humans called ichthyosis with confetti is an example. It is caused by the inheritance of a dominant mutation in one of the keratins (which make intermediate filaments). At birth, infants with the disease have uniformly reddened skin whose cells are heterozygous for the dominant mutant allele and a normal keratin allele. But as the years go by, an increasing number of patches of normal, whitened, skin appear (like "confetti"). Genomic analysis reveals that each patch develops from a skin stem cell that by mitotic recombination has undergone "reduction to homozygosity". In this case, the daughter cell that inherits two normal keratin alleles goes on to generate a patch of normal skin. This work is described in Choate, K.A., et al., Science 330:94-97 (1 October 2010).
Mutation is not the only way to inactivate tumor suppressor genes.
Their function can also be blocked by methylation of their promoter.
Cancer cells often contain a methylated promoter on one tumor suppressor gene accompanied by
• a similarly blocked promoter on the other allele (producing the same effect as #2 above);
• a loss of that locus on the other chromosome (like the LOH in #1 above);
• an inactivating mutation in the other allele.
Tumor suppressor genes = anti-oncogenes
Genes like RB and p53 are also called anti-oncogenes. They were first given this name because they reverse, at least in cell culture, the action of known oncogenes. This image (courtesy of Moshe Oren, from Cell 62:671, 1990) shows petri dishes which were seeded with the same number of mouse cells that had been transformed by two oncogenes: myc and ras. Many of those on the left have grown into colonies of cells. However, the cells plated on the right also contained the tumor suppressor p53 gene. Only a few have been able to grow into colonies.
Human Papilloma Viruses (HPV)
The name anti-oncogene may be even more appropriate than originally thought. Both the Rb protein and the p53 protein turn out to complex directly in the cell with a gene product of some human papilloma viruses.
Once inside the cells of their host, these viruses synthesize a protein designated E7 and another designated E6.
Of the >30 strains of HPV that infect humans, several, especially HVP-16 and HPV-18, have been implicated as a risk factor for cervical cancer and also cancers of the throat. Their E7 protein binds to the Rb protein preventing it from binding to the host transcription factor E2F.
Result: E2F is now free to bind to the promoters of genes (like c-myc) that cause the cell to enter the cell cycle (right). Thus this version of E7 is an oncogene product.
The E6 protein binds the p53 protein targeting it for destruction by proteasomes and thus removing the block on the host cell's entering the cell cycle.
Although the figure shows the "off" promoters as empty, it is now clear that being "off" involves both
• the absence of activators of transcription and
• the presence of repressors of transcription.
A cell cannot remain in G0 of the cell cycle without these repressors. Perhaps mutant versions of them are another cause of cancer (cancer cells are never in G0). | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.04%3A_Tumor_Suppressor_Genes.txt |
BCL-2 is a human proto-oncogene located on chromosome 18. Its product is an integral membrane protein (called Bcl-2) located in the membranes of the endoplasmic reticulum (ER), nuclear envelope, and in the outer membranes of mitochondria. The gene was discovered as the translocated locus in a B-cell leukemia (hence the name). This translocation is also found in some B-cell lymphomas.
In the cancerous B cells, the portion of chromosome 18 containing the BCL-2 locus has undergone a reciprocal translocation with the portion of chromosome 14 containing the antibody heavy chain locus. This t(14;18) translocation places the BCL-2 gene close to the heavy chain gene enhancer. This enhancer is very active in B cells (whose job it is to synthesize large amounts of antibody). So it is not surprising to find that the Bcl-2 protein is expressed at high levels in these t(14;18) cells.
What makes BCL-2 a proto-oncogene?
B cells, like all activated lymphocytes, die a few days after they have had a chance to do their job. This ensures that they do not linger around after the threat has been dealt with and turn their attack against self components. Aging B cells kill themselves by apoptosis. However, high levels of the Bcl-2 protein protect the cells from early death by apoptosis. The Bcl-2 protein suppresses apoptosis by preventing the activation of the caspases that carry out the process. So genes encoding inhibitors of apoptosis must be added to the list of genes that can act as oncogenes. In this case the effect is not achieved by increasing the rate of cell proliferation but by reducing the rate of cell death.
Although the t(14:18) translocation is found in B-cell lymphomas and leukemias, something else must contribute to creating the cancer because over 50% of us have small numbers of B-cells with that translocation that never progress to cancer. The antibody gene loci are dangerous places for proto-oncogenes to take up residence. Translocation of the proto-oncogene c-myc close to the enhancer of the antibody heavy chain genes also produces cancerous B cells resulting in Burkitt's lymphoma. The translocation of the BCL-2 locus is just one of many mutations that can give rise to a malignant clone of B cells. All of the resulting leukemias are designated chronic lymphocytic leukemia or CLL.
12.06: Burkitt's Lymphoma
Burkitt's lymphoma is a solid tumor of B lymphocytes, the lymphocytes that the immune system uses to make antibodies. The genes for making antibodies are located on chromosomes 14 (the heavy [H] chains), 2 (kappa light chains), and 22 (lambda light chains). These genes are expressed only in B lymphocytes because only B cells have the necessary transcription factors for the promoters and enhancers needed to turn these antibody genes "on". In most (approximately 90%) of the cases of Burkitt's lymphoma, a reciprocal translocation (designated t(8;14) has moved the proto-oncogene c-myc from its normal position on chromosome 8 to a location close to the enhancers of the antibody heavy chain genes on chromosome 14.
In all the other cases, c-myc has been translocated close to the antibody genes on chromosome 2 or 22. In every case, c-myc now finds itself in a region of vigorous gene transcription, and it may simply be the overproduction of the c-myc product (a transcription factor essential for mitosis of mammalian cells) that turns the lymphocyte cancerous. Uncontrolled mitosis of this cell results in a clone of cancer cells, Burkitt's lymphoma. Many other human cancers involve chromosome aberrations, such as translocations, at the loci of known proto-oncogenes.
Figure \(2\) is an actual karyotype (courtesy of Janet Finan and C. M. Croce) of a cell from the tumor of a patient with Burkitt's lymphoma. The long (q) arm of the resulting chromosome 8 is shorter (8q) than its normal homologue; the long arm of translocated chromosome 14 longer (14q+). The heavy chain gene locus on chromosome 14 is a dangerous place. Several other proto-oncogenes produce cancerous B cells — leukemias, lymphomas, and multiple myelomas — when translocated into this locus. The risk of translocations involving the heavy chain gene locus is probably especially high because breaks in its DNA occur naturally during the synthesis of antibodies. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.05%3A_BCL-2.txt |
Leukemia is an uncontrolled proliferation of one kind of white blood cell (or leukocyte). Like all cancers (probably), all the leukemic cells are descended from a single cell that lost the ability to maintain normal control over the cell cycle. There are a number of types of leukemia, as you would expect from the number of types of white blood cells (5) and the number of stages they pass through as they mature. One of the most common is chronic myelogenous leukemia or CML.
Chronic Myelogenous Leukemia (CML) arises in a bone marrow stem cell that is the precursor to all the types of blood cells. However, it usually affects the so-called myeloid lineage (hence the name) that produces granulocytes and macrophages. As the name suggests, the disease often exists for years with only moderately elevated numbers of leukemic cells (descended from the stem cells) and few symptoms. At some point, however, the patient goes through a "blast crisis" when the leukemic granulocyte-macrophage progenitors begin to divide by themselves — increasing their numbers enormously while failing to continue their differentiation.
The Philadelphia Chromosome (Ph1)
In most cases of CML, the leukemic cells share a chromosome abnormality not found in any nonleukemic white blood cells, nor in any other cells of the patient's body. This abnormality is a reciprocal translocation between one chromosome 9 and one chromosome 22. This translocation is designated t(9;22). It results in one chromosome 9 longer than normal and one chromosome 22 shorter than normal. The latter is called the Philadelphia chromosome and designated \(Ph^1\).
The DNA removed from chromosome 9 contains most of the proto-oncogene designated c-ABL. The break in chromosome 22 occurs in the middle of a gene designated BCR. The resulting Philadelphia chromosome has the 5' section of BCR fused with most of c-ABL.
The micrograph in Figure \(2\) uses fluorescence in situ hybridization (FISH) to reveal the ABL DNA (red) and the BCR DNA (green) in the interphase nuclei of the leukemic cells of a patient with CML. The red dot at left center reveals the location of ABL on the normal chromosome 9; the green dot (top center) shows BCR on the normal chromosome 22. The combined dots (red + green = yellow) at the lower right reveal the fused BCR-ABL gene on the Philadelphia chromosome. Figure 12.7.3 is a schematic which can help you interpret the micrograph.
Transcription and translation of the hybrid BCR-ABL gene produces an abnormal ("fusion") protein that activates constitutively (all the time) a number of cell activities that normally are turned on only when the cell is stimulated by a growth factor, such as platelet-derived growth factor (PDGF).
This unrestrained activation increases the rate of mitosis and protects the cell from apoptosis. The outcome is an increase in the number of Ph1-containing cells. During the chronic phase of the disease, these are still able to exit the cell cycle and to differentiate into mature cells that perform their normal functions. At some point, however, another mutation in a proto-oncogene (RAS, for example) or in a tumor-suppressor gene (p53, for example), will occur in one of these cells. The additional mutation causes the rate of mitosis in that cell and its descendants to rise sharply. The daughter cells fail to differentiate and the patient enters the crisis phase of the disease.
A Promising Treatment
Until recently, the only successful treatment of CML was to destroy the patient's bone marrow and then restore blood-cell production by infusing stem cells from the bone marrow of a healthy donor. But now treatment with the drug imatinib mesylate (Gleevec® also known STI571) appears to be able to cure the disease. This molecule fits into the active site of the ABL protein preventing ATP from binding there. Without ATP as a phosphate donor, the ABL protein cannot phosphorylate its substrate(s). A phase 2 study, found that almost 90% of the CML patients treated with the drug showed no further progression of their disease.
Gleevec also shows promise against one type of stomach cancer (gastrointestinal stromal tumors = GIST), which is a life-threatening excessive production of eosinophils. In this disease, Gleevec inhibits a different overactive tyrosine kinase. This one also results from the fusion of parts two different genes (because of the deletion of the DNA between them):
• the first 233 codons of a gene designated FIP1L1 fused to
• the final 523 codons of the gene (PDGFRα) encoding the tyrosine kinase domain of a receptor for platelet-derived growth factor. The fusion protein produced, like BCR-ABL, is hyperactive. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.07%3A_Chronic_Myelogenous_Leukemia_%28CML%29.txt |
Once a nest of cancer cells reaches a certain size (1–2 mm in diameter), it must develop a blood supply in order to grow larger. Diffusion is no longer adequate to supply the cells with oxygen and nutrients and to take away wastes. Cancer cells (probably like all tissues) secrete substances that promote the formation of new blood vessels — a process called angiogenesis. Over a dozen substances have been identified that promote angiogenesis. A few examples are angiopoietin-1, the basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF).
Curiously, some tumors also secrete substances that inhibit angiogenesis. This explains a clinical phenomenon that has been known for decades:
• A patient has a tumor, the so-called primary tumor.
• There is no evidence that the primary tumor has metastasized.
• A surgeon removes the primary tumor.
• Some weeks later metastases of the tumor appear throughout the patient's body
• The speed of their appearance indicates that they were present all along, but too small to be detected.
This phenomenon caused Dr. Judah Folkman of Children's Hospital and the Harvard Medical School in Boston to hypothesize that a large primary tumor secretes not only stimulators of its own angiogenesis but angiogenesis inhibitors that are released into the circulation and inhibit angiogenesis — and thus further growth — of any metastases of the primary tumor. A number of inhibitors of angiogenesis have been discovered.
Angiostatin
Angiostatin is a polypeptide of approximately 200 amino acids. It is produced by the cleavage of plasminogen, a plasma protein that is important for dissolving blood clots. Angiostatin binds to subunits of ATP synthase exposed at the surface of the cell embedded in the plasma membrane. (Before this recent discovery, ATP synthase was known only as a mitochondrial protein.)
Endostatin
Endostatin is a polypeptide of 184 amino acids. It is the globular domain found at the C-terminal of Type XVIII (18) collagen (a collagen found in blood vessels) cut off from the parent molecule.
Effects of angiostatin and endostatin in mice
Injections of angiostatin inhibit the metastasis of certain (mouse) primary tumors. Injections of endostatin (made by recombinant DNA technology) cause the primary tumor to regress. In time, the primary tumor reappears, but a repeat injection causes it to regress again. Each time it reappears, the tumor is just as susceptible to treatment as before. After a few cycles of growth, treatment, and regression, the primary tumor finally stops growing (at least in the cases examined) and remains dormant at a small size.
Some human tumors can be grown in immunodeficient mice. (Being immunodeficient, they cannot reject this foreign tissue). Treatment with endostatin caused these human tumor masses to shrink in their mouse host. Combined treatment with both angiostatin and endostatin has caused some primary mouse tumors to disappear entirely.
This is not seen with conventional chemotherapy. Repeated exposure to chemotherapeutic drugs selects for the appearance of drug-resistant tumor cells. Eventually, further drug treatment is worthless. Why the difference in response? Chemotherapy works directly on tumor cells which mutate easily. Angiogenesis inhibitors don't work on the tumor cells but on normal cells involved in the formation of blood vessels.
Other Angiogenesis Inhibitors
Epithelial cells express transmembrane proteins on their surface — called integrins — by which they anchor themselves to the extracellular matrix. It turns out that the new blood vessels in tumors express a vascular integrin — designated alpha-v/beta-3 — that is not found on the old blood vessels of normal tissues.
Vitaxin®, a humanized monoclonal antibody directed against the alpha-v/beta-3 vascular integrin, shrinks tumors in mice without harming them. In Phase II clinical trials in humans, Vitaxin has shown some promise in shrinking solid tumors without harmful side effects.
What does the future hold for angiogenesis inhibitors?
Clinical trials of endostatin (manufactured by recombinant DNA technology), in combination with standard chemotherapy have shown some benefit in one type of lung cancer.
Bevacizumab (Avastin®). This is a humanized monoclonal antibody that binds to VEGF thus keeping it from binding to its receptors. Approved by the US FDA in February 2004 for the treatment of colorectal cancers.
Ranibizumab (Lucentis®) is a modified version of Avastin® that is showing great promise in inhibiting the formation of new blood vessels in the retina — the cause "wet" macular degeneration.
Trials are also scheduled to begin on a synthetic ribozyme that blocks synthesis of the VEGF receptor. These are only a few examples of the ~50 antiangiogenesis drugs now in clinical trials.
But proceed with caution.
In animal studies, some cancers — notably pancreatic cancer — have turned out to resist chemotherapy because of their poor blood supply. Perhaps such cancers need to have angiogenesis promoted; inhibiting it could make a bad problem worse. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.08%3A_Fighting_Cancer_with_Inhibitors_of_Angiogenesis.txt |
Most cancer patients are treated with some combination of surgery, radiation, and chemotherapy. Radiation and chemotherapy have the disadvantage of destroying healthy as well as malignant cells and thus can cause severe side-effects. What is needed are more precisely-targeted therapies. One long-held dream is that the specificity of immune mechanisms could be harnessed against tumor cells. This might use the patient's own immune system or the transfer of antibodies or T cells from an outside source (i.e. passive immunization). Ideally, these agents would be targeted to molecules expressed on the cancer cells but not on healthy cells. However, such tumor-specific antigens have been hard to find, and so many of the immune agents now in use do target healthy cells as well.
Immunostimulants
There is considerable evidence that cancer patients have T cells that are capable of attacking their tumor cells. In fact, it may be that the appearance of cancer is a failure of immune surveillance: the ability of one's own immune system to destroy cancer cells as soon as they appear. But what to do if they fail? Immunostimulants are nonspecific agents that tune-up the body's immune defenses. There have been some successes with
• injecting adjuvant-like agents directly into the tumor. The only one that succeeds often enough to remain in use is the bacterial preparation BCG. Introduced into the bladder, it can help eradicate early-stage bladder tumors.
• Oral therapy with levamisole, a drug widely-used for deworming (people as well as animals), has been used to treat a variety of cancers but with inconsistent results.
• interleukin-2 (IL-2), a potent growth factor for T cells;
• alpha-interferon (IFN-α)
Cancer Therapy with Monoclonal Antibodies
A number of monoclonal antibodies show promise against cancer, especially cancers of white blood cells (leukemias, lymphomas, and multiple myeloma). Some examples:
• Rituximab (trade name = Rituxan®). Used to treat B-cell lymphomas. The CD20 molecule to which it binds is present on most B-cells, healthy as well as malignant, but over the months following treatment, new healthy B cells are formed from precursors that do not have CD20 and thus were not destroyed by the treatment.
• Trastuzumab (trade name = Herceptin®). Binds HER2, a growth factor receptor found on some tumor cells (some breast cancers, lymphomas). The only monoclonal so far that seems to be effective against solid tumors.
• Alemtuzumab (MabCampath®). Binds to CD52, a molecule found on white blood cells. Has produced remission of chronic lymphocytic leukemia.
• Lym-1 (Oncolym®). Binds to the HLA-DR-encoded histocompatibility antigen that can be expressed at high levels on lymphoma cells.
• Bevacizumab (Avastin®). Binds to vascular endothelial growth factor (VEGF) thus blocking its action and depriving the tumor of its blood supply.
• Cetuximab (Erbitux®). Used to treat colorectal cancers.
• A monoclonal antibody against CD47. CD47 is a cell-surface protein expressed at high levels in many different human cancers. CD47 blocks any effort that macrophages and dendritic cells might make to phagocytose the cancer cells; that is, CD47 is a "don't eat me" signal. A variety of human cancers transplanted into immunodeficient mice have their growth suppressed and metastases prevented when the mice are given a monoclonal antibody against CD47 thus unleashing the ability of phagocytes to destroy the cancer cells. The success in mice will soon lead to clinical trials in humans.
• Ipilimumab (Yervoy®). Unlike the other monoclonals listed here, ipilimumab acts as an immunostimulant. It does so by binding to the CTLA-4 molecules on the T cell so that they cannot bind to the B7 molecules on the antigen-presenting cell. This frees the T cell's CD28 molecules to bind B7 thus receiving the stimulatory "signal 2" from the antigen-presenting cell. Ipilimumab was approved by the U.S. Food and Drug Administration on 25 March 2011 for use against metastatic melanoma. Because its double-negative effect works to enhance the body's overall T-cell responses, it may well turn out to be useful against other cancers as well (and explains some of the autoimmune-like side effects it produces). It also provides perhaps the best evidence yet for the existence of immune surveillance; that is, the presence in the patient's body of an innate population of T cells specific for the tumor.
• Pembrolizumab and nivolumab
• Blinatumomab is a synthetic monoclonal antibody each arm of which carries a binding site with a different specificity:
• one arm binds to CD19, an antigen found on the surface of B cells and B-cell lymphomas
• the other arm binds to CD3, a cell-surface molecule on T cells, including cytotoxic T lymphocytes (CTLs)
By forming a bridge between CD3 and CD19, blinatumomab is able to attach T cells to B cells and activate the T cells to kill the B cells. Early clinical trials with blinatumomab on a small number of patients appear quite promising. Modest doses of the drug produced partial and, in a few cases, complete regression of their lymphoma.
Immunotoxins
A major problem with chemotherapy is the damage the drugs cause to all tissues where rapid cell division is going on. What is needed is a "magic bullet", a method of delivering a cytotoxic drug directly and specifically to tumor cells, sparing healthy cells. Such a magic bullet would have two parts - a monoclonal antibody specific for the cancer cell attached to a cytotoxic drug or toxin that kills the cell once it gets inside.
Some two dozen immunotoxins are in clinical trials. Two that have already received FDA approval:
1. Adcetris®. The vedotin is attached to the monoclonal antibody by a bridge that is cleaved once the conjugate is safely inside the tumor cell releasing the toxin to do its work there. In one trial, 73% of the patients with Hodgkin's lymphoma went into remission.
• a monoclonal antibody that binds CD30, a cell-surface molecule expressed by the cells of some lymphomas but not found on the normal stem cells needed to repopulate the bone marrow.
• vedotin, a drug that blocks mitosis by preventing the polymerization of tubulin (needed to form the mitotic spindle).
2. Kadcyla® The DM1 is attached to the monoclonal antibody by a bridge that is cleaved once the conjugate is safely inside the tumor cell releasing the toxin to do its work there. Kadcyla® prolongs survival in women whose breast cancer over-expresses HER2 (about 20% of breast cancer cases).
• Trastuzumab (Herceptin®), the monoclonal antibody against HER2 listed above;
• DM1, another drug that inhibits mitosis by preventing the polymerization of tubulin (needed to form the mitotic spindle).
Radioimmunotherapy
Monoclonal antibodies against tumor antigens can also be coupled to radioactive atoms. The goal with these agents is to limit the destructive power of radiation to those cells (cancerous) that have been "fingered" by the attached monoclonal antibody. Examples:
• Zevalin®. This is a monoclonal antibody against the CD20 molecule on B cells (and lymphomas) conjugated to either
• the radioactive isotope indium-111 (111In) or
• the radioactive isotope yttrium-90 (90Y)
Both are given to the lymphoma patient, the 111In version first followed by the 90Y version (in each case supplemented with Rituxan®).
• Bexxar® (tositumomab). This is a conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131 (131I). It, too, is designed as a treatment for lymphoma. Although both Bexxar® and Zevalin® kill normal B cells, they don't harm the B-cell precursors because these do not express CD20. So, in time, the precursors can repopulate the body with healthy B cells.
On 3 February 2005, the New England Journal of Medicine reported that 59% of patients with a B-cell lymphoma were disease-free 5 years after a single treatment with 131I-tositumomab (a treatment that was relatively free of the nasty side-effects, e.g., hair loss, of conventional chemotherapy).
Adoptive Cell Therapy (ACT)
Tumor destruction is done by cells. Antibodies may help, but only by identifying the cells to be destroyed, e.g., by macrophages. But T cells, e.g., cytotoxic T lymphocytes (CTL), are designed to destroy target cells. What about enlisting them in the fight?
Tumor-Infiltrating Lymphocytes (TIL)
Solid tumors contain lymphocytes that are specific for antigens expressed by the tumor. For many years, Steven A. Rosenberg and his associates at the U. S. National Cancer Institute have tried to enlist these cells in cancer therapy.
On September 19, 2002, he reported his most promising results at that time. The procedure:
• Isolate T cells — both CD4+ T-helper cells and CD8+ cytotoxic T lymphocytes (CTL) from samples of the tumor (melanoma)
• Test them in vitro to find the most efficient killers of the melanoma cells.
• Grow large numbers of them in culture (using the powerful T-cell growth factor IL-2).
• Treat the patient with modest doses of cytotoxic drugs to reduce — but not destroy — the bone marrow (called nonmyeloablative conditioning).
• Reintroduce the mix of Th cells (CD4+) and CTL (CD8+) into the patient (along with IL-2).
The results:
• The infused cells usually took up long-term residence.
• In 10 of 13 patients, their melanoma cells — including all metastases — regressed either partially or completely.
In a few cases, the TIL seemed to be reacting to tumor-specific antigens, but in most the target seems to have been antigens expressed by all melanin-containing cells. Evidence:
• Four patients lost normal melanocytes from their skin leaving white patches.
• One patient developed inflammation of the uvea, the coat of melanin-containing cells within the eye.
Adoptive transfer of a clone of the patient's own tumor-antigen-specific T cells
The 19 June 2008 issue of the New England Journal of Medicine (Naomi Hunder et al) carried a report describing the successful treatment of a man with metastasized melanoma using his own T cells. The procedure:
• His leukocytes were harvested and a mixed culture was prepared containing
• antigen-presenting dendritic cells.
• a peptide from the antigen NY-ESO-1. NY-ESO-1 is a protein that is produced by several types of tumors (e.g., melanoma, lung and breast cancers) but is not expressed by normal cells (except those in the testis).
• The patient's own T cells.
• After repeated stimulation with the antigen, responding cells were cloned by limiting dilution.
• One (of four) antigen-reactive cells was then expanded in culture until
• 5 billion (5 x 109) identical anti-NY-ESO-1 CD4+ T cells were available to infuse into the patient.
The result: complete regression of each metastatic clump of melanoma cells, and the patient has remained free of this lethal cancer for two years since this treatment.
Adoptive transfer of genetically-modified T cells
Genetically engineered with a T-cell Receptor
On April 20, 2006, the Rosenberg group reported some success with melanoma patients using a modification of the TIL procedure.
• The patient's T cells were removed and treated with a retroviral vector containing the αβ T-cell receptor (TCR) specific for a melanoma antigen.
• Large numbers of these were grown in culture.
• After nonmyeloablative conditioning to "make room" for them, the genetically-modified lymphocytes were infused into the patient.
• This application of gene therapy succeeded in eliminating the metastases and providing a disease-free period of two years in two patients.
Genetically engineered with a Chimeric Antigen Receptor (CAR)
The 10 August 2011 online version of the New England Journal of Medicine carried a report by Porter, D., et al. on their results with one (of three) patients treated for chronic lymphocytic leukemia (CLL) with an infusion of his own genetically-modified T cells.
The patient's malignant B cells expressed the surface antigen CD19 just as normal B cells do.
T cells were harvested from his blood and later treated with a vector encoding the antigen-binding site of an anti-CD19 antibody along with two other costimulatory molecules. The result: some 5% of these T cells expressed this synthetic antibody (called a chimeric antigen receptor or CAR) and were activated when they bound CD19 with it (rather than with their T cell receptor (TCR) which they would normally use).
Injected back into the patient, they proliferated by some 1000-fold and persisted for months. During this period, they eliminated all his malignant B cells (as well as his normal B cells). At the time of the report (10 months after treatment), he continued to be free of his cancer. Lacking normal B cells as well, he needed periodic infusions of immune globulin to keep infections at bay.
"One swallow does not make a summer", but these results give hope that in time immunotherapy will become an effective weapon against cancer.
Cancer Vaccines
Any response of the patient's own immune system – immune surveillance – has clearly failed in cancer patients. The purpose of cancer vaccines is to elicit a more powerful active immunity in the patient. Several approaches are being explored.
Patient-Specific Cancer Vaccines
Patient-Specific Dendritic-Cell Vaccines
Dendritic cells are the most potent antigen-presenting cells. They engulf antigen, process it into peptides, and "present" these to T cells.
To make a dendritic-cell vaccine,
• Harvest dendritic cells from the patient.
• Expose these in vitro to antigens associated with the type of tumor in the patient.
• The antigens are found in normal – as well as cancerous – cells of that tissue (e.g., tyrosinase in melanocytes, prostatic acid phosphatase [PAP] in prostate cells).
• They may be fused with a stimulatory molecule such as granulocyte-macrophage colony-stimulating factor (GM-CSF)
• Inject these "pulsed" dendritic cells back into the patient.
• Hope that they elicit an strong cell-mediated immune response, e.g. by cytotoxic T lymphocytes (CTL).
On 29 April 2010 the U.S. Food and Drug Administration approved the first anti-cancer vaccine: a patient-specific dendritic-cell vaccine for use against advanced prostate cancer. The vaccine, called sipuleucel-T (Provenge®), is produced by pulsing the patient's dendritic cells with a fusion protein coupling prostatic acid phosphatase [PAP] with GM-CSF.
Patient-Specific Tumor-Antigen Vaccines
The antigens in these vaccines are taken from the patient's own tumor cells.
• Harvest some tumor cells from the patient.
• Ship them to a company that will use them to make complexes with adjuvant materials.
• The complexes are returned to be injected into the patient.
Several of such vaccines are currently in clinical trials.
Tumor-Antigen-Specific Vaccines
These vaccines are used to immunize the patient with an antigen universally expressed by tumors of that type (but not by normal cells) mixed with some form of adjuvant that will enhance the response.
Examples:
• Many cancer patients mount an immune response — both antibody-mediated and cell-mediated — against the tumor (and testis) antigen NY-ESO-1. Deliberate immunization with this protein (plus an adjuvant) boosts this response and has shown some promise in early clinical trials. (Cells in the testis do not express HLA antigens, so are not at risk from attack by NY-ESO-1-specific cytotoxic T lymphocytes).
• MAGE-A3 is another protein common on cancer cells. A vaccine using MAGE-A3 — along with an adjuvant — is in Phase III clinical trials to assess its effectiveness against melanoma and lung cancer.
• HER2 is a protein over-expressed on 20–30% of breast cancers. NeuVax® is a vaccine that contains a peptide of HER2 along with recombinant GM-CSF as an adjuvant. It stimulates the formation of cytotoxic T lymphocytes (CTLs) that attack cells expressing HER2 and has shown promise in clinical trials.
Unlike patient-specific vaccines, these vaccines can be mass-produced for use in anyone with the appropriate tumor.
Combining Procedures #3 and #4
While tumors are immunogenic in the patient who carries them, they are only weakly so. In the hopes of improving cancer immunotherapy, clinical trials are now proceeding to test the efficacy of combining potent patient-specific cancer immunization with treating the patient with large numbers of cultured cancer-antigen-specific T cells that result.
The patient is repeatedly immunized with his or her own cancer cells along with a strong adjuvant (e.g., GM-CSF) followed by harvesting the patient's leukocytes and growing large numbers of them in the laboratory before infusing them into the patient along with interleukin-2. This combined approach — which generates large numbers of patient-cancer-specific killer T cells — has been tested against kidney and one type of brain cancer with promising results.
Blood Cancers
Cancers of blood cells, leukemias and lymphomas, arise in the bone marrow — the source of all blood cells.
One approach to curing leukemia is to treat the patient with such high doses of chemotherapy and radiation that not only are the leukemic cells killed, but the patient's bone marrow is destroyed. If the patient is to survive the treatment, called "myeloablative conditioning", he or she must be given a transplant of hematopoietic stem cells — the cells from which all blood cells are formed.
The stem cells can be
• an autograft; that is, from bone marrow harvested from the patient and stored before treatment begins. In this case, however, the marrow must also be treated to purge it of all cancer cells it may contain before it is returned to the patient. This sometimes fails.
• an allograft; that is, cells harvested from another person, usually a family member sharing the same major histocompatibility molecules.
Allografted hematopoietic stem cells also sometimes fail to cure, but in that case it is because not all of the patient's leukemic cells were destroyed. However, an infusion of T lymphocytes from the blood of the same donor that provided the cells can finish off the job.
This effect is called the graft-versus-leukemia effect.
However, most (if not all) of the donor T cells are probably attacking normal cell surface molecules, not tumor-specific ones. (Even if the donor and recipient are matched for the major histocompatibility molecules, there will be minor ones that elicit a rejection response.)
So the patient may also suffer life-threatening graft-versus-host disease (GVHD).
The graft-versus-leukemia effect lays the foundation for an approach that has shown considerable promise against various blood cancers and even some solid (e.g., kidney) tumors.
• The patient is treated to kill some — but not all — of the bone marrow cells (nonmyeloablative conditioning).
• Instead of using high doses of radiation to the entire body and chemotherapy, only the lymphoid organs (spleen, thymus, lymph nodes) are irradiated (called "total lymphoid irradiation").
• Antithymocyte globulin can also be given.
• Even though this leaves some cancer cells, it makes it possible for allogeneic bone marrow stem cells to take up long-term residence in the recipient (just as immunosuppression allows kidney transplants, etc. to avoid rejection by the recipient).
• This is followed by an infusion of T cells from the same donor. These can then go to work against the cancer cells without being threatened with rejection by the host.
• Once again, though, they will also attack normal cells of the recipient usually causing graft-versus-host disease (GVHD). However, this promises to be milder than that following myeloablative conditioning — perhaps because repeated small doses of radiation favors the survival of natural killer (NK) cells, and these appear to protect against GVHD.
In mice, the graft-versus-leukemia effect can be enjoyed without the downside of GVHD by including extra-large numbers of regulatory T cells (Treg cells) in the bone marrow infusion. Whether this approach could be helpful for humans remains to be seen.
Virotherapy
It has long been known that viral infections can occasionally (and unpredictably) cause tumors to regress. A number of viruses have been studied in the hope of developing a reliable therapy. On 27 October 2015, the U.S. FDA approved T-VEC (Imlygic®) for the treatment of melanoma. T-VEC is a mutated and engineered Herpes Simplex Virus (HSV-1 — the cause of cold sores). The alterations in the virus include incorporating the gene for GM-CSF and a mutation that prevents the virus from infecting non-dividing cells while preserving its ability to infect and replicate in cancer cells. Replication kills the cells and causes them to release:
• more viruses which spread the infection;
• tumor antigens, and
• GM-CSF which attracts dendritic cells to the site. These take up the tumor antigens and present them to T cells that go on to mount an attack against surviving tumor cells.
(Tumor cell death by HSV does not qualify it as immunotherapy, but the T-cell response that results certainly does.) | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.09%3A_Immunotherapy_of_cancer.txt |
As described by Bruce N. Ames, Professor of Biochemistry & Molecular Biology, Director of the National Institute of Environmental Health Sciences Centerm, University of California at Berkeley
Aging is in good part due to the oxidants produced as by-products of normal metabolism. These oxidants, such as superoxide and hydrogen peroxide, are the same mutagens produced by radiation, and cause damage to DNA, proteins, and lipids.
The DNA in each cell of a normal rat receives on average about 100,000 oxidative lesions per day. DNA repair enzymes constantly remove this damage, but they do not keep up: a young rat has about one million oxidative lesions in the DNA of each cell, which increases to about two million in an old rat. A human cell receives about ten times less damage than a rat cell, in agreement with the higher cancer rate and shorter lifespan of a rat.
The degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, and brain dysfunction, are increasingly found to have, in good part, an oxidative origin. It is argued that dietary antioxidants, such as Vitamins C and E and carotenoids, play a major role in minimizing this damage and that most of the world's population is receiving inadequate amounts of them, at a great cost to health.
The main source of dietary antioxidants is fruits and vegetables. Humans should eat 5 portions of fruits and vegetables per day, yet only 9% of the U.S. population eats that much. Epidemiological studies show that the incidence of most types of cancer is double among people who eat few fruits and vegetables as compared to those who eat about five portions per day. Considerable evidence indicates that oxidative damage is important in cardiovascular disease, cataracts, and brain and immune system dysfunction, and that adequate dietary antioxidants can minimize their incidence.
Men with low Vitamin C intake have low vitamin C in their seminal fluid and much more oxidative damage to the DNA in their sperm. Male smokers are particularly at risk as they have depleted antioxidant pools (cigarette smoke is extremely high in oxidants). A smoker must eat two to three times as much Vitamin C as a non-smoker to maintain an equal plasma level, yet smokers tend to eat worse diets than non-smokers. Indeed, male smokers have a considerably higher risk of having children with birth defects and childhood cancer.
The three main causes of cancer are smoking, dietary imbalances (excess fat and calories; inadequate intake of fruits, vegetables, fiber, and calcium), and chronic infections leading to chronic inflammation (hepatitis B and C viruses, Helicobacter pylori infection, schistosomiasis, etc.). Chronic inflammation is a major cause of cancer in the world because it releases powerful oxidants which both stimulate cell division and are mutagens.
Past occupational exposures might cause about 2% of current human cancer, a major part being asbestos exposure in smokers, and industrial or synthetic chemical pollution causes less than 0.1 %, in my view. The age-adjusted cancer death rate in the U.S. for all cancers combined (excluding those attributable to smoking) has been remaining steady since 1950, while life expectancy increases every year.
We are the healthiest we have ever been in human history.
Two factors are critical in the formation of mutations: lesions in DNA, formed when DNA is damaged, and cell division, which converts DNA lesions to mutations. Agents increasing either lesions or cell division increase mutations and as a consequence increase cancer incidence. Hormones stimulating cell division increase cancer incidence (e.g., levels of estrogen in breast cancer and testosterone in prostate cancer); hormones may be a risk factor in about 20% of human cancer.
Animal cancer tests, which are done at the maximum tolerated dose (MTD), are being misinterpreted to mean that low doses of the chemicals tested and found positive are thereby relevant to human cancer. Animal cancer tests are mainly done on synthetic chemicals and industrial pollutants, yet half of all natural chemicals that have been tested at the MTD are rodent carcinogens.
It is argued that the explanation for the high frequency of positive results in animal cancer tests is that high dose animal cancer tests are mainly measuring increases in cell division due to cell killing and compensatory cell division; this is a high dose effect that does not occur at low doses.
In any case 99.9% or more of the chemicals we eat are natural. For example, 99.99% of the pesticides we eat are natural chemicals that are present in plants to ward off insects and other predators. More than half of those natural pesticides tested in high dose animal tests are rodent carcinogens. There are about 10,000 or so different natural pesticides in our diet, and they are usually present at enormously higher levels than synthetic pesticides.
Cooking food also generates thousands of chemicals. There are over 1000 chemicals reported in a cup of coffee. Only 26 have been tested in animal cancer tests and more than half are rodent carcinogens; there are still a thousand chemicals left to test. The amount of potentially carcinogenic pesticide residues consumed in a year is less than the amount known of rodent carcinogens in a cup of coffee.
The reason we can eat the tremendous variety of natural chemical rodent carcinogens in our food is that animals are extremely well defended against all chemicals by many general defense systems. These enzymes, e.g., DNA repair and glutathione transferases which defend against reactive compounds such as mutagens, are all inducible (more of them are made when they are in use). They are equally effective against natural and synthetic reactive chemicals. Thus, animals are extremely well defended against low doses of chemicals. One does not expect, nor does one find, a general difference between synthetic and natural chemicals in their carcinogenicity, and though less well studied, the same would be expected for mutagenicity, teratogenicity, and acute toxicity.
The effort to eliminate synthetic pesticides because of unsubstantiated fears about residues in food will make fruits and vegetables more expensive, decrease consumption, and thus increase cancer rates. The levels of synthetic pesticide residues are trivial in comparison to natural chemicals, and thus their potential for cancer causation is extremely low. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.10%3A_Cancer-_The_Causes_and_Prevention_of_Cancer.txt |
Is there a safe dose of any mutagen or carcinogen?
We live surrounded by radiation and by chemicals that cause mutations in test organisms (like bacteria, yeast, and mice) and cause an increase in the rate of cancers in experimental animals (rats and mice). Is there any safe dose for humans of these agents (which include oxygen!) The question is exceedingly difficult to answer and, I believe, at low doses, unanswerable. Why?
Figure 12.11.1 shows several theoretical dose-response relationships. There is considerable evidence that at moderate doses of a mutagen or carcinogen, the response is linear (A). However, at very low doses of some chemicals, there may be a threshold below which the agent has no effect (B). Many workers believe that for some agents, it is likely that even the tiniest doses will have an effect (C), but the population exposed must be large enough to observe it. This is called the linear no-threshold (LNT) model. Note that even at zero dose, the line does not intercept the origin. This is because even unexposed animals (including people) show a spontaneous level of response (e.g., tumors).
There is also evidence that for some agents in some circumstances, increasing the dose (at relatively low levels) actually reduces the response below control levels (G). This phenomenon is called hormesis. At very high doses, the rate of response may increase faster than the dose (E) as, for example, the probability of a single cell suffering two mutations increases. On the other hand, very high doses may kill off damaged cells before they can develop into tumors (F).
Radiation and cancer
High doses of radiation cause cancer. Various studies, including excellent ones on the survivors of Hiroshima and Nagasaki, show that a population exposed to a dose of 100 millisieverts (mSv) will have a measurable increase (about 1%) in the incidence of cancer. Note that the measurements are made on a population, not on individuals. We can never say that a particular individual exposed to a particular dose of radiation will develop cancer. The induction of cancer is a chance ("stochastic") event unlike the induction of radiation sickness which is completely predictable. The element of chance arises because cancer is an event that occurs in a single cell unlucky enough to suffer damage to several specific genes. However, the energy needed to cause mutations is very low. So if you expose a sufficiently large number of cells to even tiny doses of radiation, some cell is going to be unlucky. How can we evaluate the risk?
Collective Dose
100 mSv causes a 1% increase in cancer in a population; i.e., it should cause an increase of 1 cancer in every 100 people in the exposed population. But if our reasoning is correct, a population of 10,000 people exposed to 1 mSv should also yield one case of radiation-induced cancer. In any population, where the product of radiation dose (in mSv) times population size equals 1 x 104, one case of cancer will be induced. The product of exposure multiplied by the size of the exposed population is known as the collective dose. Its units are (persons)x(mSv).
Example $1$
The population of the U.S. in 2009 was about 305 million. so anything that increases the annual exposure of the U.S. population by as little as 0.01 mSv (a typical chest x ray is 0.02 mSv) per year would cause an additional 305 cases of cancer.
$\dfrac{(305 \times 10^6\, persons)(0.01\, mSv)}{1 \times 10^4\, person\, mSv/cancer} = 305 \, cancers$
But consider:
• The total number of cancer deaths in the United States that year was expected to exceed 560,000.
• How can we possible detect an increase of 305 faced with these large numbers?
Hormesis?
The citizens of Colorado are exposed to background radiation of some 1.8 mSv per year; the figure for Massachusetts is only 1.02 mSv/year.
• If the linear non-threshold model is correct, we would expect to find a higher incidence of cancer in Colorado than in Massachusetts.
• If the background radiation added to other sources keeps both groups below a threshold, then we would expect no difference in cancer incidence.
• If the modest increase in background radiation in Colorado has a protective effect (hormesis), then we would expect that their cancer incidence would be lower than in Massachusetts.
What do we find? In 1999, when adjusted for the age of the population, the incidence of cancer averaged 16% higher in Massachusetts than in Colorado. (If this truly is evidence of hormesis, the mechanism is unknown.)
Some other parts of the world have background radiation levels that dwarf those in Colorado. In some houses in Ramsar, Iran, the inhabitants are exposed to an annual dose of background radiation of as much as 130 mSv per year — over 70 times that in Colorado. Nevertheless, the inhabitants of Ramsar are just as healthy as — or even healthier than — control populations exposed to far lower levels of radiation.
Chernobyl
It has been estimated (in this case, using a collective dose value of 5 x 104 person mSv/cancer) that the radioactive fallout from the nuclear accident at Chernobyl (now often spelled "Chornobyl") in 1986 will cause an increase of 17,000 cancers over the lifetime of people living in the Northern Hemisphere.
Large those this estimate seems, it is dwarfed by the 513 million cancer deaths that will occur anyway in this population. Even among those heavily exposed (rescue workers and people living in the region), the expected death toll from cancer is ~4,000 or only 3% more than their death rate from cancer would have been anyway. This is why I say above that the answer to the question of the dangers of low doses of radiation is unknowable.
As of September 2005, some 4000 children and adolescents who drank milk contaminated by the radioactive iodine [131I] released in the accident had come down with thyroid cancer. In their case, the ability of the thyroid gland to concentrate iodine within its cells resulted in those cells receiving a relatively high, not a low, dose. As of that date, however, only 15 of those cancer patients had died.
Chemicals and cancer: dioxin
At one time it was found that the chemical dioxin, which can be produced as a contaminant in the manufacture of paper and cardboard, was leaching from milk cartons into milk itself.
• the concentration in the milk averaged 0.1 part per trillion (ppt) or 0.0001 µg in a liter (109 µg) of milk. Assuming:
• 0.1 µg per day given to rats increases their rate of tumors by 1%
• the idea of collective dose applies to chemicals (that is, a single molecule in an unlucky cell can turn it cancerous)
• people are 100 times more sensitive to dioxin than rats (probably not true) and
• people are 100 times larger than rats
• we conclude that there is a risk of 10 additional cancers in every million people consuming a liter (about a quart) of milk a day from cardboard containers.
And, in fact, this was the estimate made. The uncertainties in such assumptions helps explain the controversy that has so often swirled around the test data on such chemicals as
• the artificial sweeteners cyclamate and saccharin,
• the pesticide Alar,
• the hydrocarbons in a charcoal-broiled steak,
• the chlorinated compounds in municipal water supplies.
Some chemicals appear to have a safety threshold
Cells have a number of different methods for detoxifying certain types of chemicals. So long as these mechanisms are not overwhelmed, they should provide a threshold of safety. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.11%3A_Estimating_Cancer_Risks.txt |
The LD50 is a standardized measure for expressing and comparing the toxicity of chemicals. The LD50 is the dose that kills half (50%) of the animals tested (LD = "lethal dose"). The animals are usually rats or mice, although rabbits, guinea pigs, hamsters, and so on are sometimes used. In all these tests, the dose must be calculated relative to the size of the animal. The most common units are milligrams of chemical per kilogram of test animal (mg/kg or ppm).
Table \(1\): LD50 values of common drugs
Chemical Category Oral LD50 in Rats (mg/kg)
Aldicarb ("Temik") Carbamate 1
Carbaryl ("Sevin") Carbamate 307
DDT Chlorinated hydrocarbon 87
Dieldrin Chlorinated hydrocarbon 40
Diflubenzuron ("Dimilin") Chitin inhibitor 10,000
Malathion Organophosphate 885
Methoprene JH mimic 34,600
Methoxychlor Chlorinated hydrocarbon 5,000
Parathion Organophosphate 3
Piperonyl butoxide Synergist 7,500
Pyrethrins Plant extract 200
Rotenone Plant extract 60
Table 12.12.1 gives the LD50 values for some insecticides. In each case, the chemical was fed to laboratory rats. Note that the lower the LD50, the more toxic the chemical. Even adjusting for the test animal's weight, the LD50 for one species is often quite different from that for another. Thus any LD50 value gives only a rough estimate of the risk to humans. The way in which the chemical is administered also has a marked effect on LD50 values. The chemical may be fed, injected, applied to the animal's skin, etc., and each method usually generates a different LD50.
Because a single test may kill as many as 100 animals, the United States and other members of the Organization for Economic Cooperation and Development agreed in December 2000 to phase out the LD50 test in favor of alternatives that greatly reduce (or even eliminate) deaths of the test animals.
12.13: Dioxin
Name given members of a family of closely-related chemicals. The term dioxin is often used for one of these: 2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD. This substance was present as a contaminant in the herbicide agent orange, which was so widely used during the Vietnam war.
When ingested or injected, TCDD is extremely poisonous to laboratory animals. At sub-lethal concentrations, it causes cancer and birth defects in them. Exposure to high levels of dioxins causes a severe skin disease (chloracne) in humans as well as damage to the liver and nervous system. While the evidence is still hotly debated, the U.S. Environmental Protection Agency (EPA) is convinced that dioxins cause cancer in humans. They base this conclusion on extrapolating from dose-response studies done in animals (rats) and following the health of industrial workers who were exposed to dioxins in the U.S., Germany, and the Netherlands.
Thanks to the development of delicate analytical techniques, it is possible to detect trace amounts in everyone's blood. Most of us have a few parts per trillion (ppt) of TCDD in our serum. TCDD (and other dioxins) are produced when organic matter is burned. Measurable levels are found in soot from wood-burning stoves and the ash of municipal incinerators. However, the amounts to which we are exposed have dropped some threefold since the mid-80s, and the cancer risk dioxins pose for most of us is probably close to zero.
Dioxin can prevent disease! (in mice). Experimental allergic encephalomyelitis (EAE) is a disease in experimental animals (e.g., mice, guinea pigs) that closely mimics multiple sclerosis, an autoimmune disease of humans in which the myelin sheaths of neurons are destroyed. In the 1 May 2008 issue of Nature, F. J. Quintana and colleagues reported that they could strongly suppress the induction of EAE in mice by pretreating them with 1 µg of TCDD. The protection appeared to be mediated by regulatory T cells (Treg) whose numbers rose sharply following TCDD treatment. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.12%3A_The_LD50_test.txt |
The Background
Late in the 1970s, researchers investigating a cluster of cancers in children in Colorado found an association with living near high-voltage power lines. The cancers, a form of leukemia called acute lymphoblastic leukemia (ALL), had long been associated with exposure to ionizing radiation. But power lines do not generate ionizing radiation. What they do generate is a weak magnetic field that oscillates at the frequency of the alternating current (60 hertz in the U.S.; 50 hertz in Europe). Follow-up studies elsewhere continued to find a weak association between living near power lines and the incidence of ALL in children. Note that the association was with the proximity to power lines; not to the strength of the magnetic fields. The nature of the wiring (e.g., voltage, proximity) was used as a surrogate to the actual agent under suspicion (the magnetic field).
The National Cancer Institute (NCI) Study
On July 3, 1997, The New England Journal of Medicine published the largest and best study of the question (Martha S. Linet, et al, "Residential Exposure to Magnetic Fields and Acute Lymphoblastic Leukemia in Children").
Their conclusion: "Our results provide little support for the hypothesis that living in homes with high time-weighted average magnetic fields or in homes close to electrical transmission or distribution lines is related to the risk of childhood ALL."
How the NCI study differed from earlier studies
The NCI study differed from the earlier studies in 4 important ways:
• It involved a much larger sample size (624 children with ALL and 615 children chosen at random to compare their homes with those of the patients.
• The strength of the magnetic fields in the homes were actually measured (including continuous measurement for 24 hours under the child's bed). They also evaluated the nearby power lines as the earlier studies had done.
• The collection of data was "blinded"; that is, the people doing the measurements did not know whether they were in the house of an ALL patient or in the house of a control.
• The investigators had no axe to grind. None had any connection to the power industry or to grieving parents seeking to find an explanation for the tragedy that had struck their family.
The Magnetic Field Results
Patients and controls were grouped in 7 classes ranging from a magnetic field of less than 0.065 microteslas (µT) to greater than 0.5 µT. The tesla is a unit of magnetic field strength; the earth's magnetic field, which makes a compass needle turn, is about 50 microteslas (but does not fluctuate at 60 hertz as the much smaller fields near alternating current lines do). The Odds Ratio is a calculation of how likely it is that the results for the patient group differ from that of the control group. The total number of patients and controls in each class is shown within each bar. The blue lines show the 95% confidence limits; that is, that there is a 95% probability that the "true" mean (the height of the bar) is somewhere within the range shown in blue.
Interpreting the results
Only one class of exposure (0.400 - 0.499 µT) showed a statistically significant difference between patients and controls. Is it truly significant? Perhaps. But note that only 19 children of the 1,239 enrolled in the study lived in homes with this level of magnetic field.
How do this and the earlier studies meet the 5 standards of epidemiology?
1. High Relative Risk
Not met. Every group but one had a relative risk whose 95% confidence limit included 1.00; that is, no relative risk at all.
2. Consistency
Not met.
• The earlier studies did not measure magnetic fields.
• Most of the earlier studies, which simply evaluated the nature of the nearby power lines, showed a 2–3 fold increase in relative risk whereas this study showed no increase.
3. A graded response to a graded dose
Not met. There is no steady increase in ALL with increasing exposure to magnetic fields. The possible increased risk of ALL with exposure to 0.400 - 0.499 µT is followed by no increase at exposures above 0.5 µT.
4. Temporal relationship
Met. In fact, built into the design of the study. All the patients were selected after they had developed ALL.
5. A plausible mechanism Not met. In vitro studies have failed to reveal any mechanism to explain how such weak magnetic fields could produce oncogenic changes in cells. (Note that the Y axis of this graph of representative magnetic fields is logarithmic: the magnetic field directly under a high-voltage power line is only 1/10 that of the earth's own magnetic field.)
Two papers published in 1992 claimed that weak magnetic fields increase the flow of calcium ions into lymphocytes. Such a response may trigger mitosis and thus provide a plausible mechanism for a tumor-promoting effect. However, in June 1999 the author was censured by the Office of Research Integrity for falsifying his data, and the author retracted the papers.
The Bottom Line
In the words of Edward W. Campion, M.D. (New England Journal of Medicine, 337:44, July 3, 1997):
"there is no convincing evidence that high-voltage power lines are a health hazard or a cause of cancer...18 years of research have produced considerable paranoia, but little insight and no prevention. It is time to stop wasting our research resources. We should redirect them to research that will be able to discover the true biologic causes of the leukemic clones that threaten the lives of children." | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/12%3A_Cancer/12.14%3A_Magnetic_Fields_and_Cancer.txt |
• 13.1: Aging
Aging is the progressive loss of physiological functions that increases the probability of death. The decline in function certainly occurs within cells. This is especially true of cells that are no longer in the cell cycle: (1) neurons in the brain, (2) skeletal and cardiac muscle, and (3) kidney cells.
• 13.2: Telomeres
The DNA molecule of a typical chromosome contains a linear array of genes (encoding proteins and RNAs) interspersed with much noncoding DNA. Included in the noncoding DNA are long stretches that make up the centromere and long stretches at the ends of the chromosome, the telomeres. Telomeres are crucial to the life of the cell. They keep the ends of the various chromosomes in the cell from accidentally becoming attached to each other.
13: Aging
What is Aging?
Aging is the progressive loss of physiological functions that increases the probability of death. This table gives some data.
Loss of structure and function in aging. Figures represent percentage of a given function remaining in an average 75-year-old man compared with that found in an average 30-year-old man, the latter value taken as 100%.
Weight of brain 56%
Blood supply to brain 80
Output of heart at rest 70
Number of glomeruli in kidney 56
Glomerular filtration rate 69
Speed of return to normal pH of blood after displacement 17
Number of taste buds 36
Vital capacity 56
Strength of hand grip 55
Maximum O2 uptake during exercise 40
Number of axons in spinal nerve 63
Velocity of nerve impulse 90
Body weight 88
The decline in function certainly occurs within cells. This is especially true of cells that are no longer in the cell cycle:
• neurons in the brain;
• skeletal and cardiac muscle;
• kidney cells.
Tissue and organs made of cells that are replenished by mitosis throughout life. Blood and intestinal epithelium show far fewer signs of aging.
In the natural world, very few animals live long enough to show signs of aging. Random mortality from starvation, predation, infectious disease and a harsh environment (e.g., cold) kills off most animals long before they begin to show signs of aging. Even for humans, aging has only become common in recent decades.
At the start of the 20th century, infectious diseases such as pneumonia and influenza caused more deaths in the United States than "organic" diseases like cancer. Now the situation is reversed. The availability of effective weapons against infectious disease (e.g., sanitation, antibiotics, and immunization) has greatly increased the average life span (but not maximum life span) and resulted in "organic" diseases like cardiovascular disease and cancer becoming the most common cause of death.
In 1900, a newborn child in the U.S. could look forward to an average life expectancy of only 47 years. Infectious diseases were the major causes of death, killing most people before they reached an age when aging set in. Three-quarters of a century later, life expectancy had risen to 73 years and "organic" diseases, including all the diseases of aging, had replaced infectious diseases as the major cause of death. Today, the life expectancy has risen to 80 for women (74 for men), and coping with an aging population has become a major economic and social challenge in the U.S.
The above graph shows four representative survival curves. The vertical axis represents the fraction of survivors at each age (on the horizontal axis).
• Curve A is characteristic of organisms that have low mortality until late in life. Then mortality increasingly becomes the endpoint of the aging process.
• Curve B is typical of populations in which such environmental factors as starvation and disease obscure the effects of aging (and infant mortality in high).
• Curve C is a theoretical curve for organisms for which the chance of death is equal at all ages. This might be the case for organisms that show few, if any, signs of aging (some fishes) or those (e.g., songbirds in the wild) that suffer severe random mortality from environmental causes throughout life.
• Curve D is typical of organisms, oysters for example, that produce huge numbers of offspring accompanied by high rates of infant mortality.
Organisms with survivorship curves between C and D have no opportunity to show the signs of aging.
Aging in Invertebrates
Invertebrate animals have provided some important clues about the aging process.
• Colonial invertebrates like sponges and corals don't show signs of aging. Even individual cnidarians, like the sea anemone that lived for 78 years, show little or no sign of aging. In all these cases, this is probably because there is constant replacement of old cells by new ones as the years go by.
• Lobsters also can live to a very old age with no obvious sign of a decline in fecundity or any other physiological process. But lobsters never stop growing, so once again it may be the continuous formation of new cells that keeps the animal going.
• In culture vessels, Drosophila does have a limited life span and shows signs of aging before it dies. Two factors have been found to influence the aging process and thus life span:
• Calorie restriction, that is, a semi-starvation diet. In fact, restricting food intake has been shown to increase life span (and slow aging) in all animals — including mammals — that have been tested.
• Single genes have been identified that extend life span in Drosophila (and also in the invertebrate Caenorhabditis elegans).
Aging in Vertebrates
Some cold-blooded vertebrates fishes, amphibians, reptiles have long life spans if they can survive environmental hazards (giant tortoises are known to have reached 177 years of age). These animals are cold-blooded and grow so slowly that they will probably succumb to environmental hazards before they stop growing and begin to show signs of aging. The situation is different for birds and mammals. They are warm-blooded, grow rapidly to adult size and, if protected from environmental hazards, will show signs of aging.
Why Do We Age?
Programmed in our genes
The pros
• Single genes have been found that increase life span in Drosophila, C. elegans, and mice. Genes that suppress signaling by insulin and insulin-like growth factor-1 (Igf-1) increase life span in these animals. Examples:
• Mice with one of their Igf-1 receptor genes "knocked out" live 25% longer than normal mice.
• Klotho. Cells in the kidney and brain release the extracellular portion of a cell surface transmembrane protein into the blood. This "hormone", called Klotho, binds to receptors on many target cells reducing their ability to respond to insulin and Igf-1 signaling.
• Mice homozygous for a mutant klotho gene show many signs of premature aging while
• mice expressing extra-high levels of the Klotho protein live 20–30% longer than normal.
• Long life spans clearly run in human families.
• Aging often appears sooner in animals that suffer high death rates from external causes (e.g. predation) early in life.
Why should this be? Three (interrelated) possibilities:
• The accumulation of harmful mutations (in the germline). Few individuals survive long enough for these to be selected against.
• Antagonistic pleiotropy. Genes that promote survival early in life at the expense of maintaining the body will be selected for.
Some examples:
• p53. By forcing cells with damaged DNA to stop dividing and become senescent or even to die by apoptosis, it protects the organism from the threat of those cells becoming cancerous but at the expense of reducing cell renewal (e.g., by decreasing the size of the pools of stem cells). Mice that are forced to produce higher-than-normal levels of the p53 protein show many signs of premature aging while female mice that are deficient in p53 have reduced fecundity (blastocysts fail to implant). So here is a tradeoff by a gene that promotes evolutionary success at an early age but at the expense of accelerated aging.
• In both Drosophila and C. elegans, some mutations that increase life span do so at the cost of decreased fecundity and vice versa.
• Disposable soma. Early death from external causes will select for genes that increase the chances of passing germplasm on (i.e. reproduction) at the expense of genes that might delay aging.
• There is no way that natural selection can select for genes whose only beneficial effect appears after the age of reproduction is over. But
• any genes that extend the reproductive period or
• any genes that promote fitness in youth as well as longevity
would be selected for.
The cons
High early mortality from external causes (e.g. predators) has been linked to early aging (in the survivors) in some animals, but the reverse has been found in others. These contradictory results do not negate the role of genes in aging, but indicate that other environmental factors (e.g. more food left for the survivors) may skew the outcome.
The Inevitable Consequence of an Active Life
The pros
• Many cold-blooded vertebrates (e.g., many fishes and reptiles) do not show signs of aging.
• Transgenic mice whose "thermostat" in the hypothalamus has been reset to give a lower body temperature (reduced by 0.3 – 0.5°C) live 12% (males) to 20% (females) longer than their nontransgenic littermates. This translates into adding some 3 months to the average life span of 27 months for these mice. (See Conti, B., et al., Science, 3 November 2006).)
• The effects of Calorie Restriction (CR). The life span of yeast, C. elegans, Drosophila, birds, and mammals (mice, rats, and probably monkeys) can be extended, and signs of aging delayed, if they are maintained on a semi-starvation diet. Calorie restriction in mice causes
• a drop in
• the level of circulating insulin and insulin-like growth factor-1 (Igf-1);
• the level of glucose and triglycerides in the blood;
• the level of NADH (produced by cellular respiration) within cells;
• an increase in the production of sirtuins — deacetylases that remove acetyl groups from proteins.
• apoptosis of cells to be inhibited;
• formation of adipose tissue to be suppressed;
• increased production of nitric oxide (NO) which is essential for the benefits of CR to take effect.
• greatly increased physical activity and
• lower body weight.
The life-extending and other effects of CR on rats and mice may not be as significant as they seem. All the studies have been done on laboratory rats and mice. Control animals are normally fed ad libitum, which means that they have food available all the time. But this would not likely be the case in the wild so it may be that the physiology and life span of these animals are already compromised and that the effects of CR are mainly restoring normal conditions for these species.
The problem of proper controls may also explain the divergent results in studies of CR in rhesus monkeys.
• In the 10 July 2009 issue of Science, researchers at the Wisconsin National Primate Research Center reported the status of rhesus monkeys that had been on CR for 20 years compared with a control group allowed to feed ad libitum on the same diet during that time. The results: the CR animals showed markedly fewer signs of aging (none showed any signs of diabetes), and the few that died from age-related causes did so at only one-third the rate of the controls.
• However, a 25-year study of CR in rhesus monkeys carried out at the National Institute of Aging (NIA) (and reported in Nature in August 2012) showed no increased longevity in the CR monkeys.
The difference may have arisen because the control monkeys in Wisconsin were fed ad libitum while the controls at the NIA were fed a fixed amount. Furthermore, the diet used in the Wisconsin study were much higher in sugar than the diet at the NIA, and the NIA diets also included omega-3 fatty acids and other healthy components absent from the Wisconsin diet. So perhaps the positive effect seen in the Wisconsin study resulted from the CR animals simply getting less of an unhealthy diet than the controls.
Resveratrol
Resveratrol, is a small molecule found in red wine which appears to activate sirtuins mimicking the effects of calorie restriction. Mice given daily doses of resveratrol while indulging in a high-fat diet get fat but avoid the degenerative changes and shortened life span that normally accompany a high-fat diet. But before rushing out to buy red wine, realize that the doses of resveratrol given to the mice were far higher than could be supplied by drinking it. Studies on the effect of resveratrol on extending life span in yeast, Drosophila and C. elegans have produced mixed results.
No one knows for certain why calorie restriction delay aging, but some mechanisms might be because it lowers the level of glucose in the blood and thus the speed with which lipids and proteins suffer from glycation. Advanced glycation end products (AGEs) are molecules that have reduced function because of the haphazard addition of sugars to them. For proteins like collagens and elastin, this results is increasing stiffness of the extracellular matrix (ECM) of blood vessels, joints, heart, kidney, etc. Reducing calorie intake reduces female fecundity (at least in C. elegans, Drosophila, rats and mice). The energy that would have been devoted to producing offspring can be devoted instead to tissue repair and maintenance. Calorie restriction raises the level of sirtuins:
• The SIRT1 protein CR in knockout mice lacking SIRT1 gain no increase in life span.
• plays a key role in repairing DNA damage and so helps protect the integrity of the genome which appears to be essential to longevity.
• It also inhibits the nutrient sensor TOR ("target of rapamycin") which accelerates aging in mice.
• SIRT1 also inhibits p53 activation thus protecting against mitochondrial damage.
• The SIRT3 protein is found in mitochondria where it inhibits the production of free radicals.
The Free Radical Theory of Aging
A major aspect of metabolism is the oxidation of foodstuffs by the mitochondria. Electron transport in the mitochondria generates reactive oxygen species ("ROS") such as the superoxide anion (O2), which generates hydrogen peroxide (H2O2). Although cells contain enzymes for detoxifying these reactive substances (e.g., catalase which breaks down H2O2), they eventually and inevitably damage macromolecules in the cell: proteins; lipids; and probably most important of all, DNA.
Damaged proteins and lipids accumulate in the cell, especially nondividing cells like neurons and muscle, producing aggregates of denatured proteins and an "aging pigment" called lipofuscin (a principal component of ear wax). The accumulation of protein aggregates in striated muscles reduces muscle strength. Protein aggregates accumulate more slowly in the cells of animals on a calorie restricted diet — perhaps as a result of more-efficient autophagy. However, it may be damage to DNA that is the crucial factor in the decline in cell function with age.
The DNA of the mitochondria (mtDNA) may be at special risk. ROS are produced as an inevitable byproduct of electron transport in the mitochondria and thus are generated close to the mtDNA. But the products of these genes are essential for electron transport. So perhaps a positive feedback loop is generated: ROS -> mutations in electron transport genes reducing their efficiency -> more ROS production.
Supporting evidence:
• mtDNA accumulates mutations faster than nuclear DNA, and these show the chemical characteristics of damage by ROS.
• Transgenic mice containing the human gene for catalase (but with the targeting signal that would normally send the protein to peroxisomes replaced with that for mitochondria) live 20% longer than normal for their strain. [See Schriner, S. E. et al., Science, 24 June 2005.]
• Transgenic mice whose DNA polymerase for copying mtDNA genes (DNA polymerase gamma) is defective, and introduces an elevated number of mutations in mtDNA, show many signs of premature aging — both cellular and in various organ systems — and die early.
The cons
• Neither mice genetically engineered to overproduce free radicals, nor those engineered to produce lower amounts of free radicals, have any change in their lifespan.
• Bats and mice are similar in size and metabolic rate, but bats can live ten times as long.
• Although glucose-starved yeast do live longer, they have an increased — not decreased — rate of cellular respiration.
• The metabolic rate of mice on a CR diet is no lower than that of mice on a normal diet.
• The beneficial effects of CR take hold at any time, at least in Drosophila. Even after three weeks on a rich diet (in the second half of the normal life span of adult flies), switching to a CR diet reduces mortality to the same degree as flies maintained on CR throughout their adult lives. The reverse is also true — switching from a CR diet to a rich diet quickly undoes the good work of the former. These results suggest that if a rich diet does produce irreversible and accumulating damage, its harmful effects on life span can be blunted at any time.
The Accumulation of Senescent Cells
Chronological Senescence
Once formed, some cells in a mouse or human are never replaced. A neuron formed during embryonic development may still be functioning at the end of life. However, during its life span, damage to its organelles and DNA may accumulate resulting in a loss of function. This is called chronological senescence. In other tissues, e.g., blood and epithelia, new cells replace old ones throughout life. But even though new, they may have reduced function because of replicative senescence.
Replicative Senescence
One might expect that cells removed from a mouse or human and placed in tissue culture could be cultured indefinitely, but that is not the case. When human fibroblasts, for example, are placed in culture, they proliferate at first, but eventually a time comes when their rate of mitosis slows and finally stops. The cells continue to live for a while, but cannot pass from G1 to the S phase of the cell cycle. This phenomenon is called replicative senescence. Fibroblasts taken from a young human pass through some 60–80 doublings before they reach replicative senescence.
Why should this be? Cells — unless they retain the enzyme telomerase — lose DNA from the tips of their chromosomes (telomeres) with each cell division. In general, the telomeres in the cells of old animals are much shorter than those in young animals. A recent study of short-lived versus long-lived birds showed that telomere shortening was faster in the short-lived species. And one species, a petrel which lives four times as long as other birds of its size, actually has telomeres that grew longer with age. Most somatic cells of the body cease to express telomerase. However, cells genetically manipulated to express telomerase long after they should have stopped, avoid replicative senescence. Germline cells, e.g., spermatogonia, and some stem cells continue to express the enzyme. Some 95% of cancer cells express telomerase. If telomeres get too short (less than 13 repeats in human cells), chromosome abnormalities — a hallmark of cancer — occur. Cancer can be avoided if the cell senses this dangerous condition and ceases to divide. So telomere shortening may protect against cancer at the price of cell senescence.
Two proteins encoded by tumor suppressor genes p53 and p16INK4a play pivotal roles in stopping the cell cycle. The result: replicative senescence. So replicative senescence may be the price we pay for removing cells from the cell cycle before they can accumulate the mutations that would turn them into cancer cells.
The role of the tumor suppressor proteins in replicative senescence is mirrored in the intact animal, at least in mice.
• Mice engineered to express abnormally-high levels of p53 activity show many signs of premature aging including premature reduction in the length of their telomeres.
• Mice
• that express abnormally-high levels of p16INK4a have a reduced ability to regenerate tissue while
• mice whose activity of p16INK4a is suppressed continue to repair damaged tissue as efficiently as young animals do.
• In mice, eliminating senescent cells (they are high in p16INK4a) prevents (in young mice) and partially reverses (in older mice) some of the signs of aging such as cataracts, and loss of adipose tissue and skeletal muscle mass.
• Mice genetically-engineered to express high levels of
• telomerase and
• tumor suppressor genes (e.g., p53 and p16INK4a)
have substantially-increased life spans and show fewer of the degenerative changes characteristic of aging in their skin and other epithelia. (The need to make these transgenic mice with increased tumor suppressor activity in addition to increased telomerase activity arises because an increased level of telomerase alone elevates the incidence of cancer — killing the animals before any anti-aging effects of telomerase can be measured reliably.)
The role of telomerase deficiency in mammalian aging
Mice whose genes for telomerase have been "knocked out" (either Tert−/− or Terc −/−) show many of the degenerative changes associated with aging.
• The number of mitochondria in their cells decreases as does the function of those that remain.
• Oxygen consumption and ATP production declines.
• The efficiency of the electron transport chain decreases.
• This leads to an increased generation of reactive oxygen species (ROS).
• The level of p53 activity increases.
• mitosis declines
• apoptosis of cells increases
• replicative senescence increases
• The anatomy and function of organs such as the liver and heart show the degenerative changes of age.
In the 6 January 2011 issue of Nature, Mariela Jaskelioff and her colleagues (many of the same team that found the results described in the previous section) report that reactivation of telomerase in aged mice reverses many signs of aging.
Their experimental animals were another telomerase-deficient strain of mice; that is, mice that couldn't produce telomerase even in those cells — "adult" stem cells and cells of the germline — that normally retain telomerase activity. The mice were made by "knocking-in" a gene that prevents any expression of telomere reverse transcriptase (TERT) unless an activating drug is given to the animal. Without the drug, these mice live half as long as normal, and as they get older, they display many signs of aging:
• their telomeres get shorter leading to chromosome aberrations;
• their cells undergo early replicative senescence;
• almost all their organs — testes, spleen, intestine, brain — show degenerative changes typical of aging.
BUT, if given the activating drug over a four-week period at a time when degenerative changes were already apparent (25-30 weeks), their deterioration stopped and even partially reversed.
• the length of their telomeres increased;
• replicative senescence was delayed;
• their life span was substantially increased;
• their brain, testes, liver, spleen, and intestine escaped the degenerative changes seen in untreated telomerase-deficient mice;
• they produced larger litters than untreated mice;
• there was reduced activation of p53, indicating
• reduced damage to their genome and
• reduced apoptosis in their tissues
How would replicative senescence of cells lead to the deterioration in structure and function of the aging tissues (e.g., skin) in which they reside? In tissues, e.g., skin and other epithelia, where mitosis must continue throughout life to replace the cells that are lost, the accumulation of senescent cells — incapable of further mitosis — could leading to the characteristic changes of aging in that tissue.
• One mechanism could be simply the inability of senescent cells to repair the tissue by mitosis.
• However, senescent cells remain active although the genes they express change. Perhaps the proteins they secrete (e.g., collagen-digesting enzymes) cause the aging changes in the tissue where they reside.
• Perhaps it is the senescence of adult stem cells that has the greatest effect on tissue aging. In knockout mice that cannot make the Klotho protein, stem cells and progenitor cells in various tissues undergo senescence and decline in numbers.
An unavoidable tradeoff?
Some of the data so far suggests that efforts to avoid the degenerative changes that come with age (e.g., by increasing cell renewal by means of increased telomerase activity) in the hope of increasing longevity may instead hasten death from cancer while efforts to prevent cancer (e.g., by increasing the activity of tumor suppressor genes) may hasten aging. However, other evidence paints a less-gloomy picture. Mice heterozygous for the p53 tumor suppressor gene (p53+/−) develop many cancers when exposed to ionizing radiation. With only a single copy of this tumor suppressor, a single cell is at great risk of losing the remaining copy ("loss of heterozygosity") and starting the growth of a malignant clone. However, if before being irradiated the mice are given resveratrol — to stimulate the production of the anti-aging SIRT1 protein — the incidence of some cancers is reduced and the mice live longer before succumbing to their tumors.
The Accumulation of Genetic Errors
The pros
• Mice given ionizing radiation that damages DNA show early aging.
• Transgenic mice with a defect in the "proofreading" function of the DNA polymerase responsible for copying mitochondrial DNA
• accumulate many mutations in their mitochondrial genes;
• show marked signs of premature aging.
• Cells taken from old mice (and old humans) show slightly elevated levels of somatic mutations and chromosome abnormalities like translocations and aneuploidy. Many of these changes also cause cancer so it is no accident that the incidence of cancer rises with advancing age (graph).
• The hematopoietic stem cells of "knockout" mice deficient in any one of these enzymes needed for genome maintenance
• XPD for nucleotide excision repair (NER)
• Ku80 for nonhomologous end joining (NHEJ)
• TR (telomerase RNA) needed for telomere maintenance
lose their ability to supply the various progenitor cells that produce the white blood cells.
• Most of the hematopoietic stem cells in aged mice show evidence of double-stranded breaks (DSBs) in their chromatin.
• As DSBs form, SIRT1 proteins move from their original locations (at gene promoters) to the locations of the DSBs (where they recruit DNA repair proteins). This shifts the pattern of gene expression to one typical of aging cells.
• Cells taken from old people (and people with premature aging syndromes) show marked reductions in the transcription of some genes, increases in others.
Clues from the Transcriptome of Aging Brains
A group of Harvard researchers reported (in the 26 June 2004 issue of Nature) the results of their study of gene expression in the human brain. They extracted the RNA from autopsied brain tissue of 30 people who had died at ages ranging from 26 to 106. They analyzed the RNA with DNA chips looking for the level of activity of some 11,000 different genes (the transcriptome). A clear pattern emerged.
The level of activity of some 400 genes changed over time.
• Gene expression declined in old age for many genes. Some examples:
• genes encoding proteins involved in synaptic activity in the brain (e.g., learning, memory)
• NMDA, AMPA, GABAA receptors
• calcium-calmodulin-dependent kinase II (CaMKII)
• genes involved in mitochondrial functions, such as
• production of ATP (needed for DNA repair)
• production of damaging reactive oxygen species (ROS)
Detailed examination of some of these down-regulated genes showed that they had suffered DNA damage — more often in their promoters than in their coding regions.
• Gene expression increased in old age for other genes. Some examples:
• genes involved in inflammation and other immune defenses;
• genes encoding proteins involved in defense against reactive oxygen species (ROS);
• genes encoding proteins involved in DNA repair.
The transition from the youthful transcriptome to the transcriptome of the aged brain occurred at varying times from as young as 42 to as old at 73.
A study of individual heart muscle cells in young and old mice (Bahar, R. et al., Nature, 22 June 2006) showed that the transcriptome of young cells was quite uniform from cell to cell but that of aged cells was highly variable from one cell to another. Variable gene expression from one cell to the next in a single tissue might well lead to defects in the functioning of that tissue.
Clues from Premature Aging Syndromes
Humans suffer from a number of rare genetic diseases that, among other things, produce signs of premature aging, e.g., gray hair, wrinkled skin, and shortened life span. In several cases, the mutated genes are ones that have roles to play in maintaining the integrity of the genome, that is, in DNA repair.
• Werner's syndrome. The hair of patients turns gray in their 20s and most die in their late 40s with such signs of age as osteoporosis, cataracts, and atherosclerosis. Even when young, their cells undergo replicative senescence after only ~20 doublings instead of the normal 70 or more. Caused by mutations in WRN, which encodes a helicase needed for DNA repair and maintenance of telomeres.
• Cockayne syndrome (CS). Caused by mutations in genes needed for DNA repair, especially transcription-coupled DNA repair. While these people show only some of the signs of aging, they do have a sharply-reduced life span.
• Ataxia telangiectasia (AT). These patients show signs of premature aging. They lack a functioning gene (ATM) product needed to detect DNA damage and initiate a repair response.
• Hutchinson-Gilford progeria syndrome. Children with this rare disorder show many signs of severe aging by their second birthday and die in their early teens. Caused by mutations in the gene (LMNA) for lamin the intermediate filament protein that stabilizes the inner membrane of the nuclear envelope. The machinery for DNA replication, transcription, and repair is located at the inner surface of the nuclear envelope, and the cells of these patients have increased DNA damage and other defects in gene expression.
So these syndromes suggest that aging may be the consequence not so much of mutations in general, but of mutations in those genes whose products are essential for the error-free replication, repair and transcription of all genes.
Why is a mouse as old at 2 years as a human at 70
If aging represents the inevitable consequence of a failure of DNA repair, why does it occur so much sooner in some mammals (e.g., mice) than in others (e.g., elephants and humans)?
The answer probably lies in the risk of death from external factors (e.g., predation, starvation, cold) in that species.
As noted above, few small mammals ever age because they die early of external causes. These animals are r-strategists, putting their energy into quickly
• reaching sexual maturity
• producing large numbers of offspring that can soon live independently
There is no selective advantage for them to invest in the machinery of efficient DNA repair because they are going to die before mutations become a problem.
Humans, in contrast, are K-strategists. They take a long time to reach sexual maturity. They also produce small numbers of young that must be cared for over a long period. Small wonder, then, that evolution in humans (and other long-lived mammals) has selected for genes promoting efficient DNA repair.
The table shows that the efficiency of DNA repair is directly correlated with life span in a variety of mammals.
Correlation between life span and the relative effectiveness of DNA repair in cells of certain mammals. In each case, cells growing in tissue culture were irradiated with ultraviolet light and then the efficiency with which they repaired their DNA was determined. (From the work of R. W. Hart and R. B. Setlow, 1974.)
Species Average life span, yr Relative effectiveness of DNA repair
Human 70 50
Elephant 60 47
Cow 30 43
Hamster 4 26
Rat 3 13
Mouse 2 9
Shrew 1 8
Interrelationships
Examining the various factors that have been implicated in the aging process suggests that most —perhaps all — are interrelated.
• Mitochondria disfunction with the production of
• reactive oxygen species (ROS) with their damaging effect on
• DNA and other cell constituents coupled with the
• onset of replicative senescence so that damaged cells can no longer be replaced
may all play important roles. So the factors described above are by no means mutually exclusive.
The above figure attempts to show how various factors involved in aging interact. Key players are
• The gradual shortening of telomeres with repeated cell divisions.
• p53.
• the enzyme designated TOR ("target of rapamycin"). TOR is a kinase that participates in many metabolic pathways in the cell. (It is inhibited by the antibiotic rapamycin that is used as an immunosuppressant).
Stimulatory interactions are shown with blue arrows; inhibitory interactions are shown in red.
Interactions:
• Telomere shortening activates p53 which leads to damaged mitochondria.
• The inefficient electron transport chain in damaged mitochondria produces ROS.
• Abundant nutrients (e.g. amino acids) as well as other growth stimulants activate TOR which promotes anabolism (protein and lipid synthesis) with attendant production of reactive oxygen species (ROS) and aging.
• Calorie restriction, working through SIRT1 inhibits TOR and its downstream effects.
• Inhibition of TOR relieves its inhibition of autophagy allowing the cells to scavenge, for example, damaged mitochondria.
• SIRT1 inhibits p53 activation thus protecting against mitochondrial damage.
Because of the association between telomere shortening and aging, two companies have begun (in 2011) to offer tests of telomere length. How such tests might be useful to the people asking for them remains to be seen.
The Hallmarks of Aging
In the 6 June 2013 issue of Cell, an international group of scientists developed a list of 9 features that characterize aging in animals. (This effort to bring order to such a complex subject is reminiscent of earlier papers in the same journal, The Hallmarks of Cancer.)
They expected that each hallmark would meet at least two of three criteria.
• It should be characteristic of normal aging.
• Increased expression of the hallmark should result in faster aging.
• Efforts to reduce expression of the hallmark should prolong a healthy lifespan ("healthspan").
The 9 hallmarks.
1. Genomic Instability
Meeting the criteria:
• Aged cells contain more DNA damage than young ones.
• Agents that increase unrepaired damage to DNA, including chromosomal damage (e.g., aneuploidy), hasten aging.
• Limited evidence that treatments that reduce, for example, chromosome missegregation, prolong healthspan.
2. Telomere Attrition
Meeting the criteria:
• The chromosomes of aged cells have shorter telomeres than those of young cells.
• Telomerase-deficient mice show premature aging.
• Treatments that reactivate telomerase in normal mice delay aging.
3. Epigenetic Alterations
Meeting the criteria:
• The patterns of DNA methylation and histone modifications changes as a mammal ages.
• Mice that are deficient in the sirtuin SIRT6, an enzyme that deacetylates histones, age more rapidly than normal.
• Treatments that increase the activity of sirtuins increase healthspan in mice.
Note
As we humans age, the DNA in our cells accumulates an ever-increasing number of epigenetic changes as measured by the methylation of CpGs. This is true for a wide variety of cell types even those that have been formed recently; that is, the number of epigenetic changes reflects the age of the donor not the age of the cell. The correlation is so good that analysis of these changes in a cell can predict the donor's age sometimes within a matter of months.
4. Loss of Proteostasis
Proteostasis is the homeostasis of the proteome — the proper balance of the synthesis and degradation of proteins in the cell.
Meeting the criteria:
• The clearance of denatured (unfolded) proteins by autophagy and proteasomes, as well as the ability to refolded them with chaperones, all decline with age. The result: toxic protein aggregates that accumulate in aged cells.
• Mutant mice with defective chaperone activity age more quickly.
• Transgenic Drosophila and C. elegans that overexpress chaperones have increased life spans.
5. Derugulated Nutrient Sensing
Meeting the criteria:
• The nutrient sensor TOR ("target of rapamycin"), which promotes anabolism, increases during normal aging (and produces obesity, at least in mice).
• Increased activity of TOR accelerates aging in mice.
• Examples:
• Genetic suppression of TOR signaling extends lifespan in Drosophila and C. elegans.
• Calorie Restriction (CR), which inhibits TOR, increases healthspan in all animals in which it has been tested.
• Rapamycin, which inhibits TOR, extends lifespan in Drosophila, C. elegans, and mice.
6. Mitochondrial Dysfunction
Meeting the criteria:
• The production and efficiency of mitochondria decreases in aging, otherwise normal, mice.
• Deleterious mutations in mitochondrial DNA and other defects in mitochondrial function all accelerate aging in mice.
• No compelling evidence yet that treatments to improve mitochondrial function increase life span.
7. Cellular Senescence
Meeting the criteria:
• Replicative senescence (cells no longer able to enter the cell cycle) sets in much sooner in the cells of aged animals than it does in young animals.
• Mice engineered to express abnormally-high levels of p53, a protein that blocks entry into the cell cycle, show many signs of premature aging.
• In mice, eliminating senescent cells prevents (in young mice) and partially reverses (in older mice) some of the signs of aging such as cataracts, and loss of adipose tissue and skeletal muscle mass.
8. Stem Cell Exhaustion
Meeting the criteria:
• The proliferative capacity of adult stem cells declines with age in the tissues that have been examined.
• Deliberately exhausting the pool of stem cells in the Drosophila intestine leads to premature aging.
• Transplantation of stem cells from young mice into aged mice improves the degenerative changes of aging and prolongs their life.
9. Altered Intercellular Communication
All cells respond to chemical signals in their environment. These include cytokines secreted by nearby cells (paracrine stimulation).
Meeting the criteria:
• Inflammation in various tissues — mediated by the secretion of a variety of cytokines — increases in the aged.
• Genetically engineered mice that are unable to down-regulate the mRNAs synthesizing pro-inflammatory cytokines show accelerated aging.
• Inhibition of the pro-inflammatory cytokine NF-κB delays aging in mice. Even such a simple anti-inflammatory agent as aspirin seems to prolong life in mice.
Relationships of the Hallmarks
• The first 4 hallmarks appear to represent the initiating events leading to aging.
• Hallmarks 5, 6, and 7 appear to represent damage produced as the cell attempts to respond to the damage caused by the first 4 hallmarks.
• Taken together, hallmarks 1 through 7 produce the aging phenotype seen in hallmarks 8 and 9, which are ultimately responsible for the decline with age in the function of cells and the organism of which they are a part.
An Elixir of Youth?
Despite years of research, only three interventions have been discovered that slow the aging process and/or prolong life.
• Calorie Restriction (CR). Works in all animals tested.
• Rapamycin. Extends lifespan in mice, Drosophila, and C. elegans but does not appear to reverse or even halt the degenerative changes of aging.
• Parabiosis. When the circulatory system of a young mouse is joined to that of an old mouse (the technique is called parabiosis), various tissues in the old mouse, e.g., its skeletal muscle, cardiac muscle, liver, and central nervous system, become rejuvenated. A promising candidate for mediating this effect is a protein called GDF11 ("Growth Differentiation Factor 11"). (GDF11 is also known as BMP-11). Injections of recombinant GDF11 are almost as effective as parabiosis. GDF11 probably acts by stimulating the activity of stem cells. However, there is no evidence yet that it increases the lifespan of the mice.
Aging in Unicellular Organisms
It used to be thought that many unicellular organisms, such as yeast and bacteria, were immortal; that is, they
• never aged but simply kept dividing to produce new individuals;
• did not have the distinction between germline (immortal) and soma (mortal) that multicellular organisms have.
If true, every time a cell divides, the two daughter cells would be identical in all respects to the parent (symmetrical division).
But at least for yeast and E. coli, this is not the case.
Yeast cells do age and, as discussed above, have proved useful for studying the aging process. Placing a single yeast cell on solid medium and removing its daughter (the bud) each time one is produced, it turns out that the number of times the mother cell can form a new bud by mitosis is limited. After producing a bud some 20–30 times, the mother cell shows a number of harmful cellular changes (e.g., defective mitochondria) and dies.
But, at least early in her life, the buds are born with the potential of a full life span. So the mitotic division must be asymmetrical with the properties of the bud different from those of its parent. Several mechanisms by which this occurs have now been demonstrated.
• A diffusion barrier is formed at the neck between the mother cell and her bud. This barrier prevents the passage from the mother into the bud of damaged nuclear components, e.g., plasmid-like fragments of DNA produced during the life of the mother. In contrast to the situation in most eukaryotes, in yeast there is no breakdown of the nuclear envelope during mitosis. During anaphase, the nuclear envelope grows and the portion enclosing the daughter chromosomes enters the bud. However, the barrier at the neck keeps all the preexisting nuclear pore complexes (NPCs) within the mother cell and these retain the DNA fragments and perhaps other damaged nuclear components.
• Most of any damaged cytoplasmic components, such as proteins denatured by reactive oxygen species (ROS) resulting in the formation of nonfunctional aggregates, become attached to aged mitochondria and the endoplasmic reticulum both of which are retained in the mother cell. Any protein aggregates that do get through are either dissolved by chaperones in the bud or, if that fails, actin filaments in the bud move the aggregates back into the mother cell. This latter mechanism requires the presence of a number of proteins, including a sirtuin found in yeast called Sir2 ("Silent information regulator 2").
Asymmetric division in stem cells.
Stem cells are cells that divide asymmetrically to produce a daughter cell that goes on to differentiate and a daughter cell that remains a stem cell. A number of examples have been found — in Drosophila and in mammals — where aging stem cells preferentially deposit their damaged cellular components, e.g. aggregated proteins, in the daughter that will go on to differentiate while keeping undamaged components in the daughter that will remain a stem cell. So like yeast, these stem cells have a mechanism that preferentially protects the "immortal" cell from the inevitable effects of aging.
Aging in E. coli
A similar aging phenomenon has been found in E. coli. When E. coli divides, a septum forms in the middle of the dividing cell and then the two daughter cells are pinched apart. As the cell wall seals the break, the two daughter cells end up with one "old" end and one newly-formed end. When the two daughters go on to divide, the process is repeated. The original old ends gets passed on from generation to generation (rather like immortal strands of DNA).
The diagram shows how during cell division, two new poles are formed, one in each of the progeny cells (new poles shown in green for the first generation; magenta for the second). The other ends of those cells were formed during a previous division.
It has been shown (Stewart EJ, Madden R, Paul G, Taddei F (2005) Aging and Death in an Organism That Reproduces by Morphologically Symmetric Division. PLoS Biol 3(2): e45 doi:10.1371/journal.pbio.0030045) that the cells that inherit an increasingly old pole exhibit a diminished growth rate, decreased offspring production, and an increased incidence of death.
So it appears that the phenomenon — characteristic of all multicellular organisms — of an aging and mortal soma producing germplasm (sperm and eggs) that starts a new youthful generation may have its counterpart in unicellular organisms. Perhaps no single cell can escape the ravages of time on the integrity of its organelles and the molecules.
Aging in Plants
Annuals and Biennials
Annuals, such as many grasses and "weeds"
• grow vigorously for a period;
• then form flowers followed by fruits.
• Fruiting is followed by a slowing of growth accompanied by physiological and morphological changes such as
• an increase in the rate of respiration (catabolism)
• loss of chlorophyll
• These changes constitute aging and end in the death of the plant. Biennials follow the same pattern, but take two years to do it.
This pattern in clearly programmed in the genes. Even with plentiful moisture, soil minerals, sunlight, and warm temperatures, the plants age and die.
Perennials
The situation is quite different in perennials. Throughout their lives, woody perennials (trees) produce new vascular tissue, leaves, and flowers each year. They do not show marked signs of aging, although their rate of growth may decline over the years. Finally, disease or inability to support their ever-increasing size against wind or snow load lead to their death.
This picture (courtesy of Walter Gierasch) is of bristlecone pines (Pinus longaeva) growing in the White Mountains of eastern California. Tree-ring analysis shows that some of these trees are almost 5000 years old. But note that no living cells in the tree are more than a few years old.
Even so, how have long-lived plants like these avoided the accumulation — over years of DNA replication as their cells divided — of deleterious mutations that would reduce fitness and life span? Perhaps it is because the cells in plant meristems, where all growth begins are stem cells which divide slowly and like all stem cells, asymmetrically; that is, producing one daughter that will remain a stem cell and one that will begin a phase of rapid mitosis and eventually differentiate into the mature tissues of the plant. If (and this is as yet only a speculation) the division of a meristematic stem cell is asymmetric with respect to the segregation of DNA strands; that is, the stem cell retains the immortal strands of DNA while the cell destined to produce more tissues receives the newly-replicated strands, this would provide an additional mechanism to protect the genome as the years go by. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/13%3A_Aging/13.01%3A_Aging.txt |
Each eukaryotic chromosome consists of a single molecule of DNA associated with a variety of proteins. The DNA molecules in eukaryotic chromosomes are linear; i.e., have two ends. (This is in contrast to such bacterial chromosomes as that in E. coli that is a closed circle, i.e. has no ends.)
The DNA molecule of a typical chromosome contains a linear array of genes (encoding proteins and RNAs) interspersed with much noncoding DNA. Included in the noncoding DNA are long stretches that make up the centromere and long stretches at the ends of the chromosome, the telomeres. Telomeres are crucial to the life of the cell. They keep the ends of the various chromosomes in the cell from accidentally becoming attached to each other. The telomeres of humans consist of as many as 2000 repeats of the sequence 5' GGTTAG 3'
5'...GGTTAG GGTTAG GGTTAG GGTTAG GGTTAG GGTTAG..3'
3'...CCAATC CCAATC CCAATC CCAATC CCAATC CCAATC..5'
Replication of linear chromosomes presents a special problem
DNA polymerase can only synthesize a new strand of DNA as it moves along the template strand in the 3' –> 5' direction. This works fine for the 3' –> 5' strand of a chromosome as the DNA polymerase can move uninterruptedly from an origin of replication until it meets another bubble of replication or the end of the chromosome. However, synthesis using the 5' –> 3' strand as the template has to be discontinuous. When the replication fork opens sufficiently, DNA polymerase can begin to synthesize a section of complementary strand — called an Okazaki fragment — working in the opposite direction. Later, a DNA ligase ("DNA ligase I") stitches the Okazaki fragments together.
In Figure \(3\), the horizontal black arrows show the direction that the replication forks are moving. Wherever the replication fork of a strand is moving towards the 3' end, the newly-synthesized DNA (red) begins as Okazaki fragments (red dashes). This continues until close to the end of the chromosome. Then, as the replication fork nears the end of the DNA, there is no longer enough template to continue forming Okazaki fragments. So the 5' end of each newly-synthesized strand cannot be completed. Thus each of the daughter chromosomes will have a shortened telomere.
It is estimated that human telomeres lose about 100 base pairs from their telomeric DNA at each mitosis. This represents about 16 GGTTAG repeats. At this rate, after 125 mitotic divisions, the telomeres would be completely gone. Is this why normal somatic cells are limited in the number of mitotic divisions before they die out?
Telomeres and Cellular Aging
Telomeres are important so their steady shrinking with each mitosis might impose a finite life span on cells. This, in fact, is the case. Normal (non-cancerous) cells do not grow indefinitely when placed in culture. Cells removed from a newborn infant and placed in culture will go on to divide almost 100 times. Well before the end, however, their rate of mitosis declines (to less than once every two weeks). Were my cells to be cultured (I am 81 years old), they would manage only a couple of dozen mitoses before they ceased dividing and died out. This phenomenon is called replicative senescence. Could shrinkage of telomeres be a clock that determines the longevity of a cell lineage and thus is responsible for replicative senescence?
Evidence
Some cells do not undergo replicative senescence:
• the cells of the germline (the germplasm)
• unicellular eukaryotes like Tetrahymena thermophila
• stem cells, including "adult" stem cells and cancer stem cells.
It turns out that these cells are able to maintain the length of their telomeres. They do so with the aid of an enzyme telomerase.
Telomerase
Telomerase is an enzyme that adds telomere repeat sequences to the 3' end of DNA strands. By lengthening this strand, DNA polymerase is able to complete the synthesis of the "incomplete ends" of the opposite strand. Telomerase is a ribonucleoprotein. Its single snoRNA molecule — called TERC ("TElomere RNA Component") — provides an CCAAUC (in mammals) template to guide the insertion of GGTTAG. Its protein component — called TERT ("TElomere Reverse Transcriptase") — provides the catalytic action. Thus telomerase is a reverse transcriptase; synthesizing DNA from an RNA template.
Telomerase is generally found only in the cells of the germline, including embryonic stem (ES) cells; unicellular eukaryotes like Tetrahymena thermophila; and some, perhaps all, "adult" stem cells (including cancer stem cells) and "progenitor" cells enabling them to proliferate. When normal somatic cells are transformed in the laboratory with DNA expressing high levels of telomerase, they continue to divide by mitosis long after replicative senescence should have set in. And they do so without any further shortening of their telomeres. This remarkable demonstration (reported by Bodnar et. al. in the 16 January 1998 issue of Science) provides the most compelling evidence yet that telomerase and maintenance of telomere length are the key to cell immortality.
Telomere Deficiency Syndromes
A number of rare human diseases are caused by mutations in TERT or TERC or several other genes involved in telomere maintenance. The severity of the disease, the organs it affects, and the age of onset vary widely. But all are characterized by abnormally short telomeres. Patients with telomere deficiency are also at increased risk of developing cancer.
Telomerase and Cancer
The crucial feature that distinguishes a cancer from normal tissue is its ability to grow indefinitely. Most (85–90%) cancers express telomerase — at least in the population of cancer stem cells that divide uncontrollably causing the tumor to grow. Perhaps agents that prevent the expression of the gene for telomerase — or prevent the action of the enzyme — will provide a new class of weapons in the fight against cancer. But if telomerase activity — however brief — is essential for all cells, we had better be careful, and if lack of telomerase hastens replicative senescence, it may also hasten the aging of the tissues that depend on newly-formed cells for continued health — a tradeoff that may not be worth making.
Telomerase and Transplanted Cells
One approach to gene therapy it to remove cells from the patient, transform them with the gene for the product that the patient has been unable to synthesize, return them to the patient. One problem with this approach is that the cells — like all normal somatic cells — are mortal. After a series of mitotic divisions, they die out. That is the reason the children described in the link above required periodic fresh infusions of their transformed T cells.
What if their cells could be transformed not only with the therapeutic gene, but also with an active telomerase gene? This should give them an unlimited life span. But if cancer cells regain the ability to make telomerase, might not the reverse be true; that cells transformed with an active telomerase gene might become cancerous? Perhaps not. The cells described by Bodnar et. al. in the 16 January 1998 issue of Science have continued to grow in culture and have been subjected to a number of tests to see if they have acquired any properties of cancer cells in culture.
The results are encouraging. While these cells continue to divide indefinitely as cancer cells do,
• They still show contact inhibition as normal cells do when grown in culture.
• They do not grow into tumors when injected into immunodeficient mice (as cancer cells do).
• They are still fussy about their diet — unable to grow on the simple media that supports cancer cells in culture.
• They still retain a normal karyotype; something that cancer cells seldom do.
However, studies with whole animals — transgenic mice that express abnormally high levels of TERT — reveal that they do suffer an elevated incidence of cancer.
Telomeres and Cloning
The now-famous sheep Dolly was cloned using a nucleus taken from an adult sheep cell that had been growing in culture. The cell donor was 6 years old, and its cells had been growing in culture for several weeks. What about Dolly's telomeres? Analysis of telomere length in Dolly's cells reveals that they were only 80% as long as in a normal one-year-old sheep. Not surprising, since the nucleus that created Dolly had been deprived of telomerase for many generations. Two other sheep — cloned from embryonic, not adult, cells — also had shortened telomeres although not as short as Dolly's. Perhaps the length of time the cells spent in culture before they were used accounts for this.
Dolly. (Cc BY-SA 2.0 Toni Barros).
Does this mean that Dolly is doomed to a shortened life? She seemed healthy at first and even had babies of her own. But medical problems — probably unrelated to her telomeres — ended with her being euthanized at a relatively young age. But her short telomeres do add another question to the debate about cloning mammals from adult cells. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/13%3A_Aging/13.02%3A_Telomeres.txt |
Embryogenesis is the process by which the embryo forms and develops. In mammals, the term refers chiefly to early stages of prenatal development, whereas the terms fetus and fetal developmentdescribe later stages. Embryogenesis starts with the fertilization of the egg cell (ovum) by a sperm cell, (spermatozoon).
• 14.1: Embryonic Development
The genome of the zygote contains all the genes needed to make the hundreds of different types of cells that will make up the complete animal. There are two major categories of these genes: "housekeeping" genes and tissue-specific genes. However, every cell descended from the zygote has been produced by mitosis and thus contains the complete genome of the organism (with a very few exceptions).
• 14.2: Frog Embryology
The frog egg is a huge cell; its volume is over 1.6 million times larger than a normal frog cell. During embryonic development, the egg will be converted into a tadpole containing millions of cells but containing the same amount of organic matter.
• 14.3: Cleavage
Cleavage refers to the early cell divisions that occur as a fertilized egg begins to develop into an embryo.
• 14.4: The Organizer
In the embryonic development of a zygote, gradients of mRNAs and proteins, deposited in the egg by the mother as she formed it, give rise to cells of diverse fates despite their identical genomes. But is the embryo fully patterned in the fertilized egg? It is difficult to imagine that the relatively simple gradients in the egg could account for all the complex migration and differentiation of cells during embryonic development. And, in fact, the answer is no.
• 14.5: Segmentation - Organizing the Embryo
Insects, like all arthropods, are segmented. The body of Drosophila melanogaster is built from 14 segments, but what signals guide segment formation? The process begins with the gradients of messenger RNA (mRNA) that the mother deposited in her egg before it was fertilized. Shortly after fertilization, these are translated into their proteins with a gradient of bicoid diminishing from anterior to posterior and a gradient of nanos diminishing from posterior to anterior.
• 14.6: Homeobox Genes
Insect (Drosophila) and frog (Xenopus) development passes through three rather different (although often overlapping) phases.
• 14.7: Stem Cells
Stem cells are cells that divide by mitosis to form either two stem cells, thus increasing the size of the stem cell "pool", or one daughter that goes on to differentiate, and one daughter that retains its stem-cell properties. How the choice is made is still unknown. However, several genes have been found whose activity prevents a daughter cell from differentiating.
• 14.8: Embryonic Stem Cells
a research team led by James Thomson of the University of Wisconsin reported (in the 6 November 1998 issue of Science) that they were able to grow human embryonic stem (ES) cells in culture. At the time of implantation, the mammalian embryo is a blastocyst. It consists of the trophoblast — a hollow sphere of cells that will go on to implant in the uterus and develop into the placenta and umbilical cord. inner cell mass (ICM) that will develop into the baby as well as the extraembryonic amnion
• 14.9: Germline vs. Soma
Could a mutation in one of your liver cells ever be passed on to your children? No! Why not? The fusion of one sperm cell and one egg cell represents the only genetic link between the bodies of parents and the body of their child and the cells destined to produce sperm and eggs are set aside very early in embryonic life.
• 14.10: Regeneration
Regeneration is the ability to replace lost or damaged body parts. This ability varies greatly among living things.
Thumbnail: Human embryo, 8-9 weeks, 38 mm. (CC BY-SA 3.0; Anatomist90).
14: Embryonic Development and its Regulation
In animals, one can usually distinguish 4 stages of embryonic development.
• Cleavage
• Patterning
• Differentiation
• Growth
Cleavage
Mitosis and cytokinesis of the zygote, an unusually large cell, produces an increasing number of smaller cells, each with an exact copy of the genome present in the zygote. However, the genes of the zygote are not expressed at first. The early activities of cleavage are controlled by the mother's genome; that is, by mRNAs and proteins she deposited in the unfertilized egg. In humans, the switch-over occurs after 4–8 cells have been produced; in frogs not until thousands of cells have been produced. Cleavage ends with the formation of a blastula.
Patterning
During this phase, the cells produced by cleavage organize themselves in layers and masses, a process called gastrulation. The pattern of the future animal appears:
• front to rear (the anterior-posterior axis)
• back side and belly side (its dorsal-ventral axis)
• left and right sides.
The genome of the zygote contains all the genes needed to make the hundreds of different types of cells that will make up the complete animal. There are two major categories of these genes:
• "housekeeping" genes = those that encode the RNAs and proteins needed by all kinds of cells. Examples:
• genes for tRNAs, rRNAs
• genes encoding the enzymes of glycolysis.
• tissue-specific genes = those that encode mRNAs and hence proteins that are used by one or a few specific kinds of cell. Examples:
• genes for hemoglobin expressed in the precursors of red blood cells
• the gene for insulin expressed in the beta cells of the islets of Langerhans
However, every cell descended from the zygote has been produced by mitosis and thus contains the complete genome of the organism (with a very few exceptions).
Two pieces of evidence:
• Dolly - Dolly is the sheep that was formed by inserting a nucleus from a single cell of an adult sheep into an enucleated sheep egg. She proves that the cell from the adult had lost none of the genes needed to build all the tissues of a sheep.
• Spemann's egg-tying experiments - Many years earlier, the German embryologist Hans Spemann demonstrated the same truth. He used strands of baby hair to tie loops around fertilized salamander eggs. Although the egg half with the nucleus began cleaving normally, the other side did not begin cleavage until a nucleus finally slipped through the knot. So long as the egg was tied so that both halves contained some of the gray crescent, the second half began normal cleavage and ultimately produced a second tadpole (right). Even after 5 mitotic division of the zygote nucleus (the 32-cell stage), the entire genome was still available in each descendant nucleus.
1. A fertilized egg is much larger than the normal cells of an animal's body. Some (e.g., a hen's egg) are truly huge. The frog egg has a volume 1.6 millions times larger than a typical frog cell. The photo is of a 16-cell frog embryo. This mass of cells is no larger than the original egg. The eggs of mammals are smaller, but even they are larger than their descendant cells will be.
2. The cytoplasm of the fertilized egg is not homogeneous. It contains gradients of mRNAs and proteins. These are the products of the mother's genes and were deposited in the egg by her.
3. Cleavage of the fertilized egg partitions it into thousands of cells of normal size. Each contains a nucleus descended from the zygote nucleus.
4. But each nucleus finds itself partitioned off in cytoplasm containing a particular mix of mRNAs and proteins.
5. When the frog blastula has produced some 4,000 cells, transcription and translation of its nuclear genes begins (and the mother's mRNA molecules, that up to now have been the source of all protein synthesis, are destroyed).
6. The genes that are expressed by the nucleus in a given cell are regulated by the molecules, mostly protein transcription factors and microRNAs (miRNAs), found in the cytoplasm surrounding that nucleus.
7. Once a cell-specific pattern of gene expression is launched, that cell may release molecules that regulate the genes of nearby cells.
8. In this way, the foundation is laid for the building of an organism with hundreds of types of differentiated cells — each in its correct location and performing its correct functions.
Xenopus
• During egg formation, molecules of mRNA encoding the protein VegT are deposited at the vegetal pole of the cell.
• Cells that form there during cleavage translate the mRNA into the VegT protein.
• VegT is a transcription factor that turns on genes that produce members of the transforming growth factor-beta (TGF-β) family (e.g., activin).
• These proteins are needed for cells to start down the path to becoming mesoderm.
• Some of those cells will, in turn, become the Spemann organizer.
• Later, the Spemann organizer will secrete molecules that induce the ectodermal cells above them to develop into the tissues of the brain and spinal cord.
Demonstration
Inject the anterior of the fertilized egg with nanos mRNA. The result: another double-posterior larva.
Make female fruit flies that are transgenic for a recombinant gene containing:
• the gene for nanos
• coupled to the 3´ anterior-directing signal of the bicoid gene.
A normal larva is shown on the right. The bright object at the right end of the normal larva and at both ends of the double posterior larva is the tip of the tail. These micrographs are courtesy of Elizabeth Gavis and Ruth Lehmann, in whose lab the third demonstration was performed.
The Mud Snail
The mud snail, Ilyanassa obsoleta, is a small gastropod that lives in mud flats along the Atlantic coast.
Like other protostomes, cleavage of the zygote produces daughter cells that are already committed to their fate. In other words, even as early as the two-cell stage, the cells are no longer totipotent. Unlike humans and other deuterostomes, then, identical twins cannot form.
In the 12 December 2002 issue of Nature, J. David Lambert and Lisa Nagy reported another mechanism by which two daughter cells become committed to different fates even though they have inherited the same genome.
They traced the distribution in the cells of early embryos of the messenger RNAs (mRNAs) encoding 3 proteins that are known to be important in the development of other animals such as Xenopus and Drosophila.
• IoEve, which is Ilyanassa obsoleta's version of even-skipped (eve) in Drosophila;
• IoDpp, which is the snail's version of
• decapentaplegic (dpp) in Drosophila and the genes encoding
• bone morphogenic proteins (BMP2 and BMP4) in vertebrates
• IoTld, which encodes the snail's version of a protein called tolloid in Drosophila.
Lambert and Nagy found that
• in interphase the messenger RNAs were distributed diffusely throughout the cytosol, but
• as the cell got ready for cleavage, the mRNAs collected at only one of the now pair of centrosomes. They were collected there by traveling along the microtubules that radiate out from the centrosome.
• As cleavage continued, the mRNAs moved from the centrosome to a spot on the inner surface of the plasma membrane. They got there by traveling along actin filaments.
• At cytokinesis, this patch of accumulated mRNAs was incorporated exclusively into the smaller daughter cell.
Centrosome sorting (of proteins in this case) also plays a role in determining whether embryonic cells of Caenorhabditis elegans remain in the germline or become the somatic cells of the worm.
What comes next?
Development in Xenopus and Drosophila passes through three rather different (although often overlapping) phases:
• Establishing the main axes (anterior-posterior; dorsal-ventral; left-right). This is done by gradients of mRNAs and proteins encoded by the mother's genes and placed in the egg by her. It has been discussed here.
• Establishing the main body parts such as the notochord and central nervous system in vertebratesand the segments in DrosophilaThese are run by genes of the zygote itself.
• Filling in the details; that is, building the various organs of the animal. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/14%3A_Embryonic_Development_and_its_Regulation/14.01%3A_Embryonic_Development.txt |
The Egg
The frog egg is a huge cell; its volume is over 1.6 million times larger than a normal frog cell. During embryonic development, the egg will be converted into a tadpole containing millions of cells but containing the same amount of organic matter.
• The upper hemisphere of the egg — the animal pole — is dark.
• The lower hemisphere — the vegetal pole — is light.
• When deposited in the water and ready for fertilization, the haploid egg is at metaphase of meiosis II.
Fertilization
Entrance of the sperm initiates a sequence of events:
• Meiosis II is completed.
• The cytoplasm of the egg rotates about 30 degrees relative to the poles.
• In some amphibians (including Xenopus), this is revealed by the appearance of a light-colored band, the gray crescent.
• The gray crescent forms opposite the point where the sperm entered.
• It foretells the future pattern of the animal: its dorsal (D) and ventral (V) surfaces; its anterior (A) and posterior (P); its left and right sides.
• The haploid sperm and egg nuclei fuse to form the diploid zygote nucleus.
Cleavage
The zygote nucleus undergoes a series of mitoses, with the resulting daughter nuclei becoming partitioned off, by cytokinesis, in separate, and ever-smaller, cells. The first cleavage occurs shortly after the zygote nucleus forms. A furrow appears that runs longitudinally through the poles of the egg, passing through the point at which the sperm entered and bisecting the gray crescent. This divides the egg into two halves forming the 2-cell stage. The second cleavage forms the 4-cell stage. The cleavage furrow again runs through the poles but at right angles to the first furrow. The furrow in the third cleavage runs horizontally but in a plane closer to the animal than to the vegetal pole. It produces the 8-cell stage.
The next few cleavages also proceed in synchrony, producing a 16-cell and then a 32-cell embryo. However, as cleavage continues, the cells in the animal pole begin dividing more rapidly than those in the vegetal pole and thus become smaller and more numerous. By the next day, continued cleavage has produced a hollow ball of thousands of cells called the blastula. A fluid-filled cavity, the blastocoel, forms within it.
During this entire process there has been no growth of the embryo. In fact, because the cells of the blastula are so small, the blastula looks just like the original egg to the unaided eye. Not until the blastula contains some 4,000 cells is there any transcription of zygote genes. All of the activities up to now have been run by gene products (mRNA and proteins) deposited by the mother when she formed the egg.
Gastrulation
The start of gastrulation is marked by the pushing inward ("invagination") of cells in the region of the embryo once occupied by the middle of the gray crescent.
This produces an opening (the blastopore) that will be the future anus. a cluster of cells that develops into the Spemann organizer (named after one of the German embryologists who discovered its remarkable inductive properties).
As gastrulation continues, three distinct "germ layers" are formed:
• ectoderm
• mesoderm
• endoderm
Each of these will have special roles to play in building the complete animal. Some are listed in the table.
Germ-layer origin of various body tissues
Ectoderm Mesoderm Endoderm
skin notochord inner lining of gut, liver, pancreas
brain muscles inner lining of lungs
spinal cord blood inner lining of bladder
all other neurons bone thyroid and parathyroid glands
sense receptors sex organs thymus
The Spemann organizer (mostly mesoderm) will develop into the notochord, which is the precursor of the backbone and induce the ectoderm lying above it to begin to form neural tissue instead of skin. This ectoderm grows up into two longitudinal folds, forming the neural folds stage. In time the lips of the folds fuse to form the neural tube. The neural tube eventually develops into the brain and spinal cord.
Differentiation
Although the various layers of cells in the frog gastrula have definite and different fates in store for them, these are not readily apparent in their structure. Only by probing for different patterns of gene expression (e.g., looking for tissue-specific proteins) can their differences be detected. In due course, however, the cells of the embryo take on the specialized structures and functions that they have in the tadpole, forming neurons, blood cells, muscle cells, epithelial cells, etc., etc.
Growth
At the time the tadpole hatches, it is a fully-formed organism. However, it has no more organic matter in it than the original frog egg had. Once able to feed, however, the tadpole can grow. It gains additional molecules with which it can increase the number of cells that make up its various tissues.
14.03: Cleavage
Cleavage refers to the early cell divisions that occur as a fertilized egg begins to develop into an embryo.
Holoblastic Cleavage
In eggs that contain no (mammals) or only moderate amounts (frog) of yolk, cytokinesis divides the cells completely. The figure shows the results of the first two cleavages in the frog embryo.
Meroblastic Cleavage
In eggs that contain a large amount of yolk, cytokinesis does not divide the egg completely.
The hen's egg consists of just a tiny patch of cytoplasm resting on the surface of a large ball of yolk (the "white" of the egg is noncellular accessory protein). When the first cleavages occur in the hen's egg, the cleavage furrows do not continue down through the mass of yolk. Therefore, each of the cells produced in the earliest stages is bound on the top and on the sides by a plasma membrane, but the bottom of the cell is in direct contact with yolk.
This type of meroblastic cleavage is also found in the eggs of fish, reptiles, and 4 species of mammals — the monotremes. This photo, courtesy of H. W. Beames and Richard G. Kessel, shows the zebrafish (Danio) embryo at the 32-cell stage. Note that the cleavage furrows have not continued down through the yolk of the egg.
Insects use a different type of meroblastic cleavage.
The yolk of the eggs of insects is concentrated in the center of the egg. The daughter nuclei produced by mitosis of the zygote nucleus remain suspended within the single egg compartment. After several thousand nuclei have been produced, they migrate to the cytoplasm-rich margin of the egg. Only then does a plasma membrane form around each one.
What does cleavage accomplish in the development of the organism? First, it provides a stockpile of cells out of which the embryo will be constructed. Second, cleavage establishes a normal relationship between the nucleus and the volume of cytoplasm it regulates (and which in turn regulates it). Even small eggs are enormous when compared with other kinds of cells. The volume of the frog egg is about 1.6 million times larger than that of a normal frog cell. But it, too, contains only a single nucleus. During cleavage, thousands of new nuclei are produced by mitosis all of which finally end up in a cell of normal dimensions. The frog blastula, with its thousands of cells is no larger than the original fertilized egg. | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/14%3A_Embryonic_Development_and_its_Regulation/14.02%3A_Frog_Embryology.txt |
In the embryonic development of a zygote, gradients of mRNAs and proteins, deposited in the egg by the mother as she formed it, give rise to cells of diverse fates despite their identical genomes. But is the embryo fully patterned in the fertilized egg? It is difficult to imagine that the relatively simple gradients in the egg could account for all the complex migration and differentiation of cells during embryonic development. And, in fact, the answer is no. However, once these gradients have sent certain cells along a particular path of gene expression, the stage is set for those cells to begin influencing nearby cells to become increasingly diversified.
In other words, cell-intrinsic signals (established between a nucleus and the particular cytoplasmic environment that cleavage has placed it in) lay the foundation for cell-cell interactions to further guide the cells of the embryo to assume their proper position in the embryo and to differentiate into their final specialized form and function.
Cell-cell interactions could — and probably do — occur in several ways:
• diffusion of a signaling molecule out of one cell and into other cells in the vicinity;
• diffusion of a signaling molecule from one cell into an adjacent cell that then secretes the same molecule to diffuse to the next cell and so on (a "cell-relay" mechanism);
• extension of projections from the plasma membrane of one cell until they make direct contact with nearby cells. This enables proteins embedded in the plasma membrane to serve as signaling molecules.
The Spemann Organizer
In 1924, the Ph.D. student Hilde Mangold working in the laboratory of German embryologist Hans Spemann performed an experiment that demonstrated that the pattern of development of cells is influenced by the activities of other cells and stimulated a search, which continues to this day, for the signals at work. Spemann and Mangold knew that the cells that develop in the region of the gray crescent migrate into the embryo during gastrulation and form the notochord (the future backbone; made of mesoderm). She cut out a piece of tissue from the gray crescent region of one newt gastrula and transplanted it into the ventral side of a second newt gastrula. To make it easier to follow the fate of the transplant, she used the embryo of one variety of newt as the donor and a second variety as the recipient.
The remarkable results:
• the transplanted tissue developed into a second notochord
• neural folds developed above the extra notochord
• these went on to form a second central nervous system (portions of brain and spinal cord) and eventually
• a two-headed tadpole.
The most remarkable finding of all was that the neural folds were built from recipient cells, not donor cells. In other words, the transplant had altered the fate of the overlying cells (which normally would have ended up forming skin [epidermis] on the side of the animal) so that they produced a second head instead!
Spemann and Mangold used the term induction for the ability of one group of cells to influence the fate of another. And because of the remarkable inductive power of the gray crescent cells, they called this region the organizer. Ever since then, vigorous searches have been made to identify the molecules liberated by the organizer that induce overlying cells to become nerve tissue. One candidate after another has been put forward and then found not to be responsible. Part of the problem has been that not until just recently has it become clear that the organizer does NOT induce the central nervous system but instead it prevents signals originating from the ventral side of the blastula from inducing skin (epidermis) there.
This is how it works:
• Cells on the ventral side of the blastula secrete a variety of proteins such as bone morphogenetic protein-4 (BMP-4)
• These induce the ectoderm above to become epidermis.
• If their action is blocked, the ectodermal cells are allowed to follow their default pathway, which is to become nerve tissue of the brain and spinal cord.
• The Spemann organizer blocks the action of BMP-4 by secreting molecules of the proteins chordin and noggin
• Both of these physically bind to BMP-4 molecules in the extracellular space and thus prevent BMP-4 from binding to receptors on the surface of the overlying ectoderm cells.
• This allows the ectodermal cells to follow their intrinsic path to forming neural folds and, eventually, the brain and spinal cord.
In the Spemann/Mangold experiment, transplanting an organizer to the ventral side provided a second source of chordin. This blocked BMP-4 binding to the overlying ectoderm and thus changed the fate of those cells to forming a second central nervous system rather than skin.
What Organizes the Organizer?
Protein synthesis by the cells of the organizer requires transcription of the relevant genes (e.g., chordin). Expression of organizer genes depends first on Wnt transcription factors. Their messenger RNAs were deposited by the mother in the vegetal pole of the egg. After fertilization and formation of the gray crescent, they migrated into the gray crescent region (destined to become the organizer) where they were translated into Wnt protein.
Its accumulation on the dorsal side of the embryo unleashes the activity of Nodal — a member of the Transforming Growth Factor-beta (TGF-β) family. Nodal induces these dorsal cells to begin expressing the proteins of Spemann's organizer.
A Tail Organizer
One of the distinguishing features of vertebrates is their tail, which extends out behind the anus. French researchers have reported (in the 24 July 2003 issue of Nature) their discovery of a tail "organizer", that is, a cluster of cells in the embryo that induces nearby cells to contribute to the formation of the tail. They worked with the zebrafish, Danio rerio (which also has a head organizer like that of newts). They removed tiny clusters of cells from the ventral part of the blastula (a region roughly opposite where the Spemann-like organizer forms) and transplanted this into a region of the host embryo that would normally form flank. The result: a second tail.
Using a fluorescent label, they were able to show that the extra tail was made not only from descendants of the transplanted cells but also from host cells that would normally have made flank. Three proteins were essential:
• a Wnt protein (establishes the anterior-posterior axis in all bilaterians)
• BMP (establishes the dorsal-ventral axis in all bilaterians)
• Nodal (establishes the left-right axis in all bilaterians)
Patterning the central nervous system in Drosophila
Remarkably, it turns out that proteins similar in structure to the bone morphogenetic proteins and also to chordin are found in Drosophila. The role of BMP-4 is taken by a related protein encoded by the decapentaplegic gene (dpp) and the role of chordin is taken by a related protein called SOG encoded by the gene called short gastrulation.
In fact, these proteins and their mRNAs are completely interchangeable! An injection of the mRNAs for BMP-4 or chordin into the blastoderm of the Drosophila embryo can replace the function of DPP and SOG respectively, and conversely, injections of mRNA for DPP or SOG into the Xenopus embryo mimics the functions of BMP-4 and chordin respectively.
Table \(1\): A selection of antagonistic pairs of proteins that guide the patterning of the embryo.
Xenopus blocked by chordin
and also by noggin
Drosophila Decapentaplegic (DPP) blocked by short gastrulation (SOG)
and also by a noggin homolog?
Dorsal vs Ventral Nerve Cords
Although their actions are similar, the distribution of these proteins in Drosophila differs from that in Xenopus (as well as in mammals and other vertebrates). In Drosophila, DPP is produced in the dorsal region of the embryo and SOG is produced in the ventral region.
However, their actions on overlying cells are the same as in Xenopus; that is, the SOG protein prevents the DPP protein from blocking the formation of the central nervous system. The result in Drosophila is that its central nervous system forms on the ventral side of the embryo, not on the dorsal! And, you may remember that one of the distinguishing traits of all arthropods (insects, crustaceans, arachnids) as well as many other invertebrates, such as the annelid worms, is a ventral nerve cord. Chordates, including all vertebrates, have a dorsal (spinal) nerve cord.
We're halfway done!
Xenopus development (and probably that of animals in general) passes through three rather different (although often overlapping) phases:
• establishing the main axes (dorsal-ventral; anterior-posterior; left-right). This is done by gradients of mRNAs and proteins encoded by the mother's genes and placed in the egg by her.
• establishing the main body parts such as
• the notochord and central nervous system in vertebrates (discussed here and also described in Fro.g Embryology)
• and the segments in Drosophila
These are run by genes of the zygote itself.
• filling in the details; that is, building the various organs of the animal. (Our examples will include the wings, legs, and eyes of Drosophila.) | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/14%3A_Embryonic_Development_and_its_Regulation/14.04%3A_The_Organizer.txt |
Insects, like all arthropods, are segmented. The body of Drosophila melanogaster is built from 14 segments:
• 3 segments make up the head with its antennae and mouth parts.
• 3 segments make up the thorax. Each thoracic segment has a pair of legs (insects are the six-legged creatures). In Drosophila (and other flies), the middle thoracic segment carries a single pair of wings; the hind segment a pair of halteres.
• 8 abdominal segments.
What signals guide segment formation? The process begins with the gradients of messenger RNA (mRNA) that the mother deposited in her egg before it was fertilized. Shortly after fertilization, these are translated into their proteins with a gradient of bicoid diminishing from anterior to posterior and a gradient of nanos diminishing from posterior to anterior.
• Bicoid protein is a transcription factor. It binds to the promoter of a gene called hunchback (hb), turning it ON (red arrow).
• Nanos protein binds to hunchback mRNAs, inhibiting their translation (blue bar).
• These effects combine to produce a high level of hunchback protein at the anterior of the embryo; with a sharp cut-off toward the posterior.
• The hunchback protein is also a transcription factor (as we shall see).
• These concentration gradients regulate the turning on and off of other genes in sharply-defined regions of the embryo.
• These establish the various segments of the body.
Eve stripe 2
The gene even-skipped (eve) is expressed in 7 bands or stripes corresponding to 7 of Drosophila's 14 segments (skipping the even-numbered ones). The photo (courtesy of Peter A. Lawrence and Blackwell Scientific Publications) shows the 7 stripes of eve activation.
At first the gene is expressed in fairly broad zones, but in time its expression becomes restricted to ever-narrower stripes. The mechanism by which this occurs is known for the second stripe.
The eve promoter has binding sites for the proteins encoded by bicoid (bcd), hunchback (hb), giant (gt) and Krüppel (Kr).
• Binding of bicoid and hunchback proteins stimulates transcription of eve.
• Binding of giant and Krüppel represses transcription.
Trapped in a valley between high levels of the giant and Krüppel proteins, expression of eve in the second stripe finally becomes limited to a band of cells only one cell thick. (A different set of promoter sites is used in the third eve stripe so expression is not repressed there.)In principle, then, such a system of interacting gradients of transcription factors could act as on-off switches, which in time partition the embryo into its future segments.
Drosophila development (and probably that of animals in general) passes through three rather different (although often overlapping) phases:
• establishing the main axes (dorsal-ventral; anterior-posterior; left-right). This is done by gradients of mRNAs and proteins encoded by the mother's genes and placed in the egg by her.
• establishing the main body parts such as the notochord and central nervous system in vertebrates.and the segments in Drosophila (discussed here). These are run by genes of the zygote itself.
• filling in the details; that is, building the various organs of the animal. (Our example will include the wings, legs, and eyes of Drosophila.) | textbooks/bio/Introductory_and_General_Biology/Biology_(Kimball)/14%3A_Embryonic_Development_and_its_Regulation/14.05%3A_Segmentation_-_Organizing_the_Embryo.txt |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.