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Learning Objectives
• Describe how light energy is converted into ATP and NADPH.
How Light-Dependent Reactions Work
The overall function of light-dependent reactions, the first stage of photosynthesis, is to convert solar energy into chemical energy in the form of NADPH and ATP, which are used in light-independent reactions and fuel the assembly of sugar molecules. Protein complexes and pigment molecules work together to produce NADPH and ATP.
Producing Chemical Energy
Light energy is converted into chemical energy in a multiprotein complex called a photosystem. Two types of photosystems, photosystem I (PSI) and photosystem II (PSII), are found in the thylakoid membrane inside the chloroplast. Each photosystem consists of multiple antenna proteins that contain a mixture of 300–400 chlorophyll a and b molecules, as well as other pigments like carotenoids. Cytochrome b6f complex and ATP synthase are also major protein complexes in the thylakoid membrane that work with the photosystems to create ATP and NADPH.
The two photosystems absorb light energy through proteins containing pigments, such as chlorophyll. The light-dependent reactions begin in photosystem II. In PSII, energy from sunlight is used to split water, which releases two electrons, two hydrogen atoms, and one oxygen atom. When a chlorophyll a molecule within the reaction center of PSII absorbs a photon, the electron in this molecule attains a higher energy level. Because this state of an electron is very unstable, the electron is transferred to another molecule creating a chain of redox reactions called an electron transport chain (ETC). The electron flow goes from PSII to cytochrome b6f to PSI; as electrons move between these two photosystems, they lose energy. Because the electrons have lost energy prior to their arrival at PSI, they must be re-energized by PSI. Therefore, another photon is absorbed by the PSI antenna. That energy is transmitted to the PSI reaction center. This reaction center, known as P700, is oxidized and sends a high-energy electron to reduce NADP+ to NADPH. This process illustrates oxygenic photosynthesis, wherein the first electron donor is water and oxygen is created as a waste product.
Cytochrome b6f and ATP synthase work together to create ATP. This process, called photophosphorylation, occurs in two different ways. In non-cyclic photophosphorylation, cytochrome b6f uses the energy of electrons from PSII to pump hydrogen ions from the lumen (an area of high concentration) to the stroma (an area of low concentration). The energy released by the hydrogen ion stream allows ATP synthase to attach a third phosphate group to ADP, which forms ATP. This flow of hydrogen ions through ATP synthase is called chemiosmosis because the ions move from an area of high to an area of low concentration through a semi-permeable structure. In cyclic photophosphorylation, cytochrome b6f uses the energy of electrons from both PSII and PSI to create more ATP and to stop the production of NADPH. Cyclic phosphorylation is important to maintain the right proportions of NADPH and ATP, which will carry out light-independent reactions later on.
The net-reaction of all light-dependent reactions in oxygenic photosynthesis is: 2H2O + 2NADP+ + 3ADP + 3Pi → O2 + 2NADPH + 3ATP
Key Points
• Light energy splits water and extracts electrons in photosystem II (PSII); then electrons are moved from PSII to cytochrome b6f to photosystem I (PSI) and reduce in energy.
• Electrons are re-energized in PSI and those high energy electrons reduce NADP+ to NADPH.
• In non-cyclic photophosphorylation, cytochrome b6f uses the energy of electrons from PSII to pump hydrogen ions from the lumen to the stroma; this energy allows ATP synthase to attach a third phosphate group to ADP, which forms ATP.
• In cyclic photophosphorylation, cytochrome b6f uses the energy of electrons from both PSII and PSI to create more ATP and to stop the production of NADPH, maintaining the right proportions of NADPH and ATP.
Key Terms
• photosystem: Either of two biochemical systems, active in chloroplasts, that are part of photosynthesis.
• photophosphorylation: The addition of a phosphate (PO43-) group to a protein or other organic molecule by photosynthesis.
• chemiosmosis: The movement of ions across a selectively permeable membrane, down their electrochemical gradient.
Contributions and Attributions
• visible light. Provided by: Wiktionary. Located at: http://en.wiktionary.org/wiki/visible_light. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...ol11448/latest. License: CC BY: Attribution
• Boundless. Provided by: Boundless Learning. Located at: www.boundless.com//biology/de...netic-spectrum. License: CC BY-SA: Attribution-ShareAlike
• wavelength. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/wavelength. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_03.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_02.jpg. License: CC BY: Attribution
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...ol11448/latest. License: CC BY: Attribution
• spectrophotometer. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/spectrophotometer. License: CC BY-SA: Attribution-ShareAlike
• carotenoid. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/carotenoid. License: CC BY-SA: Attribution-ShareAlike
• chlorophyll. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/chlorophyll. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_03.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_02.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_04.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest..._02_05abcd.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_06.jpg. License: CC BY: Attribution
• photosystem. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/photosystem. License: CC BY-SA: Attribution-ShareAlike
• Cell Biology/Energy supply/Light Dependent Reactions. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Cell_Bi...dent_Reactions. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...ol11448/latest. License: CC BY: Attribution
• photophosphorylation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/photophosphorylation. License: CC BY-SA: Attribution-ShareAlike
• chemiosmosis. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/chemiosmosis. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_03.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_02.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_04.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest..._02_05abcd.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_06.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...e_08_02_08.jpg. License: CC BY: Attribution
• OpenStax College, The Light-Dependent Reactions of Photosynthesis. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44448/latest...08_02_07ab.png. License: CC BY: Attribution | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/08%3A_Photosynthesis/8.05%3A_The_Light-Dependent_Reactions/8.5C%3A_Processes_of_the_Light-Dependent_Reactions.txt |
After the energy from the sun is converted and packaged into ATP and NADPH, the cell has the fuel needed to build food in the form of carbohydrate molecules. The carbohydrate molecules made will have a backbone of carbon atoms. Where does the carbon come from? The carbon atoms used to build carbohydrate molecules come from carbon dioxide, the gas that animals exhale with each breath. The Calvin cycle is the term used for the reactions of photosynthesis that use the energy stored by the light-dependent reactions to form glucose and other carbohydrate molecules.
The Interworkings of the Calvin Cycle
In plants, carbon dioxide (CO2) enters the chloroplast through the stomata and diffuses into the stroma of the chloroplast—the site of the Calvin cycle reactions where sugar is synthesized. The reactions are named after the scientist who discovered them, and reference the fact that the reactions function as a cycle. Others call it the Calvin-Benson cycle to include the name of another scientist involved in its discovery (Figure $1$).
The Calvin cycle reactions (Figure $2$) can be organized into three basic stages: fixation, reduction, and regeneration. In the stroma, in addition to CO2, two other chemicals are present to initiate the Calvin cycle: an enzyme abbreviated RuBisCO, and the molecule ribulose bisphosphate (RuBP). RuBP has five atoms of carbon and a phosphate group on each end.
RuBisCO catalyzes a reaction between CO2 and RuBP, which forms a six-carbon compound that is immediately converted into two three-carbon compounds. This process is called carbon fixation, because CO2 is “fixed” from its inorganic form into organic molecules.
ATP and NADPH use their stored energy to convert the three-carbon compound, 3-PGA, into another three-carbon compound called G3P. This type of reaction is called a reduction reaction, because it involves the gain of electrons. A reduction is the gain of an electron by an atom or molecule. The molecules of ADP and NAD+, resulting from the reduction reaction, return to the light-dependent reactions to be re-energized.
One of the G3P molecules leaves the Calvin cycle to contribute to the formation of the carbohydrate molecule, which is commonly glucose (C6H12O6). Because the carbohydrate molecule has six carbon atoms, it takes six turns of the Calvin cycle to make one carbohydrate molecule (one for each carbon dioxide molecule fixed). The remaining G3P molecules regenerate RuBP, which enables the system to prepare for the carbon-fixation step. ATP is also used in the regeneration of RuBP.
In summary, it takes six turns of the Calvin cycle to fix six carbon atoms from CO2. These six turns require energy input from 12 ATP molecules and 12 NADPH molecules in the reduction step and 6 ATP molecules in the regeneration step.
CONCEPT IN ACTION
The following is a link to an animation of the Calvin cycle. Click Stage 1, Stage 2, and then Stage 3 to see G3P and ATP regenerate to form RuBP.
EVOLUTION IN ACTION: Photosynthesis
The shared evolutionary history of all photosynthetic organisms is conspicuous, as the basic process has changed little over eras of time. Even between the giant tropical leaves in the rainforest and tiny cyanobacteria, the process and components of photosynthesis that use water as an electron donor remain largely the same. Photosystems function to absorb light and use electron transport chains to convert energy. The Calvin cycle reactions assemble carbohydrate molecules with this energy.
However, as with all biochemical pathways, a variety of conditions leads to varied adaptations that affect the basic pattern. Photosynthesis in dry-climate plants (Figure $3$) has evolved with adaptations that conserve water. In the harsh dry heat, every drop of water and precious energy must be used to survive. Two adaptations have evolved in such plants. In one form, a more efficient use of CO2 allows plants to photosynthesize even when CO2 is in short supply, as when the stomata are closed on hot days. The other adaptation performs preliminary reactions of the Calvin cycle at night, because opening the stomata at this time conserves water due to cooler temperatures. In addition, this adaptation has allowed plants to carry out low levels of photosynthesis without opening stomata at all, an extreme mechanism to face extremely dry periods.
Photosynthesis in Prokaryotes
The two parts of photosynthesis—the light-dependent reactions and the Calvin cycle—have been described, as they take place in chloroplasts. However, prokaryotes, such as cyanobacteria, lack membrane-bound organelles. Prokaryotic photosynthetic autotrophic organisms have infoldings of the plasma membrane for chlorophyll attachment and photosynthesis (Figure $4$). It is here that organisms like cyanobacteria can carry out photosynthesis.
The Energy Cycle
Living things access energy by breaking down carbohydrate molecules. However, if plants make carbohydrate molecules, why would they need to break them down? Carbohydrates are storage molecules for energy in all living things. Although energy can be stored in molecules like ATP, carbohydrates are much more stable and efficient reservoirs for chemical energy. Photosynthetic organisms also carry out the reactions of respiration to harvest the energy that they have stored in carbohydrates, for example, plants have mitochondria in addition to chloroplasts.
You may have noticed that the overall reaction for photosynthesis:
$\ce{6CO2 + 6H2O→C6H12O6 + 6O2}\nonumber$
is the reverse of the overall reaction for cellular respiration:
$\ce{6O2 + C6H12O6→6CO2 + 6H2O}\nonumber$
Photosynthesis produces oxygen as a byproduct, and respiration produces carbon dioxide as a byproduct.
In nature, there is no such thing as waste. Every single atom of matter is conserved, recycling indefinitely. Substances change form or move from one type of molecule to another, but never disappear (Figure $5$).
CO2 is no more a form of waste produced by respiration than oxygen is a waste product of photosynthesis. Both are byproducts of reactions that move on to other reactions. Photosynthesis absorbs energy to build carbohydrates in chloroplasts, and aerobic cellular respiration releases energy by using oxygen to break down carbohydrates. Both organelles use electron transport chains to generate the energy necessary to drive other reactions. Photosynthesis and cellular respiration function in a biological cycle, allowing organisms to access life-sustaining energy that originates millions of miles away in a star.
Summary
Using the energy carriers formed in the first stage of photosynthesis, the Calvin cycle reactions fix CO2 from the environment to build carbohydrate molecules. An enzyme, RuBisCO, catalyzes the fixation reaction, by combining CO2 with RuBP. The resulting six-carbon compound is broken down into two three-carbon compounds, and the energy in ATP and NADPH is used to convert these molecules into G3P. One of the three-carbon molecules of G3P leaves the cycle to become a part of a carbohydrate molecule. The remaining G3P molecules stay in the cycle to be formed back into RuBP, which is ready to react with more CO2. Photosynthesis forms a balanced energy cycle with the process of cellular respiration. Plants are capable of both photosynthesis and cellular respiration, since they contain both chloroplasts and mitochondria.
Glossary
Calvin cycle
the reactions of photosynthesis that use the energy stored by the light-dependent reactions to form glucose and other carbohydrate molecules
carbon fixation
the process of converting inorganic CO2 gas into organic compounds
8.06: Carbon Fixation- The Calvin Cycle
Learning Objectives
• Describe the Calvin Cycle
The Calvin Cycle
In plants, carbon dioxide (CO2) enters the leaves through stomata, where it diffuses over short distances through intercellular spaces until it reaches the mesophyll cells. Once in the mesophyll cells, CO2 diffuses into the stroma of the chloroplast, the site of light-independent reactions of photosynthesis. These reactions actually have several names associated with them. Other names for light-independent reactions include the Calvin cycle, the Calvin-Benson cycle, and dark reactions. The most outdated name is dark reactions, which can be misleading because it implies incorrectly that the reaction only occurs at night or is independent of light, which is why most scientists and instructors no longer use it.
The light-independent reactions of the Calvin cycle can be organized into three basic stages: fixation, reduction, and regeneration.
Stage 1: Fixation
In the stroma, in addition to CO2,two other components are present to initiate the light-independent reactions: an enzyme called ribulose bisphosphate carboxylase (RuBisCO) and three molecules of ribulose bisphosphate (RuBP). RuBP has five atoms of carbon, flanked by two phosphates. RuBisCO catalyzes a reaction between CO2 and RuBP. For each CO2 molecule that reacts with one RuBP, two molecules of 3-phosphoglyceric acid (3-PGA) form. 3-PGA has three carbons and one phosphate. Each turn of the cycle involves only one RuBP and one carbon dioxide and forms two molecules of 3-PGA. The number of carbon atoms remains the same, as the atoms move to form new bonds during the reactions (3 atoms from 3CO2 + 15 atoms from 3RuBP = 18 atoms in 3 atoms of 3-PGA). This process is called carbon fixation because CO2 is “fixed” from an inorganic form into organic molecules.
Stage 2: Reduction
ATP and NADPH are used to convert the six molecules of 3-PGA into six molecules of a chemical called glyceraldehyde 3-phosphate (G3P). This is a reduction reaction because it involves the gain of electrons by 3-PGA. Recall that a reduction is the gain of an electron by an atom or molecule. Six molecules of both ATP and NADPH are used. For ATP, energy is released with the loss of the terminal phosphate atom, converting it to ADP; for NADPH, both energy and a hydrogen atom are lost, converting it into NADP+. Both of these molecules return to the nearby light-dependent reactions to be reused and reenergized.
Stage 3: Regeneration
At this point, only one of the G3P molecules leaves the Calvin cycle and is sent to the cytoplasm to contribute to the formation of other compounds needed by the plant. Because the G3P exported from the chloroplast has three carbon atoms, it takes three “turns” of the Calvin cycle to fix enough net carbon to export one G3P. But each turn makes two G3Ps, thus three turns make six G3Ps. One is exported while the remaining five G3P molecules remain in the cycle and are used to regenerate RuBP, which enables the system to prepare for more CO2 to be fixed. Three more molecules of ATP are used in these regeneration reactions.
Key Points
• The Calvin cycle refers to the light-independent reactions in photosynthesis that take place in three key steps.
• Although the Calvin Cycle is not directly dependent on light, it is indirectly dependent on light since the necessary energy carriers ( ATP and NADPH) are products of light-dependent reactions.
• In fixation, the first stage of the Calvin cycle, light-independent reactions are initiated; CO2 is fixed from an inorganic to an organic molecule.
• In the second stage, ATP and NADPH are used to reduce 3-PGA into G3P; then ATP and NADPH are converted to ADP and NADP+, respectively.
• In the last stage of the Calvin Cycle, RuBP is regenerated, which enables the system to prepare for more CO2 to be fixed.
Key Terms
• light-independent reaction: chemical reactions during photosynthesis that convert carbon dioxide and other compounds into glucose, taking place in the stroma
• rubisco: (ribulose bisphosphate carboxylase) a plant enzyme which catalyzes the fixing of atmospheric carbon dioxide during photosynthesis by catalyzing the reaction between carbon dioxide and RuBP
• ribulose bisphosphate: an organic substance that is involved in photosynthesis, reacts with carbon dioxide to form 3-PGA | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/08%3A_Photosynthesis/8.06%3A_Carbon_Fixation-_The_Calvin_Cycle/8.6B%3A_The_Calvin_Cycle.txt |
Learning Objectives
• Compare C4 and CAM photosynthesis
Photosynthesis in desert plants has evolved adaptations that conserve water. In harsh, dry heat, every drop of water must be used to survive. Because stomata must open to allow for the uptake of CO2, water escapes from the leaf during active photosynthesis. Desert plants have evolved processes to conserve water and deal with harsh conditions. A more efficient use of CO2 allows plants to adapt to living with less water.
Some plants such as cacti can prepare materials for photosynthesis during the night by a temporary carbon fixation and storage process, because opening the stomata at this time conserves water due to cooler temperatures. In addition, cacti have evolved the ability to carry out low levels of photosynthesis without opening stomata at all, a mechanism for surviving extremely dry periods.
CAM Photosynthesis
Xerophytes, such as cacti and most succulents, also use
phosphoenolpyruvate (PEP) carboxylase to capture carbon dioxide in a process called crassulacean acid metabolism (CAM). In contrast to C4 metabolism, which physically separates the CO2 fixation to PEP from the Calvin cycle, CAM temporally separates these two processes.
CAM plants have a different leaf anatomy from C3 plants, and fix the CO2 at night, when their stomata are open. CAM plants store the CO2 mostly in the form of malic acid via carboxylation of phosphoenolpyruvate to oxaloacetate, which is then reduced to malate. Decarboxylation of malate during the day releases CO2 inside the leaves, thus allowing carbon fixation to 3-phosphoglycerate by RuBisCO. Sixteen thousand species of plants use CAM.
C4 Carbon Fixation
The C4 pathway bears resemblance to CAM; both act to concentrate CO2 around RuBisCO, thereby increasing its efficiency. CAM concentrates it temporally, providing CO2 during the day and not at night, when respiration is the dominant reaction.
C4 plants, in contrast, concentrate CO2 spatially, with a RuBisCO reaction centre in a “bundle sheath cell” that is inundated with CO2. Due to the inactivity required by the CAM mechanism, C4 carbon fixation has a greater efficiency in terms of PGA synthesis.
Cross section of maize, a C4 plant
Cross section of a C4 plant, specifically of a maize leaf. Drawing based on microscopic images courtesy of Cambridge University Plant Sciences Department.
C4 plants can produce more sugar than C3 plants in conditions of high light and temperature. Many important crop plants are C4 plants, including maize, sorghum, sugarcane, and millet. Plants that do not use PEP-carboxylase in carbon fixation are called C3 plants because the primary carboxylation reaction, catalyzed by RuBisCO, produces the three-carbon 3-phosphoglyceric acids directly in the Calvin-Benson cycle. Over 90% of plants use C3 carbon fixation, compared to 3% that use C4 carbon fixation; however, the evolution of C4 in over 60 plant lineages makes it a striking example of convergent evolution.
Key Points
• The process of photosynthesis in desert plants has evolved mechanisms to conserve water.
• Plants that use crassulacean acid metabolism (CAM) photosynthesis fix CO2 at night, when their stomata are open.
• Plants that use C4 carbon fixation concentrate carbon dioxide spatially, using “bundle sheath cells” which are inundated with CO2.
Key Terms
• crassulacean acid metabolism: A carbon fixation pathway that evolved in some plants as an adaptation to arid conditions, in which the stomata in the leaves remain shut during the day to reduce evapotranspiration, but open at night to collect carbon dioxide (CO2).
• C4 carbon fixation: A form of photosynthesis in which plants concentrate CO2 spatially, with a RuBisCO reaction centre in a “bundle sheath cell” that is inundated with CO2 | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/08%3A_Photosynthesis/8.07%3A_Photorespiration/8.7A%3A_CAM_and_C4_Photosynthesis.txt |
Skills to Develop
• Describe four types of signaling found in multicellular organisms
• Compare internal receptors with cell-surface receptors
• Recognize the relationship between a ligand’s structure and its mechanism of action
There are two kinds of communication in the world of living cells. Communication between cells is called intercellular signaling, and communication within a cell is called intracellular signaling. An easy way to remember the distinction is by understanding the Latin origin of the prefixes: inter- means "between" (for example, intersecting lines are those that cross each other) and intra- means "inside" (like intravenous).
Chemical signals are released by signaling cells in the form of small, usually volatile or soluble molecules called ligands. A ligand is a molecule that binds another specific molecule, in some cases, delivering a signal in the process. Ligands can thus be thought of as signaling molecules. Ligands interact with proteins in target cells, which are cells that are affected by chemical signals; these proteins are also called receptors. Ligands and receptors exist in several varieties; however, a specific ligand will have a specific receptor that typically binds only that ligand.
Forms of Signaling
There are four categories of chemical signaling found in multicellular organisms: paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across gap junctions (Figure \(1\)). The main difference between the different categories of signaling is the distance that the signal travels through the organism to reach the target cell. Not all cells are affected by the same signals.
Paracrine Signaling
Signals that act locally between cells that are close together are called paracrine signals. Paracrine signals move by diffusion through the extracellular matrix. These types of signals usually elicit quick responses that last only a short amount of time. In order to keep the response localized, paracrine ligand molecules are normally quickly degraded by enzymes or removed by neighboring cells. Removing the signals will reestablish the concentration gradient for the signal, allowing them to quickly diffuse through the intracellular space if released again.
One example of paracrine signaling is the transfer of signals across synapses between nerve cells. A nerve cell consists of a cell body, several short, branched extensions called dendrites that receive stimuli, and a long extension called an axon, which transmits signals to other nerve cells or muscle cells. The junction between nerve cells where signal transmission occurs is called a synapse. A synaptic signal is a chemical signal that travels between nerve cells. Signals within the nerve cells are propagated by fast-moving electrical impulses. When these impulses reach the end of the axon, the signal continues on to a dendrite of the next cell by the release of chemical ligands called neurotransmitters by the presynaptic cell (the cell emitting the signal). The neurotransmitters are transported across the very small distances between nerve cells, which are called chemical synapses (Figure \(2\)). The small distance between nerve cells allows the signal to travel quickly; this enables an immediate response, such as, Take your hand off the stove!
When the neurotransmitter binds the receptor on the surface of the postsynaptic cell, the electrochemical potential of the target cell changes, and the next electrical impulse is launched. The neurotransmitters that are released into the chemical synapse are degraded quickly or get reabsorbed by the presynaptic cell so that the recipient nerve cell can recover quickly and be prepared to respond rapidly to the next synaptic signal.
Endocrine Signaling
Signals from distant cells are called endocrine signals, and they originate from endocrine cells. (In the body, many endocrine cells are located in endocrine glands, such as the thyroid gland, the hypothalamus, and the pituitary gland.) These types of signals usually produce a slower response but have a longer-lasting effect. The ligands released in endocrine signaling are called hormones, signaling molecules that are produced in one part of the body but affect other body regions some distance away.
Hormones travel the large distances between endocrine cells and their target cells via the bloodstream, which is a relatively slow way to move throughout the body. Because of their form of transport, hormones get diluted and are present in low concentrations when they act on their target cells. This is different from paracrine signaling, in which local concentrations of ligands can be very high.
Autocrine Signaling
Autocrine signals are produced by signaling cells that can also bind to the ligand that is released. This means the signaling cell and the target cell can be the same or a similar cell (the prefix auto- means self, a reminder that the signaling cell sends a signal to itself). This type of signaling often occurs during the early development of an organism to ensure that cells develop into the correct tissues and take on the proper function. Autocrine signaling also regulates pain sensation and inflammatory responses. Further, if a cell is infected with a virus, the cell can signal itself to undergo programmed cell death, killing the virus in the process. In some cases, neighboring cells of the same type are also influenced by the released ligand. In embryological development, this process of stimulating a group of neighboring cells may help to direct the differentiation of identical cells into the same cell type, thus ensuring the proper developmental outcome.
Direct Signaling Across Gap Junctions
Gap junctions in animals and plasmodesmata in plants are connections between the plasma membranes of neighboring cells. These water-filled channels allow small signaling molecules, called intracellular mediators, to diffuse between the two cells. Small molecules, such as calcium ions (Ca2+), are able to move between cells, but large molecules like proteins and DNA cannot fit through the channels. The specificity of the channels ensures that the cells remain independent but can quickly and easily transmit signals. The transfer of signaling molecules communicates the current state of the cell that is directly next to the target cell; this allows a group of cells to coordinate their response to a signal that only one of them may have received. In plants, plasmodesmata are ubiquitous, making the entire plant into a giant, communication network.
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligand. There are two types of receptors, internal receptors and cell-surface receptors.
Internal receptors
Internal receptors, also known as intracellular or cytoplasmic receptors, are found in the cytoplasm of the cell and respond to hydrophobic ligand molecules that are able to travel across the plasma membrane. Once inside the cell, many of these molecules bind to proteins that act as regulators of mRNA synthesis (transcription) to mediate gene expression. Gene expression is the cellular process of transforming the information in a cell's DNA into a sequence of amino acids, which ultimately forms a protein. When the ligand binds to the internal receptor, a conformational change is triggered that exposes a DNA-binding site on the protein. The ligand-receptor complex moves into the nucleus, then binds to specific regulatory regions of the chromosomal DNA and promotes the initiation of transcription (Figure \(3\)). Transcription is the process of copying the information in a cells DNA into a special form of RNA called messenger RNA (mRNA); the cell uses information in the mRNA (which moves out into the cytoplasm and associates with ribosomes) to link specific amino acids in the correct order, producing a protein. Internal receptors can directly influence gene expression without having to pass the signal on to other receptors or messengers.
Cell-Surface Receptors
Cell-surface receptors, also known as transmembrane receptors, are cell surface, membrane-anchored (integral) proteins that bind to external ligand molecules. This type of receptor spans the plasma membrane and performs signal transduction, in which an extracellular signal is converted into an intercellular signal. Ligands that interact with cell-surface receptors do not have to enter the cell that they affect. Cell-surface receptors are also called cell-specific proteins or markers because they are specific to individual cell types.
Because cell-surface receptor proteins are fundamental to normal cell functioning, it should come as no surprise that a malfunction in any one of these proteins could have severe consequences. Errors in the protein structures of certain receptor molecules have been shown to play a role in hypertension (high blood pressure), asthma, heart disease, and cancer.
Each cell-surface receptor has three main components: an external ligand-binding domain, a hydrophobic membrane-spanning region, and an intracellular domain inside the cell. The ligand-binding domain is also called the extracellular domain. The size and extent of each of these domains vary widely, depending on the type of receptor.
Evolution Connection: How Viruses Recognize a Host
Unlike living cells, many viruses do not have a plasma membrane or any of the structures necessary to sustain life. Some viruses are simply composed of an inert protein shell containing DNA or RNA. To reproduce, viruses must invade a living cell, which serves as a host, and then take over the hosts cellular apparatus. But how does a virus recognize its host?
Viruses often bind to cell-surface receptors on the host cell. For example, the virus that causes human influenza (flu) binds specifically to receptors on membranes of cells of the respiratory system. Chemical differences in the cell-surface receptors among hosts mean that a virus that infects a specific species (for example, humans) cannot infect another species (for example, chickens).
However, viruses have very small amounts of DNA or RNA compared to humans, and, as a result, viral reproduction can occur rapidly. Viral reproduction invariably produces errors that can lead to changes in newly produced viruses; these changes mean that the viral proteins that interact with cell-surface receptors may evolve in such a way that they can bind to receptors in a new host. Such changes happen randomly and quite often in the reproductive cycle of a virus, but the changes only matter if a virus with new binding properties comes into contact with a suitable host. In the case of influenza, this situation can occur in settings where animals and people are in close contact, such as poultry and swine farms.1 Once a virus jumps to a new host, it can spread quickly. Scientists watch newly appearing viruses (called emerging viruses) closely in the hope that such monitoring can reduce the likelihood of global viral epidemics.
Cell-surface receptors are involved in most of the signaling in multicellular organisms. There are three general categories of cell-surface receptors: ion channel-linked receptors, G-protein-linked receptors, and enzyme-linked receptors.
Ion channel-linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through. To form a channel, this type of cell-surface receptor has an extensive membrane-spanning region. In order to interact with the phospholipid fatty acid tails that form the center of the plasma membrane, many of the amino acids in the membrane-spanning region are hydrophobic in nature. Conversely, the amino acids that line the inside of the channel are hydrophilic to allow for the passage of water or ions. When a ligand binds to the extracellular region of the channel, there is a conformational change in the proteins structure that allows ions such as sodium, calcium, magnesium, and hydrogen to pass through (Figure \(4\)).
G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane (Figure \(5\)). All G-protein-linked receptors have seven transmembrane domains, but each receptor has its own specific extracellular domain and G-protein-binding site.
Cell signaling using G-protein-linked receptors occurs as a cyclic series of events. Before the ligand binds, the inactive G-protein can bind to a newly revealed site on the receptor specific for its binding. Once the G-protein binds to the receptor, the resultant shape change activates the G-protein, which releases GDP and picks up GTP. The subunits of the G-protein then split into the α subunit and the βγ subunit. One or both of these G-protein fragments may be able to activate other proteins as a result. After awhile, the GTP on the active α subunit of the G-protein is hydrolyzed to GDP and the βγ subunit is deactivated. The subunits reassociate to form the inactive G-protein and the cycle begins anew.
G-protein-linked receptors have been extensively studied and much has been learned about their roles in maintaining health. Bacteria that are pathogenic to humans can release poisons that interrupt specific G-protein-linked receptor function, leading to illnesses such as pertussis, botulism, and cholera. In cholera (Figure \(6\)), for example, the water-borne bacterium Vibrio cholerae produces a toxin, choleragen, that binds to cells lining the small intestine. The toxin then enters these intestinal cells, where it modifies a G-protein that controls the opening of a chloride channel and causes it to remain continuously active, resulting in large losses of fluids from the body and potentially fatal dehydration as a result.
Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme. In some cases, the intracellular domain of the receptor itself is an enzyme. Other enzyme-linked receptors have a small intracellular domain that interacts directly with an enzyme. The enzyme-linked receptors normally have large extracellular and intracellular domains, but the membrane-spanning region consists of a single alpha-helical region of the peptide strand. When a ligand binds to the extracellular domain, a signal is transferred through the membrane, activating the enzyme. Activation of the enzyme sets off a chain of events within the cell that eventually leads to a response. One example of this type of enzyme-linked receptor is the tyrosine kinase receptor (Figure \(7\)). A kinase is an enzyme that transfers phosphate groups from ATP to another protein. The tyrosine kinase receptor transfers phosphate groups to tyrosine molecules (tyrosine residues). First, signaling molecules bind to the extracellular domain of two nearby tyrosine kinase receptors. The two neighboring receptors then bond together, or dimerize. Phosphates are then added to tyrosine residues on the intracellular domain of the receptors (phosphorylation). The phosphorylated residues can then transmit the signal to the next messenger within the cytoplasm.
Art Connection
HER2 is a receptor tyrosine kinase. In 30 percent of human breast cancers, HER2 is permanently activated, resulting in unregulated cell division. Lapatinib, a drug used to treat breast cancer, inhibits HER2 receptor tyrosine kinase autophosphorylation (the process by which the receptor adds phosphates onto itself), thus reducing tumor growth by 50 percent. Besides autophosphorylation, which of the following steps would be inhibited by Lapatinib?
1. Signaling molecule binding, dimerization, and the downstream cellular response
2. Dimerization, and the downstream cellular response
3. The downstream cellular response
4. Phosphatase activity, dimerization, and the downsteam cellular response
Signaling Molecules
Produced by signaling cells and the subsequent binding to receptors in target cells, ligands act as chemical signals that travel to the target cells to coordinate responses. The types of molecules that serve as ligands are incredibly varied and range from small proteins to small ions like calcium (Ca2+).
Small Hydrophobic Ligands
Small hydrophobic ligands can directly diffuse through the plasma membrane and interact with internal receptors. Important members of this class of ligands are the steroid hormones. Steroids are lipids that have a hydrocarbon skeleton with four fused rings; different steroids have different functional groups attached to the carbon skeleton. Steroid hormones include the female sex hormone, estradiol, which is a type of estrogen; the male sex hormone, testosterone; and cholesterol, which is an important structural component of biological membranes and a precursor of steroid hormones (Figure \(8\)). Other hydrophobic hormones include thyroid hormones and vitamin D. In order to be soluble in blood, hydrophobic ligands must bind to carrier proteins while they are being transported through the bloodstream.
Water-Soluble Ligands
Water-soluble ligands are polar and therefore cannot pass through the plasma membrane unaided; sometimes, they are too large to pass through the membrane at all. Instead, most water-soluble ligands bind to the extracellular domain of cell-surface receptors. This group of ligands is quite diverse and includes small molecules, peptides, and proteins.
Other Ligands
Nitric oxide (NO) is a gas that also acts as a ligand. It is able to diffuse directly across the plasma membrane, and one of its roles is to interact with receptors in smooth muscle and induce relaxation of the tissue. NO has a very short half-life and therefore only functions over short distances. Nitroglycerin, a treatment for heart disease, acts by triggering the release of NO, which causes blood vessels to dilate (expand), thus restoring blood flow to the heart. NO has become better known recently because the pathway that it affects is targeted by prescription medications for erectile dysfunction, such as Viagra (erection involves dilated blood vessels).
Summary
Cells communicate by both inter- and intracellular signaling. Signaling cells secrete ligands that bind to target cells and initiate a chain of events within the target cell. The four categories of signaling in multicellular organisms are paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across gap junctions. Paracrine signaling takes place over short distances. Endocrine signals are carried long distances through the bloodstream by hormones, and autocrine signals are received by the same cell that sent the signal or other nearby cells of the same kind. Gap junctions allow small molecules, including signaling molecules, to flow between neighboring cells.
Internal receptors are found in the cell cytoplasm. Here, they bind ligand molecules that cross the plasma membrane; these receptor-ligand complexes move to the nucleus and interact directly with cellular DNA. Cell-surface receptors transmit a signal from outside the cell to the cytoplasm. Ion channel-linked receptors, when bound to their ligands, form a pore through the plasma membrane through which certain ions can pass. G-protein-linked receptors interact with a G-protein on the cytoplasmic side of the plasma membrane, promoting the exchange of bound GDP for GTP and interacting with other enzymes or ion channels to transmit a signal. Enzyme-linked receptors transmit a signal from outside the cell to an intracellular domain of a membrane-bound enzyme. Ligand binding causes activation of the enzyme. Small hydrophobic ligands (like steroids) are able to penetrate the plasma membrane and bind to internal receptors. Water-soluble hydrophilic ligands are unable to pass through the membrane; instead, they bind to cell-surface receptors, which transmit the signal to the inside of the cell.
Art Connections
Figure \(7\): HER2 is a receptor tyrosine kinase. In 30 percent of human breast cancers, HER2 is permanently activated, resulting in unregulated cell division. Lapatinib, a drug used to treat breast cancer, inhibits HER2 receptor tyrosine kinase autophosphorylation (the process by which the receptor adds phosphates onto itself), thus reducing tumor growth by 50 percent. Besides autophosphorylation, which of the following steps would be inhibited by Lapatinib?
1. Signaling molecule binding, dimerization, and the downstream cellular response.
2. Dimerization, and the downstream cellular response.
3. The downstream cellular response.
4. Phosphatase activity, dimerization, and the downsteam cellular response.
Answer
C. The downstream cellular response would be inhibited.
Footnotes
1. 1 A. B. Sigalov, The School of Nature. IV. Learning from Viruses, Self/Nonself 1, no. 4 (2010): 282-298. Y. Cao, X. Koh, L. Dong, X. Du, A. Wu, X. Ding, H. Deng, Y. Shu, J. Chen, T. Jiang, Rapid Estimation of Binding Activity of Influenza Virus Hemagglutinin to Human and Avian Receptors, PLoS One 6, no. 4 (2011): e18664.
Glossary
autocrine signal
signal that is sent and received by the same or similar nearby cells
cell-surface receptor
cell-surface protein that transmits a signal from the exterior of the cell to the interior, even though the ligand does not enter the cell
chemical synapse
small space between axon terminals and dendrites of nerve cells where neurotransmitters function
endocrine cell
cell that releases ligands involved in endocrine signaling (hormones)
endocrine signal
long-distance signal that is delivered by ligands (hormones) traveling through an organisms circulatory system from the signaling cell to the target cell
enzyme-linked receptor
cell-surface receptor with intracellular domains that are associated with membrane-bound enzymes
extracellular domain
region of a cell-surface receptor that is located on the cell surface
G-protein-linked receptor
cell-surface receptor that activates membrane-bound G-proteins to transmit a signal from the receptor to nearby membrane components
intercellular signaling
communication between cells
internal receptor
(also, intracellular receptor) receptor protein that is located in the cytosol of a cell and binds to ligands that pass through the plasma membrane
intracellular mediator
(also, second messenger) small molecule that transmits signals within a cell
intracellular signaling
communication within cells
ion channel-linked receptor
cell-surface receptor that forms a plasma membrane channel, which opens when a ligand binds to the extracellular domain (ligand-gated channels)
ligand
molecule produced by a signaling cell that binds with a specific receptor, delivering a signal in the process
neurotransmitter
chemical ligand that carries a signal from one nerve cell to the next
paracrine signal
signal between nearby cells that is delivered by ligands traveling in the liquid medium in the space between the cells
receptor
protein in or on a target cell that bind to ligands
signaling cell
cell that releases signal molecules that allow communication with another cell
synaptic signal
chemical signal (neurotransmitter) that travels between nerve cells
target cell
cell that has a receptor for a signal or ligand from a signaling cell
9.01: Overview of Cell Communication
Learning Objectives
• Explain how the binding of a ligand initiates signal transduction throughout a cell
The induction of a signaling pathway depends on the modification of a cellular component by an enzyme. There are numerous enzymatic modifications that can occur which are recognized in turn by the next component downstream.
One of the most common chemical modifications that occurs in signaling pathways is the addition of a phosphate group (PO4–3) to a molecule such as a protein in a process called phosphorylation. The phosphate can be added to a nucleotide such as GMP to form GDP or GTP. Phosphates are also often added to serine, threonine, and tyrosine residues of proteins where they replace the hydroxyl group of the amino acid. The transfer of the phosphate is catalyzed by an enzyme called a kinase. Various kinases are named for the substrate they phosphorylate. Phosphorylation of serine and threonine residues often activates enzymes. Phosphorylation of tyrosine residues can either affect the activity of an enzyme or create a binding site that interacts with downstream components in the signaling cascade. Phosphorylation may activate or inactivate enzymes; the reversal of phosphorylation, dephosphorylation by a phosphatase, will reverse the effect.
The activation of second messengers is also a common event after the induction of a signaling pathway. They are small molecules that propagate a signal after it has been initiated by the binding of the signaling molecule to the receptor. These molecules help to spread a signal through the cytoplasm by altering the behavior of certain cellular proteins.
Calcium ion is a widely-used second messenger. The free concentration of calcium ions (Ca2+) within a cell is very low because ion pumps in the plasma membrane continuously use adenosine-5′-triphosphate ( ATP ) to remove it. For signaling purposes, Ca2+ is stored in cytoplasmic vesicles, such as the endoplasmic reticulum, or accessed from outside the cell. When signaling occurs, ligand-gated calcium ion channels allow the higher levels of Ca2+ that are present outside the cell (or in intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of cytoplasmic Ca2+. The response to the increase in Ca2+ varies, depending on the cell type involved. For example, in the β-cells of the pancreas, Ca2+ signaling leads to the release of insulin, whereas in muscle cells, an increase in Ca2+ leads to muscle contractions.
Another second messenger utilized in many different cell types is cyclic AMP (cAMP). Cyclic AMP is synthesized by the enzyme adenylyl cyclase from ATP. The main role of cAMP in cells is to bind to and activate an enzyme called cAMP-dependent kinase (A-kinase). A-kinase regulates many vital metabolic pathways. It phosphorylates serine and threonine residues of its target proteins, activating them in the process. A-kinase is found in many different types of cells; the target proteins in each kind of cell are different. Differences give rise to the variation of the responses to cAMP in different cells.
Present in small concentrations in the plasma membrane, inositol phospholipids are lipids that can also be converted into second messengers. Because these molecules are membrane components, they are located near membrane-bound receptors and can easily interact with them. Phosphatidylinositol (PI) is the main phospholipid that plays a role in cellular signaling. Enzymes known as kinases phosphorylate PI to form PI-phosphate (PIP) and PI-bisphosphate (PIP2).
Key Points
• Phosphorylation, the addition of a phosphate group to a molecule such as a protein, is one of the most common chemical modifications that occurs in signaling pathways.
• The activation of second messengers, small molecules that propagate a signal, is a common event after the induction of a signaling pathway.
• Calcium ion, cyclic AMP, and inositol phospholipids are examples of widely-used second messengers.
Key Terms
• second messenger: any substance used to transmit a signal within a cell, especially one which triggers a cascade of events by activating cellular components
• phosphorylation: the addition of a phosphate group to a compound; often catalyzed by enzymes | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/09%3A_Cell_Communication/9.01%3A_Overview_of_Cell_Communication/9.1B%3A_Methods_of_Intracellular_Signaling.txt |
Learning Objectives
• Compare internal receptors with cell-surface receptors
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligands. There are two types of receptors: internal receptors and cell-surface receptors.
Internal receptors
Internal receptors, also known as intracellular or cytoplasmic receptors, are found in the cytoplasm of the cell and respond to hydrophobic ligand molecules that are able to travel across the plasma membrane. Once inside the cell, many of these molecules bind to proteins that act as regulators of mRNA synthesis to mediate gene expression. Gene expression is the cellular process of transforming the information in a cell’s DNA into a sequence of amino acids that ultimately forms a protein. When the ligand binds to the internal receptor, a conformational change exposes a DNA-binding site on the protein. The ligand-receptor complex moves into the nucleus, binds to specific regulatory regions of the chromosomal DNA, and promotes the initiation of transcription. Internal receptors can directly influence gene expression without having to pass the signal on to other receptors or messengers.
Cell-Surface Receptors
Cell-surface receptors, also known as transmembrane receptors, are cell surface, membrane-anchored, or integral proteins that bind to external ligand molecules. This type of receptor spans the plasma membrane and performs signal transduction, converting an extracellular signal into an intracellular signal. Ligands that interact with cell-surface receptors do not have to enter the cell that they affect. Cell-surface receptors are also called cell-specific proteins or markers because they are specific to individual cell types.
Each cell-surface receptor has three main components: an external ligand-binding domain (extracellular domain), a hydrophobic membrane-spanning region, and an intracellular domain inside the cell. The size and extent of each of these domains vary widely, depending on the type of receptor.
Cell-surface receptors are involved in most of the signaling in multicellular organisms. There are three general categories of cell-surface receptors: ion channel-linked receptors, G-protein-linked receptors, and enzyme-linked receptors.
Ion Channel-Linked Receptors
Ion channel-linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through. To form a channel, this type of cell-surface receptor has an extensive membrane-spanning region. In order to interact with the phospholipid fatty acid tails that form the center of the plasma membrane, many of the amino acids in the membrane-spanning region are hydrophobic in nature. Conversely, the amino acids that line the inside of the channel are hydrophilic to allow for the passage of water or ions. When a ligand binds to the extracellular region of the channel, there is a conformational change in the protein’s structure that allows ions such as sodium, calcium, magnesium, and hydrogen to pass through.
G-Protein Linked Receptors
G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane. All G-protein-linked receptors have seven transmembrane domains, but each receptor has its own specific extracellular domain and G-protein-binding site.
Cell signaling using G-protein-linked receptors occurs as a cyclic series of events. Before the ligand binds, the inactive G-protein can bind to a newly-revealed site on the receptor specific for its binding. Once the G-protein binds to the receptor, the resultant shape change activates the G-protein, which releases GDP and picks up GTP. The subunits of the G-protein then split into the α subunit and the β subunit. One or both of these G-protein fragments may be able to activate other proteins as a result. Later, the GTP on the active α subunit of the G-protein is hydrolyzed to GDP and the β subunit is deactivated. The subunits reassociate to form the inactive G-protein, and the cycle starts over.
Enzyme-Linked Receptors
Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme. In some cases, the intracellular domain of the receptor itself is an enzyme or the enzyme-linked receptor has an intracellular domain that interacts directly with an enzyme. The enzyme-linked receptors normally have large extracellular and intracellular domains, but the membrane-spanning region consists of a single alpha-helical region of the peptide strand. When a ligand binds to the extracellular domain, a signal is transferred through the membrane and activates the enzyme, which sets off a chain of events within the cell that eventually leads to a response. An example of this type of enzyme-linked receptor is the tyrosine kinase receptor. The tyrosine kinase receptor transfers phosphate groups to tyrosine molecules. Signaling molecules bind to the extracellular domain of two nearby tyrosine kinase receptors, which then dimerize. Phosphates are then added to tyrosine residues on the intracellular domain of the receptors and can then transmit the signal to the next messenger within the cytoplasm.
Key Points
• Intracellular receptors are located in the cytoplasm of the cell and are activated by hydrophobic ligand molecules that can pass through the plasma membrane.
• Cell-surface receptors bind to an external ligand molecule and convert an extracellular signal into an intracellular signal.
• Three general categories of cell-surface receptors include: ion -channel, G- protein, and enzyme -linked protein receptors.
• Ion channel -linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through.
• G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein, which then interacts with either an ion channel or an enzyme in the membrane.
• Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme.
Key Terms
• integral protein: a protein molecule (or assembly of proteins) that is permanently attached to the biological membrane
• transcription: the synthesis of RNA under the direction of DNA | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/09%3A_Cell_Communication/9.02%3A_Receptor_Types/9.2C%3A_Types_of_Receptors.txt |
Another type of relatively simple, though much slower, signaling is seen in pathways in which the signals are steroid hormones, like estrogen or testosterone, pictured below. Steroid hormones, as you are aware, are related to cholesterol, and as hydrophobic molecules, they are able to cross the cell membrane by themselves. This is unusual, as most signals coming to cells are incapable of crossing the plasma membrane, and thus, must have cell surface receptors.
By contrast, steroid hormones have receptors inside the cell (intracellular receptors). Steroid hormone receptors are proteins that belong in a family known as the nuclear receptors. Nuclear hormone receptors are proteins with a double life: they are actually dormant transcription regulators. In the absence of signal, these receptors are in the cytoplasm, complexed with other proteins (HSP in Figure 8.3.2) and inactive. When a steroid hormone enters the cell, the nuclear hormone receptor binds the hormone and dissociates from the HSP. The receptors, then, with the hormone bound, translocate into the nucleus.
In the nucleus, Nuclear hormone receptors regulate the transcription of target genes by binding to their regulatory sequences (labeled HRE for hormone- response elements). The binding of the hormone-receptor complex to the regulatory elements of hormone-responsive genes modulates their expression. Because these responses involve gene expression, they are relatively slow. Most other signaling pathways, besides the two we have just discussed, involve multiple steps in which the original signal is passed on and amplified through a number of intermediate steps, before the cell responds to the signal.
We will now consider two signaling pathways, each mediated by a major class of cell surface receptor- the G-protein coupled receptors (GPCRs) and the receptor tyrosine kinases (RTKs). While the specific details of the signaling pathways that follow the binding of signals to each of these receptor types are different, it is easier to learn them when you can see what the pathways have in common, namely, interaction of the signal with a receptor, followed by relaying the signal through a variable number of intermediate molecules, with the last of these molecules interacting with target protein(s) to modify their activity in the cell.
9.4.01: Receptor Tyrosine Kinases (RTKs)
Receptor tyrosine kinases mediate responses to a large number of signals, including peptide hormones like insulin and growth factors like epidermal growth factor. Like the GPCRs, receptor tyrosine kinases bind a signal, then pass the message on through a series of intracellular molecules, the last of which acts on target proteins to change the state of the cell.
As the name suggests, a receptor tyrosine kinase is a cell surface receptor that also has a tyrosine kinase activity. The signal binding domain of the receptor tyrosine kinase is on the cell surface, while the tyrosine kinase enzymatic activity resides in the cytoplasmic part of the protein (see figure above). A transmembrane alpha helix connects these two regions of the receptor.
What happens when signal molecules bind to receptor tyrosine kinases?
Binding of signal molecules to the extracellular domains of receptor tyrosine kinase molecules causes two receptor molecules to dimerize (come together and associate). This brings the cytoplasmic tails of the receptors close to each other and causes the tyrosine kinase activity of these tails to be turned on. The activated tails then phosphorylate each other on several tyrosine residues. This is called autophosphorylation.
The phosphorylation of tyrosines on the receptor tails triggers the assembly of an intracellular signaling complex on the tails. The newly phosphorylated tyrosines serve as binding sites for signaling proteins that then pass the message on to yet other proteins. An important protein that is subsequently activated by the signaling complexes on the receptor tyrosine kinases is called Ras.
The Ras protein is a monomeric guanine nucleotide binding protein that is associated with the cytosolic face of the plasma membrane (in fact, it is a lot like the alpha subunit of trimeric G-proteins). Just like the alpha subunit of a G- protein, Ras is active when GTP is bound to it and inactive when GDP is bound to it.Also, like the alpha subunit, Ras can hydrolyze the GTP to GDP.
When a signal arrives at the receptor tyrosine kinase, the receptor monomers come together and phosphorylate each others' tyrosines, triggering the assembly of a complex of proteins on the cytoplasmic tail of the receptor. One of the proteins in this complex interacts with Ras and stimulates the exchange of the GDP bound to the inactive Ras for a GTP. This activates the Ras.
Activated Ras triggers a phosphorylation cascade of three protein kinases, which relay and distribute the signal. These protein kinases are members of a group called the MAP kinases (Mitogen Activated Protein Kinases). The final kinase in this cascade phosphorylates various target proteins, including enzymes and transcriptional activators that regulate gene expression.
The phosphorylation of various enzymes can alter their activities, and set off new chemical reactions in the cell, while the phosphorylation of transcriptional activators can change which genes are expressed. The combined effect of changes in gene expression and protein activity alter the cell's physiological state.
Once again, in following the path of signal transduction mediated by RTKs, it is possible to discern the same basic pattern of events: a signal is bound by the extracellular domains of receptor tyrosine kinases, resulting in receptor dimerization and autophosphorylation of the cytosolic tails, thus conveying the message to the interior of the cell.
The message is passed on via a signalling complex to Ras which then stimulates a series of kinases. The terminal kinase in the cascade acts on target proteins and brings about in changes in protein activities and gene expression.
The descriptions above provide a very simple sketch of some of the major classes of receptors and deal primarily with the mechanistic details of the steps by which signals received by various types of receptors bring about changes in cells. A major take-home lesson is the essential similarity of the different pathways.
Another point to keep in mind is that while we have looked at each individual pathway in isolation, a cell, at any given time receives multiple signals that set off a variety of different responses at once. The pathways described above show a considerable degree of "cross-talk" and the response to any given signal is affected by the other signals that the cell receives simultaneously. The multitude of different receptors, signals and the combinations thereof are the means by which cells are able to respond to an enormous variety of different circumstances. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/09%3A_Cell_Communication/9.03%3A_Intracellular_Receptors/9.3.01%3A_Nuclear_Hormone_Receptors.txt |
G-protein coupled receptors are involved in responses of cells to many different kinds of signals, from epinephrine, to odors, to light. In fact, a variety of physiological phenomena including vision, taste, smell and the fight-or-flight response are mediated by GPCRs.
What are G-protein coupled receptors?
G-protein coupled receptors are cell surface receptors that pass on the signals that they receive with the help of guanine nucleotide binding proteins (a.k.a. G-proteins). Before thinking any further about the signaling pathways downstream of GPCRs, it is necessary to know a few important facts about these receptors and the G-proteins that assist them. Though there are hundreds of different G-protein coupled receptors, they all have the same basic structure: they all consist of a single polypeptide chain that threads back and forth seven times through the lipid bilayer of the plasma membrane. For this reason, they are sometimes called seven- pass transmembrane (7TM) receptors.
One end of the polypeptide forms the extracellular domain that binds the signal while the other end is in the cytosol of the cell.
When a ligand (signal) binds the extracellular domain of a GPCR, the receptor undergoes a conformational change that allows it to interact with a G-protein that will then pass the signal on to other intermediates in the signaling pathway.
What is a G-protein?
As noted above, a G-protein is a guanine nucleotide-binding protein that can interact with a G-protein linked receptor. G-proteins are associated with the cytosolic side of the plasma membrane, where they are ideally situated to interact with the cytosolic tail of the GPCR, when a signal binds to the GPCR.
There are many different G-proteins, all of which share a characteristic structure- they are composed of three subunits called alpha, beta and gamma (aß.). Because of this, they are sometimes called heterotrimeric G proteins (hetero=different, trimeric= having three parts). The a subunit of such proteins can bind GDP or GTP and is capable of hydrolyzing a GTP molecule bound to it into GDP. In the unstimulated state of the cell, that is, in the absence of a signal bound to the GPCR, the G-proteins are found in the trimeric form (aß. bound together) and the a subunit has a GDP molecule bound to it.
With this background on the structure and general properties of the GPCRs and the G-proteins, we can now look at what happens when a signal arrives at the cell surface and binds to a GPCR. The binding of a signal molecule by the extracellular part of the G-protein linked receptor causes the cytosolic tail of the receptor to interact with, and alter the conformation of, a G-protein. This has two consequences:
• First, the alpha subunit of the G- protein loses its GDP and binds a GTP instead.
• Second, the G-protein breaks up into the GTP-bound a part and the ß. part.
These two parts can diffuse freely along the cytosolic face of the plasma membrane and act upon their targets.
What happens when G-proteins interact with their target proteins? That depends on what the target is. G-proteins interact with different kinds of target proteins, of which we will examine two major categories:
Ion Channels
We have earlier seen that some gated ion channels can be opened or closed by the direct binding of neurotransmitters to a receptor that is an ion-channel protein. In other cases, ion channels are regulated by the binding of G-proteins. That is, instead of the signal directly binding to the ion channel, it binds to a GPCR, which activates a G-protein that then binds and opens the ion channel. The change in the distribution of ions across the plasma membrane causes a change in the membrane potential.
Specific Enzymes
The interaction of G-proteins with their target enzymes can regulate the activity of the enzyme, either increasing or decreasing its activity. Often the target enzyme will pass the signal on in another form to another part of the cell. As you might imagine, this kind of response takes a little longer than the kind where an ion channel is opened instantaneously. Two well-studied examples of enzymes whose activity is regulated by a G-protein are adenylate cyclase and phospholipase C. When adenylate cyclase is activated, the molecule cAMP is produced in large amounts.
When phospholipase C is activated, the molecules inositol trisphosphate (IP3) and diacylglycerol (DAG) are made. cAMP, IP3 and DAG are second messengers, small, diffusible molecules that can "spread the message" brought by the original signal, to other parts of the cell.
In these cases, the binding of a signal to the GPCR activated a G- protein, which in turn, activated an enzyme that makes a second messenger that can amplify the message in the cell. We will first trace the effects of activating adenylate cyclase and the resulting increase in cAMP.
What is the effect of elevated cAMP levels?
cAMP molecules bind to, and activate an enzyme, protein kinase A (PKA). PKA is composed of two catalytic and two regulatory subunits that are bound tightly together. Upon binding of cAMP the catalytic subunits are released from the regulatory subunits, allowing the enzyme to carry out its function, namely phosphorylating other proteins.
Thus, cAMP can regulate the activity of PKA, which in turn, by phosphorylating other proteins can change their activity. The targets of PKA may be enzymes that are activated by phosphorylation, or they may be proteins that regulate transcription. The phosphorylation of a transcriptional activator, for example, may cause the activator to bind to a regulatory sequence on DNA and to increase the transcription of the gene it controls. The activation of previously inactive enzymes alters the state of the cell by changing the reactions that are occurring within the cell.
For example, the binding of epinephrine to its receptor on the cell surface, activates, through the action of G-proteins, and subsequent activation of PKA, the phosphorylation of glycogen phosphorylase. The resulting activation of glycogen phosphorylase leads to the breakdown of glycogen, releasing glucose (in the form of glucose-1-phosphate) for use by the cell. Changes in gene expression, likewise, lead to changes in the cell by altering the production of particular proteins in response to the signal.
Although the steps described above seem complicated, they follow the simple pattern outlined at the beginning of this section:
• Binding of signal to receptor
• Several steps where the signal is passed on through intermediate molecules (G-proteins, adenylate cyclase, cAMP, and finally, PKA)
• Phosphorylation of target proteins by the kinase, leading to changes in the cell.
Finally, if the signal binding to the receptor serves as a switch that sets these events in motion, there must be mechanisms to turn the pathway off. The first is at the level of the G-protein. Recall that the alpha subunit of the G-protein is in its free and activated state when it has GTP bound and that it associates with the beta- gamma subunits and has a GDP bound when it is inactive. We also know that the alpha subunit has an activity that enables it to hydrolyze GTP to GDP, as shown in the figure above left. This GTP-hydrolyzing activity makes it possible for the alpha subunit, once it has completed its task, to return to its GDP bound state, re-associate with the beta-gamma part and become inactive again.
The second "off switch" is further down the signaling pathway, and controls the level of cAMP. We just noted that cAMP levels increase when adenylate cyclase is activated. When its job is done, cAMP is broken down by an enzyme called phosphodiesterase. When cAMP levels drop, PKA returns to its inactive state, putting a halt to the changes brought about by the activation of adenylate cyclase by an activated G-protein.
Let us now examine the events that follow the activation of Phospholipase C (PLC) by a G-protein. As we noted earlier, the activation of PLC results in the production of the second messengers IP3 and DAG. What do these molecules do?
The IP3 and DAG produced by activated phospholipase C work together to activate a protein kinase. First, IP3 diffuses to the endoplasmic reticulum membrane where it binds to gated calcium ion channels. This causes calcium channels in the ER membrane to open and release large amounts of calcium into the cytoplasm from the ER lumen, as shown in the figure below.
The increase in cytosolic calcium ion concentration has various effects, one of which is to activate a protein kinase called protein kinase C (C for calcium), together with the DAG made in the earlier step. Like PKA, Protein kinase C phosphorylates a variety of proteins in the cell, altering their activity and thus changing the state of the cell.
The pathways leading to PKC and PKA activation following the binding of a signal to a GPCR are summarized in Figure 8.4.12. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/09%3A_Cell_Communication/9.05%3A_Signal_Transduction_Through_G_Protein_Coupled_Receptors/9.5.01%3A_G-protein_Coupled_Receptors_%28GPCRs%29.txt |
• 10.1: Bacterial Cell Division
In both prokaryotic and eukaryotic cell division, the genomic DNA is replicated and then each copy is allocated into a daughter cell. In addition, the cytoplasmic contents are divided evenly and distributed to the new cells. However, there are many differences between prokaryotic and eukaryotic cell division. Bacteria have a single, circular DNA chromosome but no nucleus. Therefore, mitosis is not necessary in bacterial cell division.
• 10.2: Eukaryotic Chromosomes
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.
• 10.3: Overview of the Eukaryotic 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).
• 10.4: Interphase- Preparation for Mitosis
• 10.5: M phase- Chromosome Segregation and the Division of Cytoplasmic Contents
• 10.6: Control of the Cell Cycle
The length of the cell cycle is highly variable, even within the cells of a single organism. In humans, the frequency of cell turnover ranges from a few hours in early embryonic development, to an average of two to five days for epithelial cells, and to an entire human lifetime spent in G0 by specialized cells, such as cortical neurons or cardiac muscle cells. There is also variation in the time that a cell spends in each phase of the cell cycle.
• 10.7: Genetics of Cancer
Cancer is the result of unchecked cell division caused by a breakdown of the mechanisms that regulate the cell cycle. The loss of control begins with a change in the DNA sequence of a gene that codes for one of the regulatory molecules. Faulty instructions lead to a protein that does not function as it should. Any disruption of the monitoring system can allow other mistakes to be passed on to the daughter cells. Each successive cell division will give rise to daughter cells with even more damage
10: How Cells Divide
Skills to Develop
• Describe the process of binary fission in prokaryotes
• Explain how FtsZ and tubulin proteins are examples of homology
Prokaryotes, such as bacteria, propagate by binary fission. For unicellular organisms, cell division is the only method to produce new individuals. In both prokaryotic and eukaryotic cells, the outcome of cell reproduction is a pair of daughter cells that are genetically identical to the parent cell. In unicellular organisms, daughter cells are individuals.
To achieve the outcome of cloned offspring, certain steps are essential. The genomic DNA must be replicated and then allocated into the daughter cells; the cytoplasmic contents must also be divided to give both new cells the machinery to sustain life. In bacterial cells, the genome consists of a single, circular DNA chromosome; therefore, the process of cell division is simplified. Karyokinesis is unnecessary because there is no nucleus and thus no need to direct one copy of the multiple chromosomes into each daughter cell. This type of cell division is called binary (prokaryotic) fission.
Binary Fission
Due to the relative simplicity of the prokaryotes, the cell division process, called binary fission, is a less complicated and much more rapid process than cell division in eukaryotes. The single, circular DNA chromosome of bacteria is not enclosed in a nucleus, but instead occupies a specific location, the nucleoid, within the cell (Figure \(1\)). Although the DNA of the nucleoid is associated with proteins that aid in packaging the molecule into a compact size, there are no histone proteins and thus no nucleosomes in prokaryotes. The packing proteins of bacteria are, however, related to the cohesin and condensin proteins involved in the chromosome compaction of eukaryotes.
The bacterial chromosome is attached to the plasma membrane at about the midpoint of the cell. The starting point of replication, the origin, is close to the binding site of the chromosome to the plasma membrane (Figure \(1\)). Replication of the DNA is bidirectional, moving away from the origin on both strands of the loop simultaneously. As the new double strands are formed, each origin point moves away from the cell wall attachment toward the opposite ends of the cell. As the cell elongates, the growing membrane aids in the transport of the chromosomes. After the chromosomes have cleared the midpoint of the elongated cell, cytoplasmic separation begins. The formation of a ring composed of repeating units of a protein called FtsZ directs the partition between the nucleoids. Formation of the FtsZ ring triggers the accumulation of other proteins that work together to recruit new membrane and cell wall materials to the site. A septum is formed between the nucleoids, extending gradually from the periphery toward the center of the cell. When the new cell walls are in place, the daughter cells separate.
Evolution Connection: Mitotic Spindle Apparatus
The precise timing and formation of the mitotic spindle is critical to the success of eukaryotic cell division. Prokaryotic cells, on the other hand, do not undergo karyokinesis and therefore have no need for a mitotic spindle. However, the FtsZ protein that plays such a vital role in prokaryotic cytokinesis is structurally and functionally very similar to tubulin, the building block of the microtubules that make up the mitotic spindle fibers that are necessary for eukaryotes. FtsZ proteins can form filaments, rings, and other three-dimensional structures that resemble the way tubulin forms microtubules, centrioles, and various cytoskeletal components. In addition, both FtsZ and tubulin employ the same energy source, GTP (guanosine triphosphate), to rapidly assemble and disassemble complex structures.
FtsZ and tubulin are homologous structures derived from common evolutionary origins. In this example, FtsZ is the ancestor protein to tubulin (a modern protein). While both proteins are found in extant organisms, tubulin function has evolved and diversified tremendously since evolving from its FtsZ prokaryotic origin. A survey of mitotic assembly components found in present-day unicellular eukaryotes reveals crucial intermediary steps to the complex membrane-enclosed genomes of multicellular eukaryotes.
Table \(1\): Cell Division Apparatus among Various Organisms
Structure of genetic material Division of nuclear material Separation of daughter cells
Prokaryotes There is no nucleus. The single, circular chromosome exists in a region of cytoplasm called the nucleoid. Occurs through binary fission. As the chromosome is replicated, the two copies move to opposite ends of the cell by an unknown mechanism. FtsZ proteins assemble into a ring that pinches the cell in two.
Some protists Linear chromosomes exist in the nucleus. Chromosomes attach to the nuclear envelope, which remains intact. The mitotic spindle passes through the envelope and elongates the cell. No centrioles exist. Microfilaments form a cleavage furrow that pinches the cell in two.
Other protists Linear chromosomes exist in the nucleus. A mitotic spindle forms from the centrioles and passes through the nuclear membrane, which remains intact. Chromosomes attach to the mitotic spindle, which separates the chromosomes and elongates the cell. Microfilaments form a cleavage furrow that pinches the cell in two.
Animal cells Linear chromosomes exist in the nucleus. A mitotic spindle forms from the centrosomes. The nuclear envelope dissolves. Chromosomes attach to the mitotic spindle, which separates the chromosomes and elongates the cell. Microfilaments form a cleavage furrow that pinches the cell in two.
Summary
In both prokaryotic and eukaryotic cell division, the genomic DNA is replicated and then each copy is allocated into a daughter cell. In addition, the cytoplasmic contents are divided evenly and distributed to the new cells. However, there are many differences between prokaryotic and eukaryotic cell division. Bacteria have a single, circular DNA chromosome but no nucleus. Therefore, mitosis is not necessary in bacterial cell division. Bacterial cytokinesis is directed by a ring composed of a protein called FtsZ. Ingrowth of membrane and cell wall material from the periphery of the cells results in the formation of a septum that eventually constructs the separate cell walls of the daughter cells.
Glossary
binary fission
prokaryotic cell division process
FtsZ
tubulin-like protein component of the prokaryotic cytoskeleton that is important in prokaryotic cytokinesis (name origin: Filamenting temperature-sensitive mutant Z)
origin
(also, ORI) region of the prokaryotic chromosome where replication begins (origin of replication)
septum
structure formed in a bacterial cell as a precursor to the separation of the cell into two daughter cells | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.01%3A_Bacterial_Cell_Division.txt |
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).
10.02: Eukaryotic Chromosomes
Learning Objectives
• Describe the levels of chromsomal structure and compaction
Eukaryotic Chromosomal Structure and Compaction
If the DNA from all 46 chromosomes in a human cell nucleus was laid out end to end, it would measure approximately two meters. However, the diameter would be only 2 nm. Considering that the size of a typical human cell is about 10 µm (100,000 cells lined up to equal one meter), DNA must be tightly packaged to fit in the cell’s nucleus. At the same time, it must also be readily accessible for the genes to be expressed. During some stages of the cell cycle, the long strands of DNA are condensed into compact chromosomes. There are a number of ways that chromosomes are compacted to fit in the cell’s nucleus and be accessible for gene expression.
In the first level of compaction, short stretches of the DNA double helix wrap around a core of eight histone proteins at regular intervals along the entire length of the chromosome. The DNA-histone complex is called chromatin. The beadlike, histone DNA complex is called a nucleosome. DNA connecting the nucleosomes is called linker DNA. A DNA molecule in this form is about seven times shorter than the double helix without the histones. The beads are about 10 nm in diameter, in contrast with the 2-nm diameter of a DNA double helix. The next level of compaction occurs as the nucleosomes and the linker DNA between them are coiled into a 30-nm chromatin fiber. This coiling further shortens the chromosome so that it is now about 50 times shorter than the extended form. In the third level of packing, a variety of fibrous proteins is used to pack the chromatin. These fibrous proteins also ensure that each chromosome in a non-dividing cell occupies a particular area of the nucleus that does not overlap with that of any other chromosome.
DNA replicates in the S phase of interphase. After replication, the chromosomes are composed of two linked sister chromatids. When fully compact, the pairs of identically-packed chromosomes are bound to each other by cohesin proteins. The connection between the sister chromatids is closest in a region called the centromere. The conjoined sister chromatids, with a diameter of about 1 µm, are visible under a light microscope. The centromeric region is highly condensed and will appear as a constricted area.
Key Points
• During some stages of the cell cycle, the long strands of DNA are condensed into compact chromosomes to fit in the cell’s nucleus.
• In the first level of compaction, short stretches of the DNA double helix wrap around a core of eight histone proteins at regular intervals along the entire length of the chromosome.
• The DNA surrouding the histone core is called a nucleosome; the DNA-histone complex is called chromatin.
• The second level of compaction occurs as the nucleosomes and the linker DNA between them are coiled into a 30-nm chromatin fiber, which shortens the chromosome so it’s about 50 times shorter than the extended form.
• After replication, the chromosomes are composed of two linked sister chromatids; when fully compact, the pairs of identically-packed chromosomes are bound to each other by cohesin proteins.
• The connection between the sister chromatids is closest in a region called the centromere; this region is highly condensed.
Key Terms
• nucleosome: any of the subunits that repeat in chromatin; a coil of DNA surrounding a histone core
• histone: any of various simple water-soluble proteins that are rich in the basic amino acids lysine and arginine and are complexed with DNA in the nucleosomes of eukaryotic chromatin
• chromatin: a complex of DNA, RNA, and proteins within the cell nucleus out of which chromosomes condense during cell division | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.02%3A_Eukaryotic_Chromosomes/10.2C%3A_Eukaryotic_Chromosomal_Structure_and_Compaction.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.
10.04: Interphase- Preparation for Mitosis
Learning Objectives
• Describe the events that occur during Interphase
Interphase
During interphase, the cell undergoes normal growth processes while also preparing for cell division. In order for a cell to move from interphase into the mitotic phase, many internal and external conditions must be met. The three stages of interphase are called G1, S, and G2 .
G1 Phase (First Gap)
The first stage of interphase is called the G1 phase (first gap) because, from a microscopic aspect, little change is visible. However, during the G1 stage, the cell is quite active at the biochemical level. The cell grows and accumulates the building blocks of chromosomal DNA and the associated proteins as well as sufficient energy reserves to complete the task of replicating each chromosome in the nucleus.
S Phase (Synthesis of DNA)
The synthesis phase of interphase takes the longest because of the complexity of the genetic material being duplicated. Throughout interphase, nuclear DNA remains in a semi-condensed chromatin configuration. In the S phase, DNA replication results in the formation of identical pairs of DNA molecules, sister chromatids, that are firmly attached to the centromeric region. The centrosome is duplicated during the S phase. The two centrosomes will give rise to the mitotic spindle, the apparatus that orchestrates the movement of chromosomes during mitosis. At the center of each animal cell, the centrosomes of animal cells are associated with a pair of rod-like objects, the centrioles, which are at right angles to each other. Centrioles help organize cell division. Centrioles are not present in the centrosomes of other eukaryotic species, such as plants and most fungi.
G2 Phase (Second Gap)
In the G2 phase, the cell replenishes its energy stores and synthesizes proteins necessary for chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic phase. There may be additional cell growth during G2. The final preparations for the mitotic phase must be completed before the cell is able to enter the first stage of mitosis.
Key Points
• There are three stages of interphase: G1 (first gap), S (synthesis of new DNA ), and G2 (second gap).
• Cells spend most of their lives in interphase, specifically in the S phase where genetic material must be copied.
• The cell grows and carries out biochemical functions, such as protein synthesis, in the G1 phase.
• During the S phase, DNA is duplicated into two sister chromatids, and centrosomes, which give rise to the mitotic spindle, are also replicated.
• In the G2 phase, energy is replenished, new proteins are synthesized, the cytoskeleton is dismantled, and additional growth occurs.
Key Terms
• interphase: the stage in the life cycle of a cell where the cell grows and DNA is replicated
• sister chromatid: either of the two identical strands of a chromosome (DNA material) that separate during mitosis
• mitotic spindle: the apparatus that orchestrates the movement of chromosomes during mitosis | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.03%3A_Overview_of_the_Eukaryotic_Cell_Cycle.txt |
Learning Objectives
• Describe the events that occur at the different stages of mitosis
The Mitotic Phase
The mitotic phase is a multistep process during which the duplicated chromosomes are aligned, separated, and move into two new, identical daughter cells. The first portion of the mitotic phase is called karyokinesis or nuclear division. The second portion of the mitotic phase, called cytokinesis, is the physical separation of the cytoplasmic components into the two daughter cells.
Karyokinesis (Mitosis)
Karyokinesis, also known as mitosis, is divided into a series of phases (prophase, prometaphase, metaphase, anaphase, and telophase) that result in the division of the cell nucleus.
During prophase, the “first phase,” the nuclear envelope starts to dissociate into small vesicles. The membranous organelles (such as the Golgi apparatus and endoplasmic reticulum) fragment and disperse toward the periphery of the cell. The nucleolus disappears and the centrosomes begin to move to opposite poles of the cell. Microtubules that will eventually form the mitotic spindle extend between the centrosomes, pushing them farther apart as the microtubule fibers lengthen. The sister chromatids begin to coil more tightly with the aid of condensin proteins and become visible under a light microscope.
During prometaphase, the “first change phase,” many processes that began in prophase continue to advance. The remnants of the nuclear envelope fragment. The mitotic spindle continues to develop as more microtubules assemble and stretch across the length of the former nuclear area. Chromosomes become more condensed and discrete. Each sister chromatid develops a protein structure called a kinetochore in the centromeric region. The proteins of the kinetochore attract and bind mitotic spindle microtubules.
During metaphase, the “change phase,” all the chromosomes are aligned on a plane called the metaphase plate, or the equatorial plane, midway between the two poles of the cell. The sister chromatids are still tightly attached to each other by cohesin proteins. At this time, the chromosomes are maximally condensed.
During anaphase, the “upward phase,” the cohesin proteins degrade, and the sister chromatids separate at the centromere. Each chromatid, now called a chromosome, is pulled rapidly toward the centrosome to which its microtubule is attached. The cell becomes visibly elongated (oval shaped) as the polar microtubules slide against each other at the metaphase plate where they overlap.
During telophase, the “distance phase,” the chromosomes reach the opposite poles and begin to decondense (unravel), relaxing into a chromatin configuration. The mitotic spindles are depolymerized into tubulin monomers that will be used to assemble cytoskeletal components for each daughter cell. Nuclear envelopes form around the chromosomes and nucleosomes appear within the nuclear area.
Cytokinesis
Cytokinesis, or “cell motion,” is the second main stage of the mitotic phase during which cell division is completed via the physical separation of the cytoplasmic components into two daughter cells. Division is not complete until the cell components have been apportioned and completely separated into the two daughter cells. Although the stages of mitosis are similar for most eukaryotes, the process of cytokinesis is quite different for eukaryotes that have cell walls, such as plant cells.
In cells such as animal cells, which lack cell walls, cytokinesis follows the onset of anaphase. A contractile ring composed of actin filaments forms just inside the plasma membrane at the former metaphase plate. The actin filaments pull the equator of the cell inward, forming a fissure. This fissure or “crack” is called the cleavage furrow. The furrow deepens as the actin ring contracts; eventually the membrane is cleaved in two.
In plant cells, a new cell wall must form between the daughter cells. During interphase, the Golgi apparatus accumulates enzymes, structural proteins, and glucose molecules prior to breaking into vesicles and dispersing throughout the dividing cell. During telophase, these Golgi vesicles are transported on microtubules to form a phragmoplast (a vesicular structure) at the metaphase plate. There, the vesicles fuse and coalesce from the center toward the cell walls; this structure is called a cell plate. As more vesicles fuse, the cell plate enlarges until it merges with the cell walls at the periphery of the cell. Enzymes use the glucose that has accumulated between the membrane layers to build a new cell wall. The Golgi membranes become parts of the plasma membrane on either side of the new cell wall.
G0 Phase
Not all cells adhere to the classic cell cycle pattern in which a newly-formed daughter cell immediately enters the preparatory phases of interphase, closely followed by the mitotic phase. Cells in G0 phase are not actively preparing to divide. The cell is in a quiescent (inactive) stage that occurs when cells exit the cell cycle. Some cells enter G0 temporarily until an external signal triggers the onset of G1. Other cells that never or rarely divide, such as mature cardiac muscle and nerve cells, remain in G0 permanently.
Key Points
• During prophase, the nucleus disappears, spindle fibers form, and DNA condenses into chromosomes ( sister chromatids ).
• During metaphase, the sister chromatids align along the equator of the cell by attaching their centromeres to the spindle fibers.
• During anaphase, sister chromatids are separated at the centromere and are pulled towards opposite poles of the cell by the mitotic spindle.
• During telophase, chromosomes arrive at opposite poles and unwind into thin strands of DNA, the spindle fibers disappear, and the nuclear membrane reappears.
• Cytokinesis is the actual splitting of the cell membrane; animal cells pinch apart, while plant cells form a cell plate that becomes the new cell wall.
• Cells enter the G0 (inactive) phase after they exit the cell cycle when they are not actively preparing to divide; some cells remain in G0 phase permanently.
Key Terms
• karyokinesis: (mitosis) the first portion of mitotic phase in which division of the cell nucleus takes place
• centrosome: an organelle near the nucleus in the cytoplasm of most organisms that controls the organization of its microtubules and gives rise to the mitotic spindle
• cytokinesis: the second portion of the mitotic phase in which the cytoplasm of a cell divides following the division of the nucleus | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.05%3A_M_phase-_Chromosome_Segregation_and_the_Division_of_Cytoplasmic_Contents.txt |
Skills to Develop
• Understand how the cell cycle is controlled by mechanisms both internal and external to the cell
• Explain how the three internal control checkpoints occur at the end of G1, at the G2/M transition, and during metaphase
• Describe the molecules that control the cell cycle through positive and negative regulation
The length of the cell cycle is highly variable, even within the cells of a single organism. In humans, the frequency of cell turnover ranges from a few hours in early embryonic development, to an average of two to five days for epithelial cells, and to an entire human lifetime spent in G0 by specialized cells, such as cortical neurons or cardiac muscle cells. There is also variation in the time that a cell spends in each phase of the cell cycle. When fast-dividing mammalian cells are grown in culture (outside the body under optimal growing conditions), the length of the cycle is about 24 hours. In rapidly dividing human cells with a 24-hour cell cycle, the G1 phase lasts approximately nine hours, the S phase lasts 10 hours, the G2 phase lasts about four and one-half hours, and the M phase lasts approximately one-half hour. In early embryos of fruit flies, the cell cycle is completed in about eight minutes. The timing of events in the cell cycle is controlled by mechanisms that are both internal and external to the cell.
Regulation of the Cell Cycle by External Events
Both the initiation and inhibition of cell division are triggered by events external to the cell when it is about to begin the replication process. An event may be as simple as the death of a nearby cell or as sweeping as the release of growth-promoting hormones, such as human growth hormone (HGH). A lack of HGH can inhibit cell division, resulting in dwarfism, whereas too much HGH can result in gigantism. Crowding of cells can also inhibit cell division. Another factor that can initiate cell division is the size of the cell; as a cell grows, it becomes inefficient due to its decreasing surface-to-volume ratio. The solution to this problem is to divide.
Whatever the source of the message, the cell receives the signal, and a series of events within the cell allows it to proceed into interphase. Moving forward from this initiation point, every parameter required during each cell cycle phase must be met or the cycle cannot progress.
Regulation at Internal Checkpoints
It is essential that the daughter cells produced be exact duplicates of the parent cell. Mistakes in the duplication or distribution of the chromosomes lead to mutations that may be passed forward to every new cell produced from an abnormal cell. To prevent a compromised cell from continuing to divide, there are internal control mechanisms that operate at three main cell cycle checkpoints. A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable. These checkpoints occur near the end of G1, at the G2/M transition, and during metaphase (Figure \(1\)).
The G1 Checkpoint
The G1 checkpoint determines whether all conditions are favorable for cell division to proceed. The G1 checkpoint, also called the restriction point (in yeast), is a point at which the cell irreversibly commits to the cell division process. External influences, such as growth factors, play a large role in carrying the cell past the G1 checkpoint. In addition to adequate reserves and cell size, there is a check for genomic DNA damage at the G1 checkpoint. A cell that does not meet all the requirements will not be allowed to progress into the S phase. The cell can halt the cycle and attempt to remedy the problematic condition, or the cell can advance into G0 and await further signals when conditions improve.
The G2 Checkpoint
The G2 checkpoint bars entry into the mitotic phase if certain conditions are not met. As at the G1 checkpoint, cell size and protein reserves are assessed. However, the most important role of the G2 checkpoint is to ensure that all of the chromosomes have been replicated and that the replicated DNA is not damaged. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the damaged DNA.
The M Checkpoint
The M checkpoint occurs near the end of the metaphase stage of karyokinesis. The M checkpoint is also known as the spindle checkpoint, because it determines whether all the sister chromatids are correctly attached to the spindle microtubules. Because the separation of the sister chromatids during anaphase is an irreversible step, the cycle will not proceed until the kinetochores of each pair of sister chromatids are firmly anchored to at least two spindle fibers arising from opposite poles of the cell.
Link to Learning
Watch what occurs at the G1, G2, and M checkpoints by visiting this website to see an animation of the cell cycle.
Regulator Molecules of the Cell Cycle
In addition to the internally controlled checkpoints, there are two groups of intracellular molecules that regulate the cell cycle. These regulatory molecules either promote progress of the cell to the next phase (positive regulation) or halt the cycle (negative regulation). Regulator molecules may act individually, or they can influence the activity or production of other regulatory proteins. Therefore, the failure of a single regulator may have almost no effect on the cell cycle, especially if more than one mechanism controls the same event. Conversely, the effect of a deficient or non-functioning regulator can be wide-ranging and possibly fatal to the cell if multiple processes are affected.
Positive Regulation of the Cell Cycle
Two groups of proteins, called cyclins and cyclin-dependent kinases (Cdks), are responsible for the progress of the cell through the various checkpoints. The levels of the four cyclin proteins fluctuate throughout the cell cycle in a predictable pattern (Figure \(2\)). Increases in the concentration of cyclin proteins are triggered by both external and internal signals. After the cell moves to the next stage of the cell cycle, the cyclins that were active in the previous stage are degraded.
Cyclins regulate the cell cycle only when they are tightly bound to Cdks. To be fully active, the Cdk/cyclin complex must also be phosphorylated in specific locations. Like all kinases, Cdks are enzymes (kinases) that phosphorylate other proteins. Phosphorylation activates the protein by changing its shape. The proteins phosphorylated by Cdks are involved in advancing the cell to the next phase. (Figure \(3\)). The levels of Cdk proteins are relatively stable throughout the cell cycle; however, the concentrations of cyclin fluctuate and determine when Cdk/cyclin complexes form. The different cyclins and Cdks bind at specific points in the cell cycle and thus regulate different checkpoints.
Since the cyclic fluctuations of cyclin levels are based on the timing of the cell cycle and not on specific events, regulation of the cell cycle usually occurs by either the Cdk molecules alone or the Cdk/cyclin complexes. Without a specific concentration of fully activated cyclin/Cdk complexes, the cell cycle cannot proceed through the checkpoints.
Although the cyclins are the main regulatory molecules that determine the forward momentum of the cell cycle, there are several other mechanisms that fine-tune the progress of the cycle with negative, rather than positive, effects. These mechanisms essentially block the progression of the cell cycle until problematic conditions are resolved. Molecules that prevent the full activation of Cdks are called Cdk inhibitors. Many of these inhibitor molecules directly or indirectly monitor a particular cell cycle event. The block placed on Cdks by inhibitor molecules will not be removed until the specific event that the inhibitor monitors is completed.
Negative Regulation of the Cell Cycle
The second group of cell cycle regulatory molecules are negative regulators. Negative regulators halt the cell cycle. Remember that in positive regulation, active molecules cause the cycle to progress.
The best understood negative regulatory molecules are retinoblastoma protein (Rb), p53, and p21. Retinoblastoma proteins are a group of tumor-suppressor proteins common in many cells. The 53 and 21 designations refer to the functional molecular masses of the proteins (p) in kilodaltons. Much of what is known about cell cycle regulation comes from research conducted with cells that have lost regulatory control. All three of these regulatory proteins were discovered to be damaged or non-functional in cells that had begun to replicate uncontrollably (became cancerous). In each case, the main cause of the unchecked progress through the cell cycle was a faulty copy of the regulatory protein.
Rb, p53, and p21 act primarily at the G1 checkpoint. p53 is a multi-functional protein that has a major impact on the commitment of a cell to division because it acts when there is damaged DNA in cells that are undergoing the preparatory processes during G1. If damaged DNA is detected, p53 halts the cell cycle and recruits enzymes to repair the DNA. If the DNA cannot be repaired, p53 can trigger apoptosis, or cell suicide, to prevent the duplication of damaged chromosomes. As p53 levels rise, the production of p21 is triggered. p21 enforces the halt in the cycle dictated by p53 by binding to and inhibiting the activity of the Cdk/cyclin complexes. As a cell is exposed to more stress, higher levels of p53 and p21 accumulate, making it less likely that the cell will move into the S phase.
Rb exerts its regulatory influence on other positive regulator proteins. Chiefly, Rb monitors cell size. In the active, dephosphorylated state, Rb binds to proteins called transcription factors, most commonly, E2F (Figure \(4\)). Transcription factors “turn on” specific genes, allowing the production of proteins encoded by that gene. When Rb is bound to E2F, production of proteins necessary for the G1/S transition is blocked. As the cell increases in size, Rb is slowly phosphorylated until it becomes inactivated. Rb releases E2F, which can now turn on the gene that produces the transition protein, and this particular block is removed. For the cell to move past each of the checkpoints, all positive regulators must be “turned on,” and all negative regulators must be “turned off.”
Exercise \(1\)
Rb and other proteins that negatively regulate the cell cycle are sometimes called tumor suppressors. Why do you think the name tumor suppressor might be appropriate for these proteins?
Answer
Rb and other negative regulatory proteins control cell division and therefore prevent the formation of tumors. Mutations that prevent these proteins from carrying out their function can result in cancer.
Summary
Each step of the cell cycle is monitored by internal controls called checkpoints. There are three major checkpoints in the cell cycle: one near the end of G1, a second at the G2/M transition, and the third during metaphase. Positive regulator molecules allow the cell cycle to advance to the next stage. Negative regulator molecules monitor cellular conditions and can halt the cycle until specific requirements are met.
Glossary
cell cycle checkpoint
mechanism that monitors the preparedness of a eukaryotic cell to advance through the various cell cycle stages
cyclin
one of a group of proteins that act in conjunction with cyclin-dependent kinases to help regulate the cell cycle by phosphorylating key proteins; the concentrations of cyclins fluctuate throughout the cell cycle
cyclin-dependent kinase
one of a group of protein kinases that helps to regulate the cell cycle when bound to cyclin; it functions to phosphorylate other proteins that are either activated or inactivated by phosphorylation
p21
cell cycle regulatory protein that inhibits the cell cycle; its levels are controlled by p53
p53
cell cycle regulatory protein that regulates cell growth and monitors DNA damage; it halts the progression of the cell cycle in cases of DNA damage and may induce apoptosis
retinoblastoma protein (Rb)
regulatory molecule that exhibits negative effects on the cell cycle by interacting with a transcription factor (E2F) | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.06%3A_Control_of_the_Cell_Cycle.txt |
Skills to Develop
• Describe how cancer is caused by uncontrolled cell growth
• Understand how proto-oncogenes are normal cell genes that, when mutated, become oncogenes
• Describe how tumor suppressors function
• Explain how mutant tumor suppressors cause cancer
Cancer comprises many different diseases caused by a common mechanism: uncontrolled cell growth. Despite the redundancy and overlapping levels of cell cycle control, errors do occur. One of the critical processes monitored by the cell cycle checkpoint surveillance mechanism is the proper replication of DNA during the S phase. Even when all of the cell cycle controls are fully functional, a small percentage of replication errors (mutations) will be passed on to the daughter cells. If changes to the DNA nucleotide sequence occur within a coding portion of a gene and are not corrected, a gene mutation results. All cancers start when a gene mutation gives rise to a faulty protein that plays a key role in cell reproduction. The change in the cell that results from the malformed protein may be minor: perhaps a slight delay in the binding of Cdk to cyclin or an Rb protein that detaches from its target DNA while still phosphorylated. Even minor mistakes, however, may allow subsequent mistakes to occur more readily. Over and over, small uncorrected errors are passed from the parent cell to the daughter cells and amplified as each generation produces more non-functional proteins from uncorrected DNA damage. Eventually, the pace of the cell cycle speeds up as the effectiveness of the control and repair mechanisms decreases. Uncontrolled growth of the mutated cells outpaces the growth of normal cells in the area, and a tumor (“-oma”) can result.
Proto-oncogenes
The genes that code for the positive cell cycle regulators are called proto-oncogenes. Proto-oncogenes are normal genes that, when mutated in certain ways, become oncogenes, genes that cause a cell to become cancerous. Consider what might happen to the cell cycle in a cell with a recently acquired oncogene. In most instances, the alteration of the DNA sequence will result in a less functional (or non-functional) protein. The result is detrimental to the cell and will likely prevent the cell from completing the cell cycle; however, the organism is not harmed because the mutation will not be carried forward. If a cell cannot reproduce, the mutation is not propagated and the damage is minimal. Occasionally, however, a gene mutation causes a change that increases the activity of a positive regulator. For example, a mutation that allows Cdk to be activated without being partnered with cyclin could push the cell cycle past a checkpoint before all of the required conditions are met. If the resulting daughter cells are too damaged to undergo further cell divisions, the mutation would not be propagated and no harm would come to the organism. However, if the atypical daughter cells are able to undergo further cell divisions, subsequent generations of cells will probably accumulate even more mutations, some possibly in additional genes that regulate the cell cycle.
The Cdk gene in the above example is only one of many genes that are considered proto-oncogenes. In addition to the cell cycle regulatory proteins, any protein that influences the cycle can be altered in such a way as to override cell cycle checkpoints. An oncogene is any gene that, when altered, leads to an increase in the rate of cell cycle progression.
Tumor Suppressor Genes
Like proto-oncogenes, many of the negative cell cycle regulatory proteins were discovered in cells that had become cancerous. Tumor suppressor genes are segments of DNA that code for negative regulator proteins, the type of regulators that, when activated, can prevent the cell from undergoing uncontrolled division. The collective function of the best-understood tumor suppressor gene proteins, Rb, p53, and p21, is to put up a roadblock to cell cycle progression until certain events are completed. A cell that carries a mutated form of a negative regulator might not be able to halt the cell cycle if there is a problem. Tumor suppressors are similar to brakes in a vehicle: Malfunctioning brakes can contribute to a car crash.
Mutated p53 genes have been identified in more than one-half of all human tumor cells. This discovery is not surprising in light of the multiple roles that the p53 protein plays at the G1 checkpoint. A cell with a faulty p53 may fail to detect errors present in the genomic DNA (Figure \(1\)). Even if a partially functional p53 does identify the mutations, it may no longer be able to signal the necessary DNA repair enzymes. Either way, damaged DNA will remain uncorrected. At this point, a functional p53 will deem the cell unsalvageable and trigger programmed cell death (apoptosis). The damaged version of p53 found in cancer cells, however, cannot trigger apoptosis.
Exercise \(1\)
Human papillomavirus can cause cervical cancer. The virus encodes E6, a protein that binds p53. Based on this fact and what you know about p53, what effect do you think E6 binding has on p53 activity?
1. E6 activates p53
2. E6 inactivates p53
3. E6 mutates p53
4. E6 binding marks p53 for degradation
Answer
D. E6 binding marks p53 for degradation.
The loss of p53 function has other repercussions for the cell cycle. Mutated p53 might lose its ability to trigger p21 production. Without adequate levels of p21, there is no effective block on Cdk activation. Essentially, without a fully functional p53, the G1 checkpoint is severely compromised and the cell proceeds directly from G1 to S regardless of internal and external conditions. At the completion of this shortened cell cycle, two daughter cells are produced that have inherited the mutated p53 gene. Given the non-optimal conditions under which the parent cell reproduced, it is likely that the daughter cells will have acquired other mutations in addition to the faulty tumor suppressor gene. Cells such as these daughter cells quickly accumulate both oncogenes and non-functional tumor suppressor genes. Again, the result is tumor growth.
Link to Learning
Go to this website to watch an animation of how cancer results from errors in the cell cycle.
Summary
Cancer is the result of unchecked cell division caused by a breakdown of the mechanisms that regulate the cell cycle. The loss of control begins with a change in the DNA sequence of a gene that codes for one of the regulatory molecules. Faulty instructions lead to a protein that does not function as it should. Any disruption of the monitoring system can allow other mistakes to be passed on to the daughter cells. Each successive cell division will give rise to daughter cells with even more accumulated damage. Eventually, all checkpoints become nonfunctional, and rapidly reproducing cells crowd out normal cells, resulting in a tumor or leukemia (blood cancer).
Glossary
oncogene
mutated version of a normal gene involved in the positive regulation of the cell cycle
proto-oncogene
normal gene that when mutated becomes an oncogene
tumor suppressor gene
segment of DNA that codes for regulator proteins that prevent the cell from undergoing uncontrolled division | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/10%3A_How_Cells_Divide/10.07%3A_Genetics_of_Cancer.txt |
• 11.1: Sexual Reproduction Requires Meiosis
Most eukaryotes undergo sexual reproduction. The variation introduced into the reproductive cells by meiosis appears to be one of the advantages of sexual reproduction. Meiosis and fertilization alternate in sexual life cycles. The process of meiosis produces genetically unique reproductive cells called gametes, which have half the number of chromosomes as the parent cell. Fertilization, the fusion of haploid gametes from two individuals, restores the diploid condition.
• 11.2: Features of Meiosis
• 11.3: The Process of Meiosis
Sexual reproduction requires that diploid organisms produce haploid cells that can fuse during fertilization to form diploid offspring. The process that results in haploid cells is called meiosis. Meiosis is a series of events that arrange and separate chromosomes into daughter cells. During the interphase of meiosis, each chromosome is duplicated. In meiosis, there are two rounds of nuclear division resulting in four nuclei and usually four haploid daughter cells.
• 11.4: Summing Up- Meiosis Versus Mitosis
11: Sexual Reproduction and Meiosis
Sexual reproduction was an early evolutionary innovation after the appearance of eukaryotic cells. The fact that most eukaryotes reproduce sexually is evidence of its evolutionary success. In many animals, it is the only mode of reproduction. And yet, scientists recognize some real disadvantages to sexual reproduction. On the surface, offspring that are genetically identical to the parent may appear to be more advantageous. If the parent organism is successfully occupying a habitat, offspring with the same traits would be similarly successful. There is also the obvious benefit to an organism that can produce offspring by asexual budding, fragmentation, or asexual eggs. These methods of reproduction do not require another organism of the opposite sex. There is no need to expend energy finding or attracting a mate. That energy can be spent on producing more offspring. Indeed, some organisms that lead a solitary lifestyle have retained the ability to reproduce asexually. In addition, asexual populations only have female individuals, so every individual is capable of reproduction. In contrast, the males in sexual populations (half the population) are not producing offspring themselves. Because of this, an asexual population can grow twice as fast as a sexual population in theory. This means that in competition, the asexual population would have the advantage. All of these advantages to asexual reproduction, which are also disadvantages to sexual reproduction, should mean that the number of species with asexual reproduction should be more common.
However, multicellular organisms that exclusively depend on asexual reproduction are exceedingly rare. Why is sexual reproduction so common? This is one of the important questions in biology and has been the focus of much research from the latter half of the twentieth century until now. A likely explanation is that the variation that sexual reproduction creates among offspring is very important to the survival and reproduction of those offspring. The only source of variation in asexual organisms is mutation. This is the ultimate source of variation in sexual organisms. In addition, those different mutations are continually reshuffled from one generation to the next when different parents combine their unique genomes, and the genes are mixed into different combinations by the process of meiosis. Meiosis is the division of the contents of the nucleus that divides the chromosomes among gametes. Variation is introduced during meiosis, as well as when the gametes combine in fertilization.
EVOLUTION IN ACTION: The Red Queen Hypothesis
There is no question that sexual reproduction provides evolutionary advantages to organisms that employ this mechanism to produce offspring. The problematic question is why, even in the face of fairly stable conditions, sexual reproduction persists when it is more difficult and produces fewer offspring for individual organisms? Variation is the outcome of sexual reproduction, but why are ongoing variations necessary? Enter the Red Queen hypothesis, first proposed by Leigh Van Valen in 1973.1 The concept was named in reference to the Red Queen's race in Lewis Carroll's book, Through the Looking-Glass, in which the Red Queen says one must run at full speed just to stay where one is.
All species coevolve with other organisms. For example, predators coevolve with their prey, and parasites coevolve with their hosts. A remarkable example of coevolution between predators and their prey is the unique coadaptation of night flying bats and their moth prey. Bats find their prey by emitting high-pitched clicks, but moths have evolved simple ears to hear these clicks so they can avoid the bats. The moths have also adapted behaviors, such as flying away from the bat when they first hear it, or dropping suddenly to the ground when the bat is upon them. Bats have evolved “quiet” clicks in an attempt to evade the moth’s hearing. Some moths have evolved the ability to respond to the bats’ clicks with their own clicks as a strategy to confuse the bats echolocation abilities.
Each tiny advantage gained by favorable variation gives a species an edge over close competitors, predators, parasites, or even prey. The only method that will allow a coevolving species to keep its own share of the resources is also to continually improve its ability to survive and produce offspring. As one species gains an advantage, other species must also develop an advantage or they will be outcompeted. No single species progresses too far ahead because genetic variation among progeny of sexual reproduction provides all species with a mechanism to produce adapted individuals. Species whose individuals cannot keep up become extinct. The Red Queen’s catchphrase was, “It takes all the running you can do to stay in the same place.” This is an apt description of coevolution between competing species.
Life Cycles of Sexually Reproducing Organisms
Fertilization and meiosis alternate in sexual life cycles. What happens between these two events depends on the organism. The process of meiosis reduces the resulting gamete’s chromosome number by half. Fertilization, the joining of two haploid gametes, restores the diploid condition. There are three main categories of life cycles in multicellular organisms: diploid-dominant, in which the multicellular diploid stage is the most obvious life stage (and there is no multicellular haploid stage), as with most animals including humans; haploid-dominant, in which the multicellular haploid stage is the most obvious life stage (and there is no multicellular diploid stage), as with all fungi and some algae; and alternation of generations, in which the two stages, haploid and diploid, are apparent to one degree or another depending on the group, as with plants and some algae.
Nearly all animals employ a diploid-dominant life-cycle strategy in which the only haploid cells produced by the organism are the gametes. The gametes are produced from diploid germ cells, a special cell line that only produces gametes. Once the haploid gametes are formed, they lose the ability to divide again. There is no multicellular haploid life stage. Fertilization occurs with the fusion of two gametes, usually from different individuals, restoring the diploid state (Figure \(1\)a).
ART CONNECTION
If a mutation occurs so that a fungus is no longer able to produce a minus mating type, will it still be able to reproduce?
Most fungi and algae employ a life-cycle strategy in which the multicellular “body” of the organism is haploid. During sexual reproduction, specialized haploid cells from two individuals join to form a diploid zygote. The zygote immediately undergoes meiosis to form four haploid cells called spores (Figure \(1\)b).
The third life-cycle type, employed by some algae and all plants, is called alternation of generations. These species have both haploid and diploid multicellular organisms as part of their life cycle. The haploid multicellular plants are called gametophytes because they produce gametes. Meiosis is not involved in the production of gametes in this case, as the organism that produces gametes is already haploid. Fertilization between the gametes forms a diploid zygote. The zygote will undergo many rounds of mitosis and give rise to a diploid multicellular plant called a sporophyte. Specialized cells of the sporophyte will undergo meiosis and produce haploid spores. The spores will develop into the gametophytes (Figure \(1\)c).
Section Summary
Nearly all eukaryotes undergo sexual reproduction. The variation introduced into the reproductive cells by meiosis appears to be one of the advantages of sexual reproduction that has made it so successful. Meiosis and fertilization alternate in sexual life cycles. The process of meiosis produces genetically unique reproductive cells called gametes, which have half the number of chromosomes as the parent cell. Fertilization, the fusion of haploid gametes from two individuals, restores the diploid condition. Thus, sexually reproducing organisms alternate between haploid and diploid stages. However, the ways in which reproductive cells are produced and the timing between meiosis and fertilization vary greatly. There are three main categories of life cycles: diploid-dominant, demonstrated by most animals; haploid-dominant, demonstrated by all fungi and some algae; and alternation of generations, demonstrated by plants and some algae.
Art Connections
Figure \(1\): If a mutation occurs so that a fungus is no longer able to produce a minus mating type, will it still be able to reproduce?
Answer
Yes, it will be able to reproduce asexually.
Footnotes
1. 1 Leigh Van Valen, “A new evolutionary law,” Evolutionary Theory 1 (1973): 1–30.
Glossary
alternation of generations
a life-cycle type in which the diploid and haploid stages alternate
diploid-dominant
a life-cycle type in which the multicellular diploid stage is prevalent
haploid-dominant
a life-cycle type in which the multicellular haploid stage is prevalent
gametophyte
a multicellular haploid life-cycle stage that produces gametes
germ cell
a specialized cell that produces gametes, such as eggs or sperm
life cycle
the sequence of events in the development of an organism and the production of cells that produce offspring
meiosis
a nuclear division process that results in four haploid cells
sporophyte
a multicellular diploid life-cycle stage that produces spores | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/11%3A_Sexual_Reproduction_and_Meiosis/11.01%3A_Sexual_Reproduction_Requires_Meiosis.txt |
Learning Objectives
• Describe the stages and results of meiosis I
Meiosis I
Meiosis is preceded by an interphase consisting of three stages. The G1 phase (also called the first gap phase) initiates this stage and is focused on cell growth. The S phase is next, during which the DNA of the chromosomes is replicated. This replication produces two identical copies, called sister chromatids, that are held together at the centromere by cohesin proteins. The centrosomes, which are the structures that organize the microtubules of the meiotic spindle, also replicate. Finally, during the G2 phase (also called the second gap phase), the cell undergoes the final preparations for meiosis.
Prophase I
During prophase I, chromosomes condense and become visible inside the nucleus. As the nuclear envelope begins to break down, homologous chromosomes move closer together. The synaptonemal complex, a lattice of proteins between the homologous chromosomes, forms at specific locations, spreading to cover the entire length of the chromosomes. The tight pairing of the homologous chromosomes is called synapsis. In synapsis, the genes on the chromatids of the homologous chromosomes are aligned with each other. The synaptonemal complex also supports the exchange of chromosomal segments between non-sister homologous chromatids in a process called crossing over. The crossover events are the first source of genetic variation produced by meiosis. A single crossover event between homologous non-sister chromatids leads to an exchange of DNA between chromosomes. Following crossover, the synaptonemal complex breaks down and the cohesin connection between homologous pairs is also removed. At the end of prophase I, the pairs are held together only at the chiasmata; they are called tetrads because the four sister chromatids of each pair of homologous chromosomes are now visible.
Prometaphase I
The key event in prometaphase I is the formation of the spindle fiber apparatus where spindle fiber microtubules attach to the kinetochore proteins at the centromeres. Microtubules grow from centrosomes placed at opposite poles of the cell. The microtubules move toward the middle of the cell and attach to one of the two fused homologous chromosomes at the kinetochores. At the end of prometaphase I, each tetrad is attached to microtubules from both poles, with one homologous chromosome facing each pole. In addition, the nuclear membrane has broken down entirely.
Metaphase I
During metaphase I, the tetrads move to the metaphase plate with kinetochores facing opposite poles. The homologous pairs orient themselves randomly at the equator. This event is the second mechanism that introduces variation into the gametes or spores. In each cell that undergoes meiosis, the arrangement of the tetrads is different. The number of variations is dependent on the number of chromosomes making up a set. There are two possibilities for orientation at the metaphase plate. The possible number of alignments, therefore, equals 2n, where n is the number of chromosomes per set. Given these two mechanisms, it is highly unlikely that any two haploid cells resulting from meiosis will have the same genetic composition.
Anaphase I
In anaphase I, the microtubules pull the attached chromosomes apart. The sister chromatids remain tightly bound together at the centromere. The chiasmata are broken in anaphase I as the microtubules attached to the fused kinetochores pull the homologous chromosomes apart.
Telophase I and Cytokinesis
In telophase I, the separated chromosomes arrive at opposite poles. In some organisms, the chromosomes decondense and nuclear envelopes form around the chromatids in telophase I. Then cytokinesis, the physical separation of the cytoplasmic components into two daughter cells, occurs without reformation of the nuclei. In nearly all species of animals and some fungi, cytokinesis separates the cell contents via a cleavage furrow (constriction of the actin ring that leads to cytoplasmic division). In plants, a cell plate is formed during cell cytokinesis by Golgi vesicles fusing at the metaphase plate. This cell plate will ultimately lead to the formation of cell walls that separate the two daughter cells.
Two haploid cells are the end result of the first meiotic division. The cells are haploid because at each pole there is just one of each pair of the homologous chromosomes. Therefore, only one full set of the chromosomes is present. Although there is only one chromosome set, each homolog still consists of two sister chromatids.
Key Points
• Meiosis is preceded by interphase which consists of the G1 phase (growth), the S phase ( DNA replication), and the G2 phase.
• During prophase I, the homologous chromosomes condense and become visible as the x shape we know, pair up to form a tetrad, and exchange genetic material by crossing over.
• During prometaphase I, microtubules attach at the chromosomes’ kinetochores and the nuclear envelope breaks down.
• In metaphase I, the tetrads line themselves up at the metaphase plate and homologous pairs orient themselves randomly.
• In anaphase I, centromeres break down and homologous chromosomes separate.
• In telophase I, chromosomes move to opposite poles; during cytokinesis the cell separates into two haploid cells.
Key Terms
• crossing over: the exchange of genetic material between homologous chromosomes that results in recombinant chromosomes
• tetrad: two pairs of sister chromatids (a dyad pair) aligned in a certain way and often on the equatorial plane during the meiosis process
• chromatid: either of the two strands of a chromosome that separate during meiosis
11.2.02: Meiosis II
Learning Objectives
• Describe the stages and results of Meiosis II
Meiosis II
Meiosis II initiates immediately after cytokinesis, usually before the chromosomes have fully decondensed. In contrast to meiosis I, meiosis II resembles a normal mitosis. In some species, cells enter a brief interphase, or interkinesis, before entering meiosis II. Interkinesis lacks an S phase, so chromosomes are not duplicated. The two cells produced in meiosis I go through the events of meiosis II together. During meiosis II, the sister chromatids within the two daughter cells separate, forming four new haploid gametes. The mechanics of meiosis II is similar to mitosis, except that each dividing cell has only one set of homologous chromosomes.
Prophase II
If the chromosomes decondensed in telophase I, they condense again. If nuclear envelopes were formed, they fragment into vesicles. The centrosomes that were duplicated during interphase I move away from each other toward opposite poles and new spindles are formed.
Prometaphase II
The nuclear envelopes are completely broken down and the spindle is fully formed. Each sister chromatid forms an individual kinetochore that attaches to microtubules from opposite poles.
Metaphase II
The sister chromatids are maximally condensed and aligned at the equator of the cell.
Anaphase II
The sister chromatids are pulled apart by the kinetochore microtubules and move toward opposite poles. Non-kinetochore microtubules elongate the cell.
Telophase II and Cytokinesis
The chromosomes arrive at opposite poles and begin to decondense. Nuclear envelopes form around the chromosomes. Cytokinesis separates the two cells into four unique haploid cells. At this point, the newly-formed nuclei are both haploid. The cells produced are genetically unique because of the random assortment of paternal and maternal homologs and because of the recombining of maternal and paternal segments of chromosomes (with their sets of genes) that occurs during crossover.
Key Points
• During prophase II, chromsomes condense again, centrosomes that were duplicated during interphase I move away from each other toward opposite poles, and new spindles are formed.
• During prometaphase II, the nuclear envelopes are completely broken down, and each sister chromatid forms an individual kinetochore that attaches to microtubules from opposite poles.
• During metaphase II, sister chromatids are condensed and aligned at the equator of the cell.
• During anaphase II sister chromatids are pulled apart by the kinetochore microtubules and move toward opposite poles.
• During telophase II and cytokinesis, chromosomes arrive at opposite poles and begin to decondense; the two cells divide into four unique haploid cells.
Key Terms
• meiosis II: the second part of the meiotic process; the end result is production of four haploid cells from the two haploid cells produced in meiosis I | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/11%3A_Sexual_Reproduction_and_Meiosis/11.02%3A_Features_of_Meiosis/11.2.01%3A_Meiosis_I.txt |
Sexual reproduction requires fertilization, a union of two cells from two individual organisms. If those two cells each contain one set of chromosomes, then the resulting cell contains two sets of chromosomes. The number of sets of chromosomes in a cell is called its ploidy level. Haploid cells contain one set of chromosomes. Cells containing two sets of chromosomes are called diploid. If the reproductive cycle is to continue, the diploid cell must somehow reduce its number of chromosome sets before fertilization can occur again, or there will be a continual doubling in the number of chromosome sets in every generation. So, in addition to fertilization, sexual reproduction includes a nuclear division, known as meiosis, that reduces the number of chromosome sets.
Most animals and plants are diploid, containing two sets of chromosomes; in each somatic cell (the nonreproductive cells of a multicellular organism), the nucleus contains two copies of each chromosome that are referred to as homologous chromosomes. Somatic cells are sometimes referred to as “body” cells. Homologous chromosomes are matched pairs containing genes for the same traits in identical locations along their length. Diploid organisms inherit one copy of each homologous chromosome from each parent; all together, they are considered a full set of chromosomes. In animals, haploid cells containing a single copy of each homologous chromosome are found only within gametes. Gametes fuse with another haploid gamete to produce a diploid cell.
The nuclear division that forms haploid cells, which is called meiosis, is related to mitosis. As you have learned, mitosis is part of a cell reproduction cycle that results in identical daughter nuclei that are also genetically identical to the original parent nucleus. In mitosis, both the parent and the daughter nuclei contain the same number of chromosome sets—diploid for most plants and animals. Meiosis employs many of the same mechanisms as mitosis. However, the starting nucleus is always diploid and the nuclei that result at the end of a meiotic cell division are haploid. To achieve the reduction in chromosome number, meiosis consists of one round of chromosome duplication and two rounds of nuclear division. Because the events that occur during each of the division stages are analogous to the events of mitosis, the same stage names are assigned. However, because there are two rounds of division, the stages are designated with a “I” or “II.” Thus, meiosis I is the first round of meiotic division and consists of prophase I, prometaphase I, and so on. Meiosis I reduces the number of chromosome sets from two to one. The genetic information is also mixed during this division to create unique recombinant chromosomes. Meiosis II, in which the second round of meiotic division takes place in a way that is similar to mitosis, includes prophase II, prometaphase II, and so on.
Interphase
Meiosis is preceded by an interphase consisting of the G1, S, and G2 phases, which are nearly identical to the phases preceding mitosis. The G1 phase is the first phase of interphase and is focused on cell growth. In the S phase, the DNA of the chromosomes is replicated. Finally, in the G2 phase, the cell undergoes the final preparations for meiosis.
During DNA duplication of the S phase, each chromosome becomes composed of two identical copies (called sister chromatids) that are held together at the centromere until they are pulled apart during meiosis II. In an animal cell, the centrosomes that organize the microtubules of the meiotic spindle also replicate. This prepares the cell for the first meiotic phase.
Meiosis I
Early in prophase I, the chromosomes can be seen clearly microscopically. As the nuclear envelope begins to break down, the proteins associated with homologous chromosomes bring the pair close to each other. The tight pairing of the homologous chromosomes is called synapsis. In synapsis, the genes on the chromatids of the homologous chromosomes are precisely aligned with each other. An exchange of chromosome segments between non-sister homologous chromatids occurs and is called crossing over. This process is revealed visually after the exchange as chiasmata (singular = chiasma) (Figure \(1\)).
As prophase I progresses, the close association between homologous chromosomes begins to break down, and the chromosomes continue to condense, although the homologous chromosomes remain attached to each other at chiasmata. The number of chiasmata varies with the species and the length of the chromosome. At the end of prophase I, the pairs are held together only at chiasmata (Figure \(1\)) and are called tetrads because the four sister chromatids of each pair of homologous chromosomes are now visible.
The crossover events are the first source of genetic variation produced by meiosis. A single crossover event between homologous non-sister chromatids leads to a reciprocal exchange of equivalent DNA between a maternal chromosome and a paternal chromosome. Now, when that sister chromatid is moved into a gamete, it will carry some DNA from one parent of the individual and some DNA from the other parent. The recombinant sister chromatid has a combination of maternal and paternal genes that did not exist before the crossover.
The key event in prometaphase I is the attachment of the spindle fiber microtubules to the kinetochore proteins at the centromeres. The microtubules assembled from centrosomes at opposite poles of the cell grow toward the middle of the cell. At the end of prometaphase I, each tetrad is attached to microtubules from both poles, with one homologous chromosome attached at one pole and the other homologous chromosome attached to the other pole. The homologous chromosomes are still held together at chiasmata. In addition, the nuclear membrane has broken down entirely.
During metaphase I, the homologous chromosomes are arranged in the center of the cell with the kinetochores facing opposite poles. The orientation of each pair of homologous chromosomes at the center of the cell is random.
This randomness, called independent assortment, is the physical basis for the generation of the second form of genetic variation in offspring. Consider that the homologous chromosomes of a sexually reproducing organism are originally inherited as two separate sets, one from each parent. Using humans as an example, one set of 23 chromosomes is present in the egg donated by the mother. The father provides the other set of 23 chromosomes in the sperm that fertilizes the egg. In metaphase I, these pairs line up at the midway point between the two poles of the cell. Because there is an equal chance that a microtubule fiber will encounter a maternally or paternally inherited chromosome, the arrangement of the tetrads at the metaphase plate is random. Any maternally inherited chromosome may face either pole. Any paternally inherited chromosome may also face either pole. The orientation of each tetrad is independent of the orientation of the other 22 tetrads.
In each cell that undergoes meiosis, the arrangement of the tetrads is different. The number of variations depends on the number of chromosomes making up a set. There are two possibilities for orientation (for each tetrad); thus, the possible number of alignments equals 2n where n is the number of chromosomes per set. Humans have 23 chromosome pairs, which results in over eight million (223) possibilities. This number does not include the variability previously created in the sister chromatids by crossover. Given these two mechanisms, it is highly unlikely that any two haploid cells resulting from meiosis will have the same genetic composition (Figure \(2\)).
To summarize the genetic consequences of meiosis I: the maternal and paternal genes are recombined by crossover events occurring on each homologous pair during prophase I; in addition, the random assortment of tetrads at metaphase produces a unique combination of maternal and paternal chromosomes that will make their way into the gametes.
In anaphase I, the spindle fibers pull the linked chromosomes apart. The sister chromatids remain tightly bound together at the centromere. It is the chiasma connections that are broken in anaphase I as the fibers attached to the fused kinetochores pull the homologous chromosomes apart (Figure \(3\)).
In telophase I, the separated chromosomes arrive at opposite poles. The remainder of the typical telophase events may or may not occur depending on the species. In some organisms, the chromosomes decondense and nuclear envelopes form around the chromatids in telophase I.
Cytokinesis, the physical separation of the cytoplasmic components into two daughter cells, occurs without reformation of the nuclei in other organisms. In nearly all species, cytokinesis separates the cell contents by either a cleavage furrow (in animals and some fungi), or a cell plate that will ultimately lead to formation of cell walls that separate the two daughter cells (in plants). At each pole, there is just one member of each pair of the homologous chromosomes, so only one full set of the chromosomes is present. This is why the cells are considered haploid—there is only one chromosome set, even though there are duplicate copies of the set because each homolog still consists of two sister chromatids that are still attached to each other. However, although the sister chromatids were once duplicates of the same chromosome, they are no longer identical at this stage because of crossovers.
CONCEPT IN ACTION
Review the process of meiosis, observing how chromosomes align and migrate, at this site.
Meiosis II
In meiosis II, the connected sister chromatids remaining in the haploid cells from meiosis I will be split to form four haploid cells. In some species, cells enter a brief interphase, or interkinesis, that lacks an S phase, before entering meiosis II. Chromosomes are not duplicated during interkinesis. The two cells produced in meiosis I go through the events of meiosis II in synchrony. Overall, meiosis II resembles the mitotic division of a haploid cell.
In prophase II, if the chromosomes decondensed in telophase I, they condense again. If nuclear envelopes were formed, they fragment into vesicles. The centrosomes duplicated during interkinesis move away from each other toward opposite poles, and new spindles are formed. In prometaphase II, the nuclear envelopes are completely broken down, and the spindle is fully formed. Each sister chromatid forms an individual kinetochore that attaches to microtubules from opposite poles. In metaphase II, the sister chromatids are maximally condensed and aligned at the center of the cell. In anaphase II, the sister chromatids are pulled apart by the spindle fibers and move toward opposite poles.
In telophase II, the chromosomes arrive at opposite poles and begin to decondense. Nuclear envelopes form around the chromosomes. Cytokinesis separates the two cells into four genetically unique haploid cells. At this point, the nuclei in the newly produced cells are both haploid and have only one copy of the single set of chromosomes. The cells produced are genetically unique because of the random assortment of paternal and maternal homologs and because of the recombination of maternal and paternal segments of chromosomes—with their sets of genes—that occurs during crossover.
Comparing Meiosis and Mitosis
Mitosis and meiosis, which are both forms of division of the nucleus in eukaryotic cells, share some similarities, but also exhibit distinct differences that lead to their very different outcomes. Mitosis is a single nuclear division that results in two nuclei, usually partitioned into two new cells. The nuclei resulting from a mitotic division are genetically identical to the original. They have the same number of sets of chromosomes: one in the case of haploid cells, and two in the case of diploid cells. On the other hand, meiosis is two nuclear divisions that result in four nuclei, usually partitioned into four new cells. The nuclei resulting from meiosis are never genetically identical, and they contain one chromosome set only—this is half the number of the original cell, which was diploid (Figure \(4\)).
The differences in the outcomes of meiosis and mitosis occur because of differences in the behavior of the chromosomes during each process. Most of these differences in the processes occur in meiosis I, which is a very different nuclear division than mitosis. In meiosis I, the homologous chromosome pairs become associated with each other, are bound together, experience chiasmata and crossover between sister chromatids, and line up along the metaphase plate in tetrads with spindle fibers from opposite spindle poles attached to each kinetochore of a homolog in a tetrad. All of these events occur only in meiosis I, never in mitosis.
Homologous chromosomes move to opposite poles during meiosis I so the number of sets of chromosomes in each nucleus-to-be is reduced from two to one. For this reason, meiosis I is referred to as a reduction division. There is no such reduction in ploidy level in mitosis.
Meiosis II is much more analogous to a mitotic division. In this case, duplicated chromosomes (only one set of them) line up at the center of the cell with divided kinetochores attached to spindle fibers from opposite poles. During anaphase II, as in mitotic anaphase, the kinetochores divide and one sister chromatid is pulled to one pole and the other sister chromatid is pulled to the other pole. If it were not for the fact that there had been crossovers, the two products of each meiosis II division would be identical as in mitosis; instead, they are different because there has always been at least one crossover per chromosome. Meiosis II is not a reduction division because, although there are fewer copies of the genome in the resulting cells, there is still one set of chromosomes, as there was at the end of meiosis I.
Cells produced by mitosis will function in different parts of the body as a part of growth or replacing dead or damaged cells. They may even be involved in asexual reproduction in some organisms. Cells produced by meiosis in a diploid-dominant organism such as an animal will only participate in sexual reproduction.
CONCEPT IN ACTION
For an animation comparing mitosis and meiosis, go to this website.
Section Summary
Sexual reproduction requires that diploid organisms produce haploid cells that can fuse during fertilization to form diploid offspring. The process that results in haploid cells is called meiosis. Meiosis is a series of events that arrange and separate chromosomes into daughter cells. During the interphase of meiosis, each chromosome is duplicated. In meiosis, there are two rounds of nuclear division resulting in four nuclei and usually four haploid daughter cells, each with half the number of chromosomes as the parent cell. During meiosis, variation in the daughter nuclei is introduced because of crossover in prophase I and random alignment at metaphase I. The cells that are produced by meiosis are genetically unique.
Meiosis and mitosis share similarities, but have distinct outcomes. Mitotic divisions are single nuclear divisions that produce daughter nuclei that are genetically identical and have the same number of chromosome sets as the original cell. Meiotic divisions are two nuclear divisions that produce four daughter nuclei that are genetically different and have one chromosome set rather than the two sets the parent cell had. The main differences between the processes occur in the first division of meiosis. The homologous chromosomes separate into different nuclei during meiosis I causing a reduction of ploidy level. The second division of meiosis is much more similar to a mitotic division.
Glossary
chiasmata
(singular = chiasma) the structure that forms at the crossover points after genetic material is exchanged
crossing over
(also, recombination) the exchange of genetic material between homologous chromosomes resulting in chromosomes that incorporate genes from both parents of the organism forming reproductive cells
fertilization
the union of two haploid cells typically from two individual organisms
interkinesis
a period of rest that may occur between meiosis I and meiosis II; there is no replication of DNA during interkinesis
meiosis I
the first round of meiotic cell division; referred to as reduction division because the resulting cells are haploid
meiosis II
the second round of meiotic cell division following meiosis I; sister chromatids are separated from each other, and the result is four unique haploid cells
recombinant
describing something composed of genetic material from two sources, such as a chromosome with both maternal and paternal segments of DNA
reduction division
a nuclear division that produces daughter nuclei each having one-half as many chromosome sets as the parental nucleus; meiosis I is a reduction division
somatic cell
all the cells of a multicellular organism except the gamete-forming cells
synapsis
the formation of a close association between homologous chromosomes during prophase I
tetrad
two duplicated homologous chromosomes (four chromatids) bound together by chiasmata during prophase I | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/11%3A_Sexual_Reproduction_and_Meiosis/11.03%3A_The_Process_of_Meiosis.txt |
Learning Objectives
• Compare and contrast mitosis and meiosis
Mitosis and meiosis are both forms of division of the nucleus in eukaryotic cells. They share some similarities, but also exhibit distinct differences that lead to very different outcomes. The purpose of mitosis is cell regeneration, growth, and asexual reproduction,while the purpose of meiosis is the production of gametes for sexual reproduction. Mitosis is a single nuclear division that results in two nuclei that are usually partitioned into two new daughter cells. The nuclei resulting from a mitotic division are genetically identical to the original nucleus. They have the same number of sets of chromosomes, one set in the case of haploid cells and two sets in the case of diploid cells. In most plants and all animal species, it is typically diploid cells that undergo mitosis to form new diploid cells. In contrast, meiosis consists of two nuclear divisions resulting in four nuclei that are usually partitioned into four new haploid daughter cells. The nuclei resulting from meiosis are not genetically identical and they contain one chromosome set only. This is half the number of chromosome sets in the original cell, which is diploid.
The main differences between mitosis and meiosis occur in meiosis I. In meiosis I, the homologous chromosome pairs become associated with each other and are bound together with the synaptonemal complex. Chiasmata develop and crossover occurs between homologous chromosomes, which then line up along the metaphase plate in tetrads with kinetochore fibers from opposite spindle poles attached to each kinetochore of a homolog in a tetrad. All of these events occur only in meiosis I.
When the tetrad is broken up and the homologous chromosomes move to opposite poles, the ploidy level is reduced from two to one. For this reason, meiosis I is referred to as a reduction division. There is no such reduction in ploidy level during mitosis.
Meiosis II is much more similar to a mitotic division. In this case, the duplicated chromosomes (only one set, as the homologous pairs have now been separated into two different cells) line up on the metaphase plate with divided kinetochores attached to kinetochore fibers from opposite poles. During anaphase II and mitotic anaphase, the kinetochores divide and sister chromatids, now referred to as chromosomes, are pulled to opposite poles. The two daughter cells of mitosis, however, are identical, unlike the daughter cells produced by meiosis. They are different because there has been at least one crossover per chromosome. Meiosis II is not a reduction division because, although there are fewer copies of the genome in the resulting cells, there is still one set of chromosomes, as there was at the end of meiosis I. Meiosis II is, therefore, referred to as equatorial division.
Key Points
• For the most part, in mitosis, diploid cells are partitioned into two new diploid cells, while in meiosis, diploid cells are partitioned into four new haploid cells.
• In mitosis, the daughter cells have the same number of chromosomes as the parent cell, while in meiosis, the daughter cells have half the number of chromosomes as the parent.
• The daughter cells produced by mitosis are identical, whereas the daughter cells produced by meiosis are different because crossing over has occurred.
• The events that occur in meiosis but not mitosis include homologous chromosomes pairing up, crossing over, and lining up along the metaphase plate in tetrads.
• Meiosis II and mitosis are not reduction division like meiosis I because the number of chromosomes remains the same; therefore, meiosis II is referred to as equatorial division.
• When the homologous chromosomes separate and move to opposite poles during meiosis I, the ploidy level is reduced from two to one, which is referred to as a reduction division.
Key Terms
• reduction division: the first of the two divisions of meiosis, a type of cell division
• ploidy: the number of homologous sets of chromosomes in a cell
• equatorial division: a process of nuclear division in which each chromosome divides equally such that the number of chromosomes remains the same from parent to daughter cells | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/11%3A_Sexual_Reproduction_and_Meiosis/11.04%3A_Summing_Up-_Meiosis_Versus_Mitosis.txt |
Learning Objectives
• Describe the traits of pea plants that were studied by Mendel
Gregor Mendel and the Study of Genetics
Genetics is the study of heredity, or the passing of traits from parents to offspring. Gregor Johann Mendel set the framework for genetics long before chromosomes or genes had been identified, at a time when meiosis was not well understood. For his work, Mendel is often referred to as the “father of modern genetics. ” Mendel selected a simple biological system, garden peas, and conducted methodical, quantitative analyses using large sample sizes.
Mendel entered the Augustinian St. Thomas’s Abbey and began his training as a priest. He began studying heredity using mice, but his bishop did not like one of his friars studying animal sex, so he switched to plants. Mendel grew and studied around 29,000 garden pea plants in a monastery’s garden, where he analyzed seven characteristics of the garden pea plants: flower color (purple or white), seed texture (wrinkled or round), seed color (yellow or green), stem length (long or short), pod color (yellow or green), pod texture (inflated or constricted), and flower position (axial or terminal). Based on the appearance, or phenotypes, of the seven traits, Mendel developed genotypes for those traits.
Because of Mendel’s work, the fundamental principles of heredity were revealed, which are often referred to as Mendel’s Laws of Inheritance. We now know that genes, carried on chromosomes, are the basic functional units of heredity with the capability to be replicated, expressed, or mutated. Today, the postulates put forth by Mendel form the basis of classical, or Mendelian, genetics. Not all genes are transmitted from parents to offspring according to Mendelian genetics, but Mendel’s experiments serve as an excellent starting point for thinking about inheritance.
Mendel made all of his observations and findings crossing individual plants. We can now view a human karyotype of all of the chromosomes in an individual to visualize chromosomal abnormalities in offspring, even before birth. Shortly after Mendel proposed that traits were determined by what are now known as genes, other researchers observed that different traits were often inherited together, and thereby deduced that the genes were physically linked by being located on the same chromosome. Mendel’s work was the beginning of many of the advances in molecular biology over the years.
Key Points
• Mendel studied seven characteristics of the garden pea plants: flower color, seed texture, seed color, stem length, pod color, pod texture, and flower position to develop his Laws of Inheritance.
• Genetics is the study of genes passed from parents to offspring.
• Genes are the basic fundamental units of heredity.
Key Terms
• genetics: The branch of biology that deals with the transmission and variation of inherited characteristics, in particular chromosomes and DNA.
12.1B: Mendels Model System
Learning Objectives
• Describe the scientific reasons for the success of Mendel’s experimental work
Mendel’s Model System
Mendel’s seminal work was accomplished using the garden pea, Pisum sativum, to study inheritance. Pea plant reproduction is easily manipulated; large quantities of garden peas could be cultivated simultaneously, allowing Mendel to conclude that his results did not occur simply by chance. The garden pea also grows to maturity within one season; several generations could be evaluated over a relatively short time.
Pea plants have both male and female parts and can easily be grown in large numbers. For this reason, garden pea plants can either self-pollinate or cross-pollinate with other pea plants. In the absence of outside manipulation, this species naturally self-fertilizes: ova (the eggs) within individual flowers are fertilized by pollen (containing the sperm cell) from the same flower. The sperm and the eggs that produce the next generation of plants both come from the same parent. What’s more, the flower petals remain sealed tightly until after pollination, preventing pollination from other plants. The result is highly inbred, or “true-breeding,” pea plants. These are plants that always produce offspring that look like the parent. Today, we know that these “true-breeding” plants are homozygous for most traits.
A gardener or researcher, such as Mendel, can cross-pollinate these same plants by manually applying sperm from one plant to the pistil (containing the ova) of another plant. Now the sperm and eggs come from different parent plants. When Mendel cross-pollinated a true-breeding plant that only produced yellow peas with a true-breeding plant that only produced green peas, he found that the first generation of offspring is always all yellow peas. The green pea trait did not show up. However, if this first generation of yellow pea plants were allowed to self-pollinate, the following or second generation had a ratio of 3:1 yellow to green peas.
In this and all the other pea plant traits Mendel followed, one form of the trait was “dominant” over another so it masked the presence of the other “recessive” form in the first generation after cross-breeding two homozygous plants.. Even if the phenotype (visible form) is hidden, the genotype (allele controlling that form of the trait) can be passed on to next generation and produce the recessive form in the second generation. By experimenting with true-breeding pea plants, Mendel avoided the appearance of unexpected (recombinant) traits in offspring that might occur if the plants were not true breeding.
Key Points
• Mendel used true-breeding plants in his experiments. These plants, when self-fertilized, always produce offspring with the same phenotype.
• Pea plants are easily manipulated, grow in one season, and can be grown in large quantities; these qualities allowed Mendel to conduct methodical, quantitative analyses using large sample sizes.
• Based on his experiments with the garden peas, Mendel found that one phenotype was always dominant over another recessive phenotype for the same trait.
Key Terms
• phenotype: the observable characteristics of an organism, often resulting from its genetic information or a combination of genetic information and environmental factors
• genotype: the specific genetic information of a cell or organism, usually a description of the allele or alleles relating to a specific gene.
• true-breeding plant: a plant that always produces offspring of the same phenotype when self-fertilized; one that is homozygous for the trait being followed. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.01%3A_The_Mystery_of_Heredity/12.1A%3A_Introduction_to_Mendelian_Inheritance.txt |
Pedigree charts are diagrams that show the phenotypes and/or genotypes for a particular organism and its ancestors. While commonly used in human families to track genetic diseases, they can be used for any species and any inherited trait. Geneticists use a standardized set of symbols to represent an individual’s sex, family relationships and phenotype. These diagrams are used to determine the mode of inheritance of a particular disease or trait, and to predict the probability of its appearance among offspring. Pedigree analysis is therefore an important tool in both basic research and genetic counseling.
Each pedigree chart represents all of the available information about the inheritance of a single trait (most often a disease) within a family. The pedigree chart is therefore drawn using factual information, but there is always some possibility of errors in this information, especially when relying on family members’ recollections or even clinical diagnoses. In real pedigrees, further complications can arise due to incomplete penetrance (including age of onset) and variable expressivity of disease alleles, but for the examples presented in this book, we will presume complete accuracy of the pedigrees. A pedigree may be drawn when trying to determine the nature of a newly discovered disease, or when an individual with a family history of a disease wants to know the probability of passing the disease on to their children. In either case, a tree is drawn, as shown in Figure \(2\), with circles to represent females, and squares to represent males. Matings are drawn as a line joining a male and female, while a consanguineous mating (closely related is two lines.
The affected individual that brings the family to the attention of a geneticist is called the proband (or propositus). If an individual is known to have symptoms of the disease (affected), the symbol is filled in. Sometimes a half-filled in symbol is used to indicate a known carrier of a disease; this is someone who does not have any symptoms of the disease, but who passed the disease on to subsequent generations because they are a heterozygote. Note that when a pedigree is constructed, it is often unknown whether a particular individual is a carrier or not, so not all carriers are always explicitly indicated in a pedigree. For simplicity, in this chapter we will assume that the pedigrees presented are accurate, and represent fully penetrant traits.
12.2.02: Inferring the Mode of Inheritance
Given a pedigree of an uncharacterized disease or trait, one of the first tasks is to determine which modes of inheritance are possible and then which mode of inheritance is most likely. This information is essential in calculating the probability that the trait will be inherited in any future offspring. We will mostly consider five major types of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked dominant (XD), X-linked recessive (XR), and Y-linked (Y).
Autosomal Dominant (AD)
When a disease is caused by a dominant allele of a gene, every person with that allele will show symptoms of the disease (assuming complete penetrance), and only one disease allele needs to be inherited for an individual to be affected. Thus, every affected individual must have an affected parent. A pedigree with affected individuals in every generation is typical of AD diseases. However, beware that other modes of inheritance can also show the disease in every generation, as described below. It is also possible for an affected individual with an AD disease to have a family without any affected children, if the affected parent is a heterozygote. This is particularly true in small families, where the probability of every child inheriting the normal, rather than disease allele is not extremely small. Note that AD diseases are usually rare in populations, therefore affected individuals with AD diseases tend to be heterozygotes (otherwise, both parents would have had to been affected with the same rare disease). Achondroplastic dwarfism, and polydactyly are both examples of human conditions that may follow an AD mode of inheritance.
X-linked dominant (XD)
In X-linked dominant inheritance, the gene responsible for the disease is located on the X-chromosome, and the allele that causes the disease is dominant to the normal allele in females. Because females have twice as many X-chromosomes as males, females tend to be more frequently affected than males in the population. However, not all pedigrees provide sufficient information to distinguish XD and AD. One definitive indication that a trait is inherited as AD, and not XD, is that an affected father passes the disease to a son; this type of transmission is not possible with XD, since males inherit their X chromosome from their mothers.
Autosomal recessive (AR)
Diseases that are inherited in an autosomal recessive pattern require that both parents of an affected individual carry at least one copy of the disease allele. With AR traits, many individuals in a pedigree can be carriers, probably without knowing it. Compared to pedigrees of dominant traits, AR pedigrees tend to show fewer affected individuals and are more likely than AD or XD to “skip a generation”. Thus, the major feature that distinguishes AR from AD or XD is that unaffected individuals can have affected offspring.
X-linked recessive (XR)
Because males have only one X-chromosome, any male that inherits an X-linked recessive disease allele will be affected by it (assuming complete penetrance). Therefore, in XR modes of inheritance, males tend to be affected more frequently than females in a population. This is in contrast to AR and AD, where both sexes tend to be affected equally, and XD, in which females are affected more frequently. Note, however, in the small sample sizes typical of human families, it is usually not possible to accurately determine whether one sex is affected more frequently than others. On the other hand, one feature of a pedigree that can be used to definitively establish that an inheritance pattern is not XR is the presence of an affected daughter from unaffected parents; because she would have had to inherit one X-chromosome from her father, he would also have been affected in XR.
Y-linked and Mitochondrial Inheritance.
Two additional modes are Y-linked and Mitochondrial inheritance.
Only males are affected in human Y-linked inheritance (and other species with the X/Y sex determining system). There is only father to son transmission. This is the easiest mode of inheritance to identify, but it is one of the rarest because there are so few genes located on the Y-chromosome. An example of Y-linked inheritance is the hairy-ear-rim phenotype seen in some Indian families. As expected this trait is passed on from father to all sons and no daughters. Y-chromosome DNA polymorphisms can be used to follow the male lineage in large families or through ancient ancestral lineages. For example, the Y-chromosome of Mongolian ruler Genghis Khan (1162-1227 CE), and his male relatives, accounts for ~8% of the Y-chromosome lineage of men in Asia (0.5% world wide).
Mutations in Mitochondrial DNA are inherited through the maternal line (from your mother). There are some human diseases associated with mutations in mitochondria genes. These mutations can affect both males and females, but males cannot pass them on as the mitochondria are inherited via the egg, not the sperm. Mitochondrial DNA polymorphisms are also used to investigate evolutionary lineages, both ancient and recent. Because of the relative similarity of sequence mtDNA is also used in species identification in ecology studies. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.02%3A_Monohybrid_Crosses-_The_Principle_of_Segregation/12.2.01%3A_Pedigree_Analysis.txt |
Learning Objectives
• Distinguish between the phenotype and the genotype of an organism
Phenotypes and Genotypes
The observable traits expressed by an organism are referred to as its phenotype. An organism’s underlying genetic makeup, consisting of both physically visible and non-expressed alleles, is called its genotype. Johann Gregor Mendel’s (1822–1884) hybridization experiments demonstrate the difference between phenotype and genotype.
Mendel crossed or mated two true-breeding (self-pollinating) garden peas, Pisum saivum, by manually transferring pollen from the anther of a mature pea plant of one variety to the stigma of a separate mature pea plant of the second variety. Plants used in first-generation crosses were called P0, or parental generation one, plants. Mendel collected the seeds belonging to the P0plants that resulted from each cross and grew them the following season. These offspring were called the F1, or the first filial (filial = offspring, daughter or son), generation. Once Mendel examined the characteristics in the F1 generation of plants, he allowed them to self-fertilize naturally. He then collected and grew the seeds from the F1 plants to produce the F2, or second filial, generation.
When true-breeding plants in which one parent had white flowers and one had violet flowers were cross-fertilized, all of the F1 hybrid offspring had violet flowers. That is, the hybrid offspring were phenotypically identical to the true-breeding parent with violet flowers. However, we know that the allele donated by the parent with white flowers was not simply lost because it reappeared in some of the F2 offspring. Therefore, the F1 plants must have been genotypically different from the parent with violet flowers.
In his 1865 publication, Mendel reported the results of his crosses involving seven different phenotypes, each with two contrasting traits. A trait is defined as a variation in the physical appearance of a heritable characteristic. The characteristics included plant height, seed texture, seed color, flower color, pea pod size, pea pod color, and flower position. To fully examine each characteristic, Mendel generated large numbers of F1 and F2 plants, reporting results from 19,959 F2 plants alone. His findings were consistent. First, Mendel confirmed that he had plants that bred true for white or violet flower color. Regardless of how many generations Mendel examined, all self-crossed offspring of parents with white flowers had white flowers, and all self-crossed offspring of parents with violet flowers had violet flowers. In addition, Mendel confirmed that, other than flower color, the pea plants were physically identical.
Key Points
• Mendel used pea plants with seven distinct traits or phenotypes to determine the pattern of inheritance and the underlying genotypes.
• Mendel found that crossing two purebred pea plants which expressed different traits resulted in an F1generation where all the pea plants expressed the same trait or phenotype.
• When Mendel allowed the F1 plants to self-fertilize, the F2 generation showed two different phenotypes, indicating that the F1 plants had different genotypes.
Key Terms
• phenotype: the appearance of an organism based on a multifactorial combination of genetic traits and environmental factors, especially used in pedigrees
• genotype: the combination of alleles, situated on corresponding chromosomes, that determines a specific trait of an individual, such as “Aa” or “aa” | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.02%3A_Monohybrid_Crosses-_The_Principle_of_Segregation/12.2B%3A_Phenotypes_and_Genotypes.txt |
Learning Objectives
• Identify Mendelian crosses
Mendelian Crosses
Mendel performed crosses, which involved mating two true-breeding individuals that have different traits. In the pea, which is a naturally self-pollinating plant, this is done by manually transferring pollen from the anther of a mature pea plant of one variety to the stigma of a separate mature pea plant of the second variety. In plants, pollen carries the male gametes (sperm) to the stigma, a sticky organ that traps pollen and allows the sperm to move down the pistil to the female gametes (ova) below. To prevent the pea plant that was receiving pollen from self-fertilizing and confounding his results, Mendel painstakingly removed all of the anthers from the plant’s flowers before they had a chance to mature.
Plants used in first-generation crosses were called P0, or parental generation one, plants. Mendel collected the seeds belonging to the P0 plants that resulted from each cross and grew them the following season. These offspring were called the F1, or the first filial (filial = offspring, daughter or son), generation. Once Mendel examined the characteristics in the F1generation of plants, he allowed them to self-fertilize naturally. He then collected and grew the seeds from the F1 plants to produce the F2, or second filial, generation. Mendel’s experiments extended beyond the F2 generation to the F3 and F4generations, and so on, but it was the ratio of characteristics in the P0−F1−F2 generations that were the most intriguing and became the basis for Mendel’s postulates.
Key Points
• Mendel carefully controlled his experiments by removing the anthers from the pea plants before they matured.
• First generation pea plants were called parental generation, P0, while the following generations were called filial, Fn, where n is the number of generations from P0.
• The ratio of characteristics in the P0−F1−F2 generations became the basis for Mendel’s postulates.
Key Terms
• filial: of a generation or generations descending from a specific previous one
• parental: of the generation of organisms that produce a hybrid
12.2C: Mendels Law of Segregation
Learning Objectives
• Apply the law of segregation to determine the chances of a particular genotype arising from a genetic cross
Equal Segregation of Alleles
Observing that true-breeding pea plants with contrasting traits gave rise to F1 generations that all expressed the dominant trait and F2 generations that expressed the dominant and recessive traits in a 3:1 ratio, Mendel proposed the law of segregation. The law of segregation states that each individual that is a diploid has a pair of alleles (copy) for a particular trait. Each parent passes an allele at random to their offspring resulting in a diploid organism. The allele that contains the dominant trait determines the phenotype of the offspring. In essence, the law states that copies of genes separate or segregate so that each gamete receives only one allele.
For the F2 generation of a monohybrid cross, the following three possible combinations of genotypes could result: homozygous dominant, heterozygous, or homozygous recessive. Because heterozygotes could arise from two different pathways (receiving one dominant and one recessive allele from either parent), and because heterozygotes and homozygous dominant individuals are phenotypically identical, the law supports Mendel’s observed 3:1 phenotypic ratio. The equal segregation of alleles is the reason we can apply the Punnett square to accurately predict the offspring of parents with known genotypes.
The physical basis of Mendel’s law of segregation is the first division of meiosis in which the homologous chromosomes with their different versions of each gene are segregated into daughter nuclei. The behavior of homologous chromosomes during meiosis can account for the segregation of the alleles at each genetic locus to different gametes. As chromosomes separate into different gametes during meiosis, the two different alleles for a particular gene also segregate so that each gamete acquires one of the two alleles. In Mendel’s experiments, the segregation and the independent assortment during meiosis in the F1 generation give rise to the F2 phenotypic ratios observed by Mendel. The role of the meiotic segregation of chromosomes in sexual reproduction was not understood by the scientific community during Mendel’s lifetime.
Key Points
• Each gamete acquires one of the two alleles as chromosomes separate into different gametes during meiosis.
• Heterozygotes, which posess one dominant and one recessive allele, can receive each allele from either parent and will look identical to homozygous dominant individuals; the Law of Segregation supports Mendel’s observed 3:1 phenotypic ratio.
• Mendel proposed the Law of Segregation after observing that pea plants with two different traits produced offspring that all expressed the dominant trait, but the following generation expressed the dominant and recessive traits in a 3:1 ratio.
Key Terms
• law of segregation: a diploid individual possesses a pair of alleles for any particular trait and each parent passes one of these randomly to its offspring | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.02%3A_Monohybrid_Crosses-_The_Principle_of_Segregation/12.2C%3A_Mendelian_Crosses.txt |
Learning Objectives
• Describe the Punnett square approach to a monohybrid cross
Punnett Square Approach to a Monohybrid Cross
When fertilization occurs between two true-breeding parents that differ in only one characteristic, the process is called a monohybrid cross, and the resulting offspring are monohybrids. Mendel performed seven monohybrid crosses involving contrasting traits for each characteristic. On the basis of his results in F1 and F2 generations, Mendel postulated that each parent in the monohybrid cross contributed one of two paired unit factors to each offspring and that every possible combination of unit factors was equally likely.
To demonstrate a monohybrid cross, consider the case of true-breeding pea plants with yellow versus green pea seeds. The dominant seed color is yellow; therefore, the parental genotypes were YY ( homozygous dominant) for the plants with yellow seeds and yy (homozygous recessive ) for the plants with green seeds, respectively. A Punnett square, devised by the British geneticist Reginald Punnett, can be drawn that applies the rules of probability to predict the possible outcomes of a genetic cross or mating and their expected frequencies.To prepare a Punnett square, all possible combinations of the parental alleles are listed along the top (for one parent) and side (for the other parent) of a grid, representing their meiotic segregation into haploid gametes. Then the combinations of egg and sperm are made in the boxes in the table to show which alleles are combining. Each box then represents the diploid genotype of a zygote, or fertilized egg, that could result from this mating. Because each possibility is equally likely, genotypic ratios can be determined from a Punnett square. If the pattern of inheritance (dominant or recessive) is known, the phenotypic ratios can be inferred as well. For a monohybrid cross of two true-breeding parents, each parent contributes one type of allele. In this case, only one genotype is possible. All offspring are Yy and have yellow seeds.
A self-cross of one of the Yy heterozygous offspring can be represented in a 2 × 2 Punnett square because each parent can donate one of two different alleles. Therefore, the offspring can potentially have one of four allele combinations: YY, Yy, yY, or yy. Notice that there are two ways to obtain the Yy genotype: a Y from the egg and a y from the sperm, or a y from the egg and a Y from the sperm. Both of these possibilities must be counted. Recall that Mendel’s pea-plant characteristics behaved in the same way in reciprocal crosses. Therefore, the two possible heterozygous combinations produce offspring that are genotypically and phenotypically identical despite their dominant and recessive alleles deriving from different parents. They are grouped together. Because fertilization is a random event, we expect each combination to be equally likely and for the offspring to exhibit a ratio of YY:Yy:yy genotypes of 1:2:1. Furthermore, because the YY and Yy offspring have yellow seeds and are phenotypically identical, applying the sum rule of probability, we expect the offspring to exhibit a phenotypic ratio of 3 yellow:1 green. Indeed, working with large sample sizes, Mendel observed approximately this ratio in every F2 generation resulting from crosses for individual traits.
Beyond predicting the offspring of a cross between known homozygous or heterozygous parents, Mendel also developed a way to determine whether an organism that expressed a dominant trait was a heterozygote or a homozygote. Called the test cross, this technique is still used by plant and animal breeders. In a test cross, the dominant-expressing organism is crossed with an organism that is homozygous recessive for the same characteristic. If the dominant-expressing organism is a homozygote, then all F1 offspring will be heterozygotes expressing the dominant trait. Alternatively, if the dominant expressing organism is a heterozygote, the F1 offspring will exhibit a 1:1 ratio of heterozygotes and recessive homozygotes. The test cross further validates Mendel’s postulate that pairs of unit factors segregate equally.
Key Points
• Fertilization between two true-breeding parents that differ in only one characteristic is called a monohybrid cross.
• For a monohybrid cross of two true-breeding parents, each parent contributes one type of allele resulting in all of the offspring with the same genotype.
• A test cross is a way to determine whether an organism that expressed a dominant trait was a heterozygote or a homozygote.
Key Terms
• monohybrid: a hybrid between two species that only have a difference of one gene
• homozygous: of an organism in which both copies of a given gene have the same allele
• heterozygous: of an organism which has two different alleles of a given gene
• Punnett square: a graphical representation used to determine the probability of an offspring expressing a particular genotype | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.02%3A_Monohybrid_Crosses-_The_Principle_of_Segregation/12.2C%3A_The_Punnett_Square_Approach_for_a_Monohybrid_Cross.txt |
Learning Objectives
• Evaluate the results of F1 and F2 generations from Mendelian crosses of peas
Garden Pea Characteristics Revealed the Basics of Heredity
To fully examine each of the seven traits in garden peas, Mendel generated large numbers of F1 and F2 plants, reporting results from 19,959 F2 plants alone. His findings were consistent.
What results did Mendel find in his crosses for flower color? First, Mendel confirmed that he had plants that bred true for white or violet flower color. Regardless of how many generations Mendel examined, all self-crossed offspring of parents with white flowers had white flowers, and all self-crossed offspring of parents with violet flowers had violet flowers. In addition, Mendel confirmed that, other than flower color, the pea plants were physically identical.
Once these validations were complete, Mendel applied the pollen from a plant with violet flowers to the stigma of a plant with white flowers. After gathering and sowing the seeds that resulted from this cross, Mendel found that 100 percent of the F1hybrid generation had violet flowers. Conventional wisdom at that time would have predicted the hybrid flowers to be pale violet or for hybrid plants to have equal numbers of white and violet flowers. In other words, the contrasting parental traits were expected to blend in the offspring. Instead, Mendel’s results demonstrated that the white flower trait in the F1 generation had completely disappeared.
Importantly, Mendel did not stop his experimentation there. He allowed the F1 plants to self-fertilize and found that, of F2-generation plants, 705 had violet flowers and 224 had white flowers. This was a ratio of 3.15 violet flowers per one white flower, or approximately 3:1. When Mendel transferred pollen from a plant with violet flowers to the stigma of a plant with white flowers and vice versa, he obtained about the same ratio regardless of which parent, male or female, contributed which trait. This is called a reciprocal cross: a paired cross in which the respective traits of the male and female in one cross become the respective traits of the female and male in the other cross. For the other six characteristics Mendel examined, the F1 and F2generations behaved in the same way as they had for flower color. One of the two traits would disappear completely from the F1 generation only to reappear in the F2 generation at a ratio of approximately 3:1.
Upon compiling his results for many thousands of plants, Mendel concluded that the characteristics could be divided into expressed and latent traits. He called these, respectively, dominant and recessive traits. Dominant traits are those that are inherited unchanged in a hybridization. Recessive traits become latent, or disappear, in the offspring of a hybridization. The recessive trait does, however, reappear in the progeny of the hybrid offspring. An example of a dominant trait is the violet-flower trait. For this same characteristic (flower color), white-colored flowers are a recessive trait. The fact that the recessive trait reappeared in the F2 generation meant that the traits remained separate (not blended) in the plants of the F1 generation. Mendel also proposed that plants possessed two copies of the trait for the flower-color characteristic and that each parent transmitted one of its two copies to its offspring, where they came together. Moreover, the physical observation of a dominant trait could mean that the genetic composition of the organism included two dominant versions of the characteristic or that it included one dominant and one recessive version. Conversely, the observation of a recessive trait meant that the organism lacked any dominant versions of this characteristic.
Key Points
• Dominant traits are inherited unchanged from one generation to the next.
• Recessive traits disappear in the first filial generation, but reappear in the second filial generation at a ratio of 3:1, dominant:recessive.
• In the F1 generation, Mendel found that one of the two options for each trait had disappeared (all offspring were identical phenotypes), while in the F2 generation, the trait reappeared in 1/4 of the offspring (a 3:1 ratio).
Key Terms
• hybrid: offspring resulting from cross-breeding different entities, e.g. two different species or two purebred parent strains
• recessive: able to be covered up by a dominant trait
• dominant: a relationship between alleles of a gene, in which one allele masks the expression (phenotype) of another allele at the same locus | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.02%3A_Monohybrid_Crosses-_The_Principle_of_Segregation/12.2D%3A_Garden_Pea_Characteristics_Revealed_the_Basics_of_Heredity.txt |
Learning Objectives
• Use the probability or forked line method to calculate the chance of any particular genotype arising from a genetic cross
Independent Assortment
Mendel’s law of independent assortment states that genes do not influence each other with regard to the sorting of alleles into gametes: every possible combination of alleles for every gene is equally likely to occur. The independent assortment of genes can be illustrated by the dihybrid cross: a cross between two true-breeding parents that express different traits for two characteristics. Consider the characteristics of seed color and seed texture for two pea plants: one that has green, wrinkled seeds (yyrr) and another that has yellow, round seeds (YYRR). Because each parent is homozygous, the law of segregation indicates that the gametes for the green/wrinkled plant all are yr, while the gametes for the yellow/round plant are all YR. Therefore, the F1 generation of offspring all are YyRr.
For the F2 generation, the law of segregation requires that each gamete receive either an R allele or an r allele along with either a Y allele or a y allele. The law of independent assortment states that a gamete into which an r allele sorted would be equally likely to contain either a Y allele or a y allele. Thus, there are four equally likely gametes that can be formed when the YyRr heterozygote is self-crossed as follows: YR, Yr, yR, and yr. Arranging these gametes along the top and left of a 4 × 4 Punnett square gives us 16 equally likely genotypic combinations. From these genotypes, we infer a phenotypic ratio of 9 round/yellow:3 round/green:3 wrinkled/yellow:1 wrinkled/green. These are the offspring ratios we would expect, assuming we performed the crosses with a large enough sample size.
Because of independent assortment and dominance, the 9:3:3:1 dihybrid phenotypic ratio can be collapsed into two 3:1 ratios, characteristic of any monohybrid cross that follows a dominant and recessive pattern. Ignoring seed color and considering only seed texture in the above dihybrid cross, we would expect that three-quarters of the F2 generation offspring would be round and one-quarter would be wrinkled. Similarly, isolating only seed color, we would assume that three-quarters of the F2offspring would be yellow and one-quarter would be green. The sorting of alleles for texture and color are independent events, so we can apply the product rule. Therefore, the proportion of round and yellow F2 offspring is expected to be (3/4) × (3/4) = 9/16, and the proportion of wrinkled and green offspring is expected to be (1/4) × (1/4) = 1/16. These proportions are identical to those obtained using a Punnett square. Round/green and wrinkled/yellow offspring can also be calculated using the product rule as each of these genotypes includes one dominant and one recessive phenotype. Therefore, the proportion of each is calculated as (3/4) × (1/4) = 3/16.
Forked-Line Method
When more than two genes are being considered, the Punnett-square method becomes unwieldy. For instance, examining a cross involving four genes would require a 16 × 16 grid containing 256 boxes. It would be extremely cumbersome to manually enter each genotype. For more complex crosses, the forked-line and probability methods are preferred.
To prepare a forked-line diagram for a cross between F1 heterozygotes resulting from a cross between AABBCC and aabbcc parents, we first create rows equal to the number of genes being considered and then segregate the alleles in each row on forked lines according to the probabilities for individual monohybrid crosses. We then multiply the values along each forked path to obtain the F2 offspring probabilities. Note that this process is a diagrammatic version of the product rule. The values along each forked pathway can be multiplied because each gene assorts independently. For a trihybrid cross, the F2phenotypic ratio is 27:9:9:9:3:3:3:1.
Probability Method
While the forked-line method is a diagrammatic approach to keeping track of probabilities in a cross, the probability method gives the proportions of offspring expected to exhibit each phenotype (or genotype) without the added visual assistance.
To fully demonstrate the power of the probability method, however, we can consider specific genetic calculations. For instance, for a tetrahybrid cross between individuals that are heterozygotes for all four genes, and in which all four genes are sorting independently in a dominant and recessive pattern, what proportion of the offspring will be expected to be homozygous recessive for all four alleles? Rather than writing out every possible genotype, we can use the probability method. We know that for each gene the fraction of homozygous recessive offspring will be 1/4. Therefore, multiplying this fraction for each of the four genes, (1/4) × (1/4) × (1/4) × (1/4), we determine that 1/256 of the offspring will be quadruply homozygous recessive.
Key Points
• Mendel’s law of independent assortment states that genes do not influence each other with regard to the sorting of alleles into gametes; every possible combination of alleles for every gene is equally likely to occur.
• The calculation of any particular genotypic combination of more than one gene is, therefore, the probability of the desired genotype at the first locus multiplied by the probability of the desired genotype at the other loci.
• The forked line method can be used to calculate the chances of all possible genotypic combinations from a cross, while the probability method can be used to calculate the chance of any one particular genotype that might result from that cross.
Key Terms
• independent assortment: separate genes for separate traits are passed independently of one another from parents to offspring | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.03%3A_Dihybrid_Crosses-_The_Principle_of_Independent_Assortment/12.3D%3A_Mendels_Law_of_Independent_Assortment.txt |
Learning Objectives
• Calculate the probability of traits of pea plants using Mendelian crosses
Probability Basics
Probabilities are mathematical measures of likelihood. The empirical probability of an event is calculated by dividing the number of times the event occurs by the total number of opportunities for the event to occur. Empirical probabilities come from observations such as those of Mendel. An example of a genetic event is a round seed produced by a pea plant. Mendel demonstrated that the probability of the event “round seed” was guaranteed to occur in the F1 offspring of true-breeding parents, one of which has round seeds and one of which has wrinkled seeds. When the F1 plants were subsequently self-crossed, the probability of any given F2 offspring having round seeds was now three out of four. In other words, in a large population of F2 offspring chosen at random, 75 percent were expected to have round seeds, whereas 25 percent were expected to have wrinkled seeds. Using large numbers of crosses, Mendel was able to calculate probabilities and use these to predict the outcomes of other crosses.
The Product Rule
Mendel demonstrated that the pea-plant characteristics he studied were transmitted as discrete units from parent to offspring. Mendel also determined that different characteristics were transmitted independently of one another and could be considered in separate probability analyses. For instance, performing a cross between a plant with green, wrinkled seeds and a plant with yellow, round seeds produced offspring that had a 3:1 ratio of green:yellow seeds and a 3:1 ratio of round:wrinkled seeds. The characteristics of color and texture did not influence each other.
The product rule of probability can be applied to this phenomenon of the independent transmission of characteristics. It states that the probability of two independent events occurring together can be calculated by multiplying the individual probabilities of each event occurring alone. Imagine that you are rolling a six-sided die (D) and flipping a penny (P) at the same time. The die may roll any number from 1–6 (D#), whereas the penny may turn up heads (PH) or tails (PT). The outcome of rolling the die has no effect on the outcome of flipping the penny and vice versa. There are 12 possible outcomes, and each is expected to occur with equal probability: D1PH, D1PT, D2PH, D2PT, D3PH, D3PT, D4PH, D4PT, D5PH, D5PT, D6PH, D6PT.
Of the 12 possible outcomes, the die has a 2/12 (or 1/6) probability of rolling a two, and the penny has a 6/12 (or 1/2) probability of coming up heads. The probability that you will obtain the combined outcome 2 and heads is: (D2) x (PH) = (1/6) x (1/2) or 1/12. The word “and” is a signal to apply the product rule. Consider how the product rule is applied to a dihybrid: the probability of having both dominant traits in the F2 progeny is the product of the probabilities of having the dominant trait for each characteristic.
The Sum Rule
The sum rule is applied when considering two mutually-exclusive outcomes that can result from more than one pathway. It states that the probability of the occurrence of one event or the other, of two mutually-exclusive events, is the sum of their individual probabilities. The word “or” indicates that you should apply the sum rule. Let’s imagine you are flipping a penny (P) and a quarter (Q). What is the probability of one coin coming up heads and one coming up tails? This can be achieved by two cases: the penny is heads (PH) and the quarter is tails (QT), or the quarter is heads (QH) and the penny is tails (PT). Either case fulfills the outcome. We calculate the probability of obtaining one head and one tail as [(PH) × (QT)] + [(QH) × (PT)] = [(1/2) × (1/2)] + [(1/2) × (1/2)] = 1/2. You should also notice that we used the product rule to calculate the probability of PH and QT and also the probability of PT and QH, before we summed them. The sum rule can be applied to show the probability of having just one dominant trait in the F2 generation of a dihybrid cross.
To use probability laws in practice, it is necessary to work with large sample sizes because small sample sizes are prone to deviations caused by chance. The large quantities of pea plants that Mendel examined allowed him to calculate the probabilities of the traits appearing in his F2 generation. This discovery meant that when parental traits were known, the offspring’s traits could be predicted accurately even before fertilization.
Key Points
• The Product Rule is used to determine the outcome of an event with two independent events; the probability of the event is the product of the probabilities of each individual event.
• The Sum Rule is used to determine the outcome of an event with two mutually exclusive events from multiple pathways; the probability of the event is the sum of the probabilities of each individual event.
• The Product Rule of probability is used to determine the probability of having both dominant traits in the F2progeny; it is the product of the probabilities of having the dominant trait for each characteristic.
• The Sum Rule of probability is used to determine the probability of having one dominant trait in the F2 generation of a dihybrid cross; it is the sum of the probabilities of each individual with that trait.
Key Terms
• sum rule: the probability of the occurrence of one event or the other event, of two mutually exclusive events, is the sum of their individual probabilities
• product rule: the probability of two independent events occurring together can be calculated by multiplying the individual probabilities of each event occurring alone
• probability: a number, between 0 and 1, expressing the precise likelihood of an event happening
Contributions and Attributions
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• Punnett square mendel flowers. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...el_flowers.svg. License: CC BY-SA: Attribution-ShareAlike | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.04%3A_Probability-_Predicting_the_Results_of_Crosses/12.4E%3A_Rules_of_Probability_for_Mendelian_Inheritance.txt |
Classical Genetics
Not only did Mendel solve the mystery of inheritance as units (genes), he also invented several testing and analysis techniques still used today. Classical genetics is the science of solving biological questions using controlled matings of model organisms. It began with Mendel in 1865 but did not take off until Thomas Morgan began working with fruit flies in 1908. Later, starting with Watson and Crick’s structure of DNA in 1953, classical genetics was joined by molecular genetics, the science of solving biological questions using DNA, RNA, and proteins isolated from organisms. The genetics of DNA cloning began in 1970 with the discovery of restriction enzymes.
True Breeding Lines
Geneticists make use of true breeding lines just as Mendel did (Figure \(6\)a). These are in-bred populations of plants or animals in which all parents and their offspring (over many generations) have the same phenotypes with respect to a particular trait. True breeding lines are useful, because they are typically assumed to be homozygous for the alleles that affect the trait of interest. When two individuals that are homozygous for the same alleles are crossed, all of their offspring will all also be homozygous. The continuation of such crosses constitutes a true breeding line or strain. A large variety of different strains, each with a different, true breeding character, can be collected and maintained for genetic research.
Monohybrid Crosses
A monohybrid cross is one in which both parents are heterozygous (or a hybrid) for a single (mono) trait. The trait might be petal colour in pea plants (Figure \(6\)b). Recall from chapter 1 that the generations in a cross are named P (parental), F1 (first filial), F2 (second filial), and so on.
Punnett Squares
Given the genotypes of any two parents, we can predict all of the possible genotypes of the offspring. Furthermore, if we also know the dominance relationships for all of the alleles, we can predict the phenotypes of the offspring. A convenient method for calculating the expected genotypic and phenotypic ratios from a cross was invented by Reginald Punnett. A Punnett square is a matrix in which all of the possible gametes produced by one parent are listed along one axis, and the gametes from the other parent are listed along the other axis. Each possible combination of gametes is listed at the intersection of each row and column. The F1 cross from Figure \(6\)b would be drawn as in Figure \(7\). Punnett squares can also be used to calculate the frequency of offspring. The frequency of each offspring is the frequency of the male gametes multiplied by the frequency of the female gamete.
A
a
A
AA
Aa
a
Aa
aa
Figure \(7\): A Punnett square showing a monohybrid cross. (Original-Deholos (Fireworks)-CC:AN)
Test Crosses
Knowing the genotypes of an individual is usually an important part of a genetic experiment. However, genotypes cannot be observed directly; they must be inferred based on phenotypes. Because of dominance, it is often not possible to distinguish between a heterozygote and a homozgyote based on phenotype alone (e.g. see the purple-flowered F2 plants in Figure \(6\)b). To determine the genotype of a specific individual, a test cross can be performed, in which the individual with an uncertain genotype is crossed with an individual that is homozygous recessive for all of the loci being tested.
For example, if you were given a pea plant with purple flowers it might be a homozygote (AA) or a heterozygote (Aa). You could cross this purple-flowered plant to a white-flowered plant as a tester, since you know the genotype of the tester is aa. Depending on the genotype of the purple-flowered parent (Figure \(8\)), you will observe different phenotypic ratios in the F1 generation. If the purple-flowered parent was a homozgyote, all of the F1 progeny will be purple. If the purple-flowered parent was a heterozygote, the F1 progeny should segregate purple-flowered and white-flowered plants in a 1:1 ratio.
A
A
a
Aa
Aa
a
Aa
Aa
A
a
a
Aa
aa
a
Aa
aa
Figure \(8\): Punnett Squares showing the two possible outcomes of a test cross. (Original-Deholos (Fireworks)-CC:AN) | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.05%3A_The_Testcross-_Revealing_Unknown_Genotypes/12.5.01%3A__Crossing_Techniques_Used_in_Classical_Genetics.txt |
Family Portrait
This photo of a South African family shows some of the variations that exist in human skin color. The color of human skin can range from very light to very dark with every possible gradation in between. As you might expect, the skin color trait has a more complex genetic basis than just one gene with two alleles, which is the type of simple trait that Mendel studied in pea plants. Like skin color, many other human traits have more complicated modes of inheritance than Mendelian traits. Such modes of inheritance are called non-Mendelian inheritance, and they include inheritance of multiple allele traits, traits with codominance or incomplete dominance, and polygenic traits, among others, all of which are described below.
Multiple Allele Traits
The majority of human genes are thought to have more than two normal versions or alleles. Traits controlled by a single gene with more than two alleles are called multiple allele traits. An example is ABO blood type. Your blood type refers to which of certain proteins called antigens are found on your red blood cells. There are three common alleles for this trait, which are represented by the letters IA, IB, and i.
Table \(1\): ABO Blood Group
Genotype Phenotype (blood type)
IAIA A
IAi A
IBIB B
IBi B
ii O
IAIB AB
As shown in the table below, there are six possible ABO genotypes because the three alleles, taken two at a time, result in six possible combinations. The IA and IB alleles are dominant to the i allele. As a result, both IAIA and IAi genotypes have the same phenotype, with the A antigen in their blood (type A blood). Similarly, both IBIB and IBi genotypes have the same phenotype, with the B antigen in their blood (type B blood). No antigen is associated with the i allele, so people with the ii genotype have no antigens for ABO blood type in their blood (type O blood).
Codominance
Look at the genotype IAIB in the ABO blood group table. Alleles IA and IB for ABO blood type are neither dominant nor recessive to one another. Instead, they are codominant to each other. Codominance occurs when two alleles for a gene are expressed equally in the phenotype of heterozygotes. In the case of ABO blood type, IAIB heterozygotes have a unique phenotype, with both A and B antigens in their blood (type AB blood).
Incomplete Dominance
Another relationship that may occur between alleles for the same gene is incomplete dominance. This occurs when the dominant allele is not completely dominant, so an intermediate phenotype results in heterozygotes who inherit both alleles. Generally, this happens when the two alleles for a given gene both produce proteins but one protein is not functional. As a result, the heterozygote individual produces only half the amount of normal protein as is produced by an individual who is homozygous for the normal allele.
An example of incomplete dominance in humans is Tay Sachs disease. The normal allele for the gene, in this case, produces an enzyme that is responsible for breaking down lipids. A defective allele for the gene results in the production of a nonfunctional enzyme. Heterozygotes who have one normal and one defective allele produce half as much functional enzyme as the normal homozygote, and this is enough for normal development. However, homozygotes who have only defective alleles produce only the nonfunctional enzyme. This leads to the accumulation of lipids in the brain beginning in utero, which causes significant brain damage. Most individuals with Tay Sachs disease die at a young age, typically by the age of five years.
Polygenic Traits
Many human traits are controlled by more than one gene. These traits are called polygenic traits. The alleles of each gene have a minor additive effect on the phenotype. There are many possible combinations of alleles, especially if each gene has multiple alleles. Therefore, a whole continuum of phenotypes is possible.
An example of a human polygenic trait is adult height. Several genes, each with more than one allele, contribute to this trait, so there are many possible adult heights. For example, one adult’s height might be 1.655 m (5.430 feet), and another adult’s height might be 1.656 m (5.433 feet). Adult height ranges from less than 5 feet to more than 6 feet, with males being somewhat taller than females on average. The majority of people fall near the middle of the range of heights for their sex, as shown in the graph in Figure \(2\).
Environmental Effects on Phenotype
Many traits are affected by the environment as well as by genes. This may be especially true for polygenic traits. For example, adult height might be negatively impacted by poor diet or illness during childhood. Skin color is another polygenic trait. There is a wide range of skin colors in people worldwide. In addition to differences in skin color genes, differences in exposure to ultraviolet (UV) light cause some of the variations. As shown in Figure \(3\), exposure to UV light darkens the skin.
Pleiotropy
Some genes affect more than one phenotypic trait. This is called pleiotropy. There are numerous examples of pleiotropy in humans. They generally involve important proteins that are needed for the normal development or functioning of more than one organ system. An example of pleiotropy in humans occurs with the gene that codes for the main protein in collagen, a substance that helps form bones. This protein is also important in the ears and eyes. Mutations in the gene result in problems not only in bones but also in these sensory organs, which is how the gene's pleiotropic effects were discovered.
Another example of pleiotropy occurs with sickle cell anemia. This recessive genetic disorder occurs when there is a mutation in the gene that normally encodes the red blood cell protein called hemoglobin. People with the disorder have two alleles for sickle-cell hemoglobin, so named for the sickle shape (Figure \(4\)) that their red blood cells take on under certain conditions such as physical exertion. The sickle-shaped red blood cells clog small blood vessels, causing multiple phenotypic effects, including stunting of physical growth, certain bone deformities, kidney failure, and strokes.
Epistasis
Some genes affect the expression of other genes. This is called epistasis. Epistasis is similar to dominance, except that it occurs between different genes rather than between different alleles for the same gene.
Albinism is an example of epistasis. A person with albinism has virtually no pigment in the skin. The condition occurs due to an entirely different gene than the genes that encode skin color. Albinism occurs because a protein called tyrosinase, which is needed for the production of normal skin pigment, is not produced due to a gene mutation. If an individual has albinism mutation, he or she will not have any skin pigment, regardless of the skin color genes that were inherited.
Feature: My Human Body
Do you know your ABO blood type? In an emergency, knowing this valuable piece of information could possibly save your life. If you ever need a blood transfusion, it is vital that you receive blood that matches your own blood type. Why? If the blood transfused into your body contains an antigen that your own blood does not contain, antibodies in your blood plasma (the liquid part of your blood) will recognize the antigen as foreign to your body and cause a reaction called agglutination. In this reaction, the transfused red blood cells will clump together, as shown in the image below. The agglutination reaction is serious and potentially fatal.
Knowing the antigens and antibodies present in each of the ABO blood types will help you understand which type(s) of blood you can safely receive if you ever need a transfusion. This information is shown in the table below for all of the ABO blood types. For example, if you have blood type A, this means that your red blood cells have the A antigen and that your blood plasma contains anti-B antibodies. If you were to receive a transfusion of type B or type AB blood, both of which have the B antigen, your anti-B antibodies would attack the transfused red blood cells, causing agglutination.
Table \(2\): Antigens and antibodies in ABO blood types
Characteristics Type A Type B Type AB Type O
Red Blood Cell
Antibodies in Plasma
Anti-B
Anti-A
None
Anti-A and Anti-B
Antigens in Red Blood Cells
A antigen
B antigens
A and B antigens
None
You may have heard that people with blood type O are called universal donors and that people with blood type AB are called universal recipients. People with type O blood have neither A nor B antigens in their blood, so if their blood is transfused into someone with a different ABO blood type, it causes no immune reaction. In other words, they can donate blood to anyone. On the other hand, people with type AB blood have no anti-A or anti-B antibodies in their blood, so they can receive a transfusion of blood from anyone. Which blood type(s) can safely receive a transfusion of type AB blood, and which blood type(s) can be safely received by those with type O blood?
Review
1. What is non-Mendelian inheritance?
2. Explain why the human ABO blood group is an example of a multiple allele trait with codominance.
3. What is incomplete dominance? Give an example of this type of non-Mendelian inheritance in humans.
4. Explain the genetic basis of human skin color.
5. How may the human trait of adult height be influenced by the environment?
6. Define pleiotropy, and give a human example.
7. What is the difference between pleiotropy and epistasis?
8. Which of the following terms best matches each trait description? Choose only the one term that best fits each trait. (codominance; multiple allele trait; Mendelian trait; polygenic trait)
1. A trait controlled by four genes.
2. A trait where each allele of a heterozygote makes an equal contribution to the phenotype.
3. A trait controlled by a single gene that has three different versions.
4. A trait controlled by a single gene where one allele is fully dominant to the only other allele.
9. People with type AB blood have:
1. anti-O antibodies
2. anti-A and anti-B antibodies
3. A and B antigens
10. True or False. People with type O blood cannot receive a blood transfusion from anyone besides others with type O blood.
11. True or False. People with type O blood can be heterozygous for this trait.
Explore More
To learn more about non-Mendelian Inheritance, check out this video:
Attributions
1. Family by Henry M. Trotter, released into the public domain via Wikimedia Commons
2. Adult height graph by Mariana Ruiz Villarreal (LadyofHats), CC BY-NC 3.0 for CK-12 Foundation
3. Skin tanning by Onetwo1, licensed CC BY 3.0 via Wikimedia Commons
4. Sickle cells by OpenStax College, licensed CC BY 3.0 via Wikimedia Commons
5. Type A Blood, public domain via Wikimedia Commons
6. Blood type table based on image of ABO Blood type, public domain via Wikimedia Commons
7. Text adapted from Human Biology by CK-12 licensed CC BY-NC 3.0 | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.06%3A_Extensions_to_Mendel/12.6.01%3A_Complex_Inheritance.txt |
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/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.06%3A_Extensions_to_Mendel/12.6.02%3A_Quantitative_Trait_Loci.txt |
Based on Mendel’s experiments, you might imagine that all genes control a single characteristic, are present in two copies, and affect some harmless aspect of an organism’s appearance (such as color, height, or shape). Although those predictions are accurate in many cases, there are also some important exceptions. For instance, how can we explain observations like the following?
• The genetic disorder Marfan syndrome is caused by a mutation in one gene, yet it affects many aspects of growth and development, including height, vision, and heart function.
To understand observations like these, we need to look more deeply at what genes are. Rather than abstract “heritable factors,” genes are stretches of DNA found on chromosomes, and most of them encode (specify the sequence of) proteins that do a certain job in the cell or body. In this article, we’ll look in more detail at genes affecting multiple characteristics (pleiotropy).
Pleiotropy
When we discussed Mendel’s experiments with purple-flowered and white-flowered plants, we didn’t mention any other phenotypes associated with the two flower colors. However, Mendel noticed that the flower colors were always correlated with two other features: the color of the seed coat (covering of the seed) and the color of the axils (junctions where the leaves met the main stem)[1]. In plants with white flowers, the seed coats and axils were colorless, while in plants with purple flowers, the seed coats were brown-gray and the axils were reddish. Thus, rather than affecting just one characteristic, the flower color gene actually affected three.
Based on similar diagram by Ingrid Lobo
Genes like this, which affect multiple, seemingly unrelated aspects of an organism’s phenotype, are said to be pleiotropic (pleio– = many, –tropic = effects)[2]. We now know that Mendel’s flower color gene encodes a regulator protein that activates pigment biosynthesis, and that it works in several different parts of the pea plant (flowers, seed coat, and leaf axils). Thus, the seemingly unrelated phenotypes can all be traced back to a defect in a single gene with several jobs.
Alleles of pleiotropic genes are transmitted in the same way as alleles of genes that affect single traits. In these cases, the difference is that the phenotype contains multiple elements. These elements are specified as a package, and there would be both a dominant and recessive version of this package of traits.
Pleiotropy in Human Genetic Disorders
Genes affected in human genetic disorders are often pleiotropic. For example, people with the hereditary disorder Marfan syndrome may have a constellation of seemingly unrelated symptoms[3]:
• Unusually tall height
• Thin fingers and toes
• Dislocation of the lens of the eye
• Heart problems (in which the aorta, the large blood vessel carrying blood away from the heart, bulges or ruptures).
These symptoms don’t appear directly related to one another, but as it turns out, they can all be traced back to the mutation of a single gene. This gene encodes a protein that assembles into chains, making elastic fibrils that give strength and flexibility to the body’s connective tissues[4]. Disease-causing mutations in the Marfan syndrome reduce the amount of functional protein produced, resulting in fewer fibrils. The eye and the aorta normally contain many fibrils that help maintain structure, explaining why these two organs are strongly affected in Marfan syndrome[5]. In addition, the fibrils serve as “storage shelves” for growth factors. When there are fewer of them in Marfan syndrome, the growth factors cannot be shelved and thus cause excess growth (leading to the characteristic tall, thin Marfan build)[6].
1. Lobo, I. (2008). Pleiotropy: One gene can affect multiple traits. Nature Education, 1(1), 10. Retrieved from http://www.nature.com/scitable/topicpage/pleiotropy-one-gene-can-affect-multiple-traits-569. ↵
2. Ibid. ↵
3. Marfan syndrome. (2012). In Genetics home reference. Retrieved from http://ghr.nlm.nih.gov/condition/marfan-syndrome. ↵
4. FBN1. (2015). In Genetics home reference. Retrieved from http://ghr.nlm.nih.gov/gene/FBN1. ↵
5. Marfan syndrome. (2015, November 3). Retrieved November 21, 2015 from Wikipedia: https://en.Wikipedia.org/wiki/Marfan_syndrome. ↵
6. FBN1. (2015). In Genetics home reference. Retrieved from http://ghr.nlm.nih.gov/gene/FBN1. ↵
Contributors and Attributions
CC licensed content, Shared previously | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.06%3A_Extensions_to_Mendel/12.6.03%3A_Pleiotropy.txt |
Learning Objectives
• Discuss incomplete dominance, codominance, and multiple alleles as alternatives to dominance and recessiveness
Alternatives to Dominance and Recessiveness
Mendel’s experiments with pea plants suggested that: (1) two “units” or alleles exist for every gene; (2) alleles maintain their integrity in each generation (no blending); and (3) in the presence of the dominant allele, the recessive allele is hidden and makes no contribution to the phenotype. Therefore, recessive alleles can be “carried” and not expressed by individuals. Such heterozygous individuals are sometimes referred to as “carriers.” Further genetic studies in other plants and animals have shown that much more complexity exists, but that the fundamental principles of Mendelian genetics still hold true.
Mendel’s results, that traits are inherited as dominant and recessive pairs, contradicted the view at that time that offspring exhibited a blend of their parents’ traits. However, the heterozygote phenotype occasionally does appear to be intermediate between the two parents. For example, in the snapdragon, Antirrhinum majus, a cross between a homozygous parent with white flowers (CWCW) and a homozygous parent with red flowers (CRCR) will produce offspring with pink flowers (CRCW). This pattern of inheritance is described as incomplete dominance, denoting the expression of two contrasting alleles such that the individual displays an intermediate phenotype. The allele for red flowers is incompletely dominant over the allele for white flowers. However, the results of a heterozygote self-cross can still be predicted, just as with Mendelian dominant and recessive crosses. In this case, the genotypic ratio would be 1 CRCR:2 CRCW:1 CWCW, and the phenotypic ratio would be 1:2:1 for red:pink:white.
A variation on incomplete dominance is codominance, in which both alleles for the same characteristic are simultaneously expressed in the heterozygote. An example of codominance is the MN blood groups of humans. The M and N alleles are expressed in the form of an M or N antigen present on the surface of red blood cells. Homozygotes (LMLMand LNLN) express either the M or the N allele, and heterozygotes (LMLN) express both alleles equally. In a self-cross between heterozygotes expressing a codominant trait, the three possible offspring genotypes are phenotypically distinct. However, the 1:2:1 genotypic ratio characteristic of a Mendelian monohybrid cross still applies.
Mendel implied that only two alleles, one dominant and one recessive, could exist for a given gene. We now know that this is an oversimplification. Although individual humans (and all diploid organisms) can only have two alleles for a given gene, multiple alleles may exist at the population level such that many combinations of two alleles are observed. Note that when many alleles exist for the same gene, the convention is to denote the most common phenotype or genotype among wild animals as the wild type (often abbreviated “+”); this is considered the standard or norm. All other phenotypes or genotypes are considered variants of this standard, meaning that they deviate from the wild type. The variant may be recessive or dominant to the wild-type allele. An example of multiple alleles is coat color in rabbits. Here, four alleles exist for the c gene. The wild-type version, C+C+, is expressed as brown fur. The chinchilla phenotype, cchcch, is expressed as black-tipped white fur. The Himalayan phenotype, chch, has black fur on the extremities and white fur elsewhere. Finally, the albino, or “colorless” phenotype, cc, is expressed as white fur. In cases of multiple alleles, dominance hierarchies can exist. In this case, the wild-type allele is dominant over all the others, chinchilla is incompletely dominant over Himalayan and albino, and Himalayan is dominant over albino. This hierarchy, or allelic series, was revealed by observing the phenotypes of each possible heterozygote offspring.
The complete dominance of a wild-type phenotype over all other mutants often occurs as an effect of “dosage” of a specific gene product, such that the wild-type allele supplies the correct amount of gene product whereas the mutant alleles cannot. For the allelic series in rabbits, the wild-type allele may supply a given dosage of fur pigment, whereas the mutants supply a lesser dosage or none at all. Alternatively, one mutant allele can be dominant over all other phenotypes, including the wild type. This may occur when the mutant allele somehow interferes with the genetic message so that even a heterozygote with one wild-type allele copy expresses the mutant phenotype. One way in which the mutant allele can interfere is by enhancing the function of the wild-type gene product or changing its distribution in the body. One example of this is the Antennapedia mutation in Drosophila. In this case, the mutant allele expands the distribution of the gene product; as a result, the Antennapedia heterozygote develops legs on its head where its antennae should be.
Key Points
• Incomplete dominance is the expression of two contrasting alleles such that the individual displays an intermediate phenotype.
• Codominance is a variation on incomplete dominance in which both alleles for the same characteristic are simultaneously expressed in the heterozygote.
• Diploid organisms can only have two alleles for a given gene; however, multiple alleles may exist at the population level such that many combinations of two alleles are observed.
• The complete dominance of a wild-type phenotype over all other mutants often occurs as an effect of “dosage” of a specific gene product: the wild-type allele supplies the correct amount of gene product whereas the mutant alleles cannot.
• One mutant allele can also be dominant over all other phenotypes, including the wild type.
Key Terms
• allele: one of a number of alternative forms of the same gene occupying a given position on a chromosome
• incomplete dominance: a condition in which the phenotype of the heterozygous genotype is distinct from and often intermediate to the phenotypes of the homozygous genotypes
• codominance: a condition in which both alleles of a gene pair in a heterozygote are fully expressed, with neither one being dominant or recessive to the other | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.06%3A_Extensions_to_Mendel/12.6D%3A_Alternatives_to_Dominance_and_Recessiveness.txt |
Learning Objectives
• Explain the phenotypic outcomes of epistatic effects between genes
Epistasis
Mendel’s studies in pea plants implied that the sum of an individual’s phenotype was controlled by genes (or as he called them, unit factors): every characteristic was distinctly and completely controlled by a single gene. In fact, single observable characteristics are almost always under the influence of multiple genes (each with two or more alleles) acting in unison. For example, at least eight genes contribute to eye color in humans.
In some cases, several genes can contribute to aspects of a common phenotype without their gene products ever directly interacting. In the case of organ development, for instance, genes may be expressed sequentially, with each gene adding to the complexity and specificity of the organ. Genes may function in complementary or synergistic fashions: two or more genes need to be expressed simultaneously to affect a phenotype. Genes may also oppose each other with one gene modifying the expression of another.
In epistasis, the interaction between genes is antagonistic: one gene masks or interferes with the expression of another. “Epistasis” is a word composed of Greek roots that mean “standing upon.” The alleles that are being masked or silenced are said to be hypostatic to the epistatic alleles that are doing the masking. Often the biochemical basis of epistasis is a gene pathway in which the expression of one gene is dependent on the function of a gene that precedes or follows it in the pathway.
An example of epistasis is pigmentation in mice. The wild-type coat color, agouti (AA), is dominant to solid-colored fur (aa). However, a separate gene (C) is necessary for pigment production. A mouse with a recessive c allele at this locus is unable to produce pigment and is albino regardless of the allele present at locus A. Therefore, the genotypes AAcc, Aacc, and aacc all produce the same albino phenotype. A cross between heterozygotes for both genes (AaCc x AaCc) would generate offspring with a phenotypic ratio of 9 agouti:3 solid color:4 albino. In this case, the C gene is epistatic to the A gene.
Epistasis can also occur when a dominant allele masks expression at a separate gene. Fruit color in summer squash is expressed in this way. Homozygous recessive expression of the W gene (ww) coupled with homozygous dominant or heterozygous expression of the Y gene (YY or Yy) generates yellow fruit, while the wwyy genotype produces green fruit. However, if a dominant copy of the W gene is present in the homozygous or heterozygous form, the summer squash will produce white fruit regardless of the Y alleles. A cross between white heterozygotes for both genes (WwYy × WwYy) would produce offspring with a phenotypic ratio of 12 white:3 yellow:1 green.
Finally, epistasis can be reciprocal: either gene, when present in the dominant (or recessive) form, expresses the same phenotype. In the shepherd’s purse plant (Capsella bursa-pastoris), the characteristic of seed shape is controlled by two genes in a dominant epistatic relationship. When the genes A and B are both homozygous recessive (aabb), the seeds are ovoid. If the dominant allele for either of these genes is present, the result is triangular seeds. That is, every possible genotype other than aabb results in triangular seeds; a cross between heterozygotes for both genes (AaBb x AaBb) would yield offspring with a phenotypic ratio of 15 triangular:1 ovoid.
Keep in mind that any single characteristic that results in a phenotypic ratio that totals 16 is typical of a two-gene interaction. Recall the phenotypic inheritance pattern for Mendel’s dihybrid cross, which considered two non-interacting genes: 9:3:3:1. Similarly, we would expect interacting gene pairs to also exhibit ratios expressed as 16 parts. Note that we are assuming the interacting genes are not linked; they are still assorting independently into gametes.
Key Points
• In many cases, several genes may contribute to a particular phenotype; when the actions of one gene masks the effects of another, this gene is said to be epistatic to the second.
• Epistasis can occur when a recessive genotype masks the actions of another gene, or when a dominant allele masks the effects of another gene.
• Epistasis can be reciprocal: either gene, when present in the dominant (or recessive) form, expresses the same phenotype.
• Any single characteristic that results in a phenotypic ratio that totals 16 (such as 12:3:1, 9:3:4, or others) is typical of a two-gene interaction.
Key Terms
• epistasis: the modification of the expression of a gene by another unrelated one | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/12%3A_Patterns_of_Inheritance/12.06%3A_Extensions_to_Mendel/12.6F%3A_Epistasis.txt |
Learning Objectives
• List the reasons that fruit flies are excellent model organisms for genetic research
Chromosomal Theory of Inheritance
The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel’s publications and re-evaluate his model in terms of the behavior of chromosomes during mitosis and meiosis. In 1902, Theodor Boveri observed that proper embryonic development of sea urchins does not occur unless chromosomes are present. That same year, Walter Sutton observed the separation of chromosomes into daughter cells during meiosis. Together, these observations led to the development of the Chromosomal Theory of Inheritance, which identified chromosomes as the genetic material responsible for Mendelian inheritance.
The Chromosomal Theory of Inheritance was consistent with Mendel’s laws and was supported by the following observations:
• During meiosis, homologous chromosome pairs migrate as discrete structures that are independent of other chromosome pairs.
• The sorting of chromosomes from each homologous pair into pre-gametes appears to be random.
• Each parent synthesizes gametes that contain only half of their chromosomal complement.
• Even though male and female gametes (sperm and egg) differ in size and morphology, they have the same number of chromosomes, suggesting equal genetic contributions from each parent.
• The gametic chromosomes combine during fertilization to produce offspring with the same chromosome number as their parents.
Despite compelling correlations between the behavior of chromosomes during meiosis and Mendel’s abstract laws, the Chromosomal Theory of Inheritance was proposed long before there was any direct evidence that traits were carried on chromosomes. Critics pointed out that individuals had far more independently segregating traits than they had chromosomes. It was only after several years of carrying out crosses with the fruit fly, Drosophila melanogaster, that Thomas Hunt Morgan provided experimental evidence to support the Chromosomal Theory of Inheritance.
In 1910, Thomas Hunt Morgan started his work with Drosophila melanogaster, a fruit fly. He chose fruit flies because they can be cultured easily, are present in large numbers, have a short generation time, and have only four pair of chromosomes that can be easily identified under the microscope. They have three pair of autosomes and a pair of sex chromosomes. At that time, he already knew that X and Y have to do with gender. He used normal flies with red eyes and mutated flies with white eyes and cross bred them. In flies, the wild type eye color is red (XW) and is dominant to white eye color (Xw). He was able to conclude that the gene for eye color was on the X chromosome. This trait was thus determined to be X-linked and was the first X-linked trait to be identified. Males are said to be hemizygous, in that they have only one allele for any X-linked characteristic.
Key Points
• Homologous chromosome pairs are independent of other chromosome pairs.
• Chromosomes from each homologous pair are sorted randomly into pre- gametes.
• Parents synthesize gametes that contain only half of their chromosomes; eggs and sperm have the same number of chromosomes.
• Gametic chromosomes combine during fertilization to produce offspring with the same chromosome number as their parents.
• Eye color in fruit flies was the first X-linked trait to be discovered; thus, Morgan’s experiments with fruit flies solidified the Chromosomal Theory of Inheritance.
Key Terms
• autosome: any chromosome other than sex chromosomes
• hemizygous: having some single copies of genes in an otherwise diploid cell or organism
• wild type: the typical form of an organism, strain, gene or characteristic as it occurs in nature | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.01%3A_Sex_Linkage_and_the_Chromosomal_Theory_of_Inheritance/13.1A%3A_Chromosomal_Theory_.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/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.02%3A_Sex_Chromosomes_and_Sex_Determination/13.2.01%3A_Sex_Chromosomes.txt |
Given a pedigree of an uncharacterized disease or trait, one of the first tasks is to determine which modes of inheritance are possible and then which mode of inheritance is most likely. This information is essential in calculating the probability that the trait will be inherited in any future offspring. We will mostly consider five major types of inheritance: autosomal dominant (AD), autosomal recessive (AR), X-linked dominant (XD), X-linked recessive (XR), and Y-linked (Y).
Autosomal Dominant (AD)
When a disease is caused by a dominant allele of a gene, every person with that allele will show symptoms of the disease (assuming complete penetrance), and only one disease allele needs to be inherited for an individual to be affected. Thus, every affected individual must have an affected parent. A pedigree with affected individuals in every generation is typical of AD diseases. However, beware that other modes of inheritance can also show the disease in every generation, as described below. It is also possible for an affected individual with an AD disease to have a family without any affected children, if the affected parent is a heterozygote. This is particularly true in small families, where the probability of every child inheriting the normal, rather than disease allele is not extremely small. Note that AD diseases are usually rare in populations, therefore affected individuals with AD diseases tend to be heterozygotes (otherwise, both parents would have had to been affected with the same rare disease). Achondroplastic dwarfism, and polydactyly are both examples of human conditions that may follow an AD mode of inheritance.
X-linked dominant (XD)
In X-linked dominant inheritance, the gene responsible for the disease is located on the X-chromosome, and the allele that causes the disease is dominant to the normal allele in females. Because females have twice as many X-chromosomes as males, females tend to be more frequently affected than males in the population. However, not all pedigrees provide sufficient information to distinguish XD and AD. One definitive indication that a trait is inherited as AD, and not XD, is that an affected father passes the disease to a son; this type of transmission is not possible with XD, since males inherit their X chromosome from their mothers.
Autosomal recessive (AR)
Diseases that are inherited in an autosomal recessive pattern require that both parents of an affected individual carry at least one copy of the disease allele. With AR traits, many individuals in a pedigree can be carriers, probably without knowing it. Compared to pedigrees of dominant traits, AR pedigrees tend to show fewer affected individuals and are more likely than AD or XD to “skip a generation”. Thus, the major feature that distinguishes AR from AD or XD is that unaffected individuals can have affected offspring.
X-linked recessive (XR)
Because males have only one X-chromosome, any male that inherits an X-linked recessive disease allele will be affected by it (assuming complete penetrance). Therefore, in XR modes of inheritance, males tend to be affected more frequently than females in a population. This is in contrast to AR and AD, where both sexes tend to be affected equally, and XD, in which females are affected more frequently. Note, however, in the small sample sizes typical of human families, it is usually not possible to accurately determine whether one sex is affected more frequently than others. On the other hand, one feature of a pedigree that can be used to definitively establish that an inheritance pattern is not XR is the presence of an affected daughter from unaffected parents; because she would have had to inherit one X-chromosome from her father, he would also have been affected in XR.
Y-linked and Mitochondrial Inheritance.
Two additional modes are Y-linked and Mitochondrial inheritance.
Only males are affected in human Y-linked inheritance (and other species with the X/Y sex determining system). There is only father to son transmission. This is the easiest mode of inheritance to identify, but it is one of the rarest because there are so few genes located on the Y-chromosome. An example of Y-linked inheritance is the hairy-ear-rim phenotype seen in some Indian families. As expected this trait is passed on from father to all sons and no daughters. Y-chromosome DNA polymorphisms can be used to follow the male lineage in large families or through ancient ancestral lineages. For example, the Y-chromosome of Mongolian ruler Genghis Khan (1162-1227 CE), and his male relatives, accounts for ~8% of the Y-chromosome lineage of men in Asia (0.5% world wide).
Mutations in Mitochondrial DNA are inherited through the maternal line (from your mother). There are some human diseases associated with mutations in mitochondria genes. These mutations can affect both males and females, but males cannot pass them on as the mitochondria are inherited via the egg, not the sperm. Mitochondrial DNA polymorphisms are also used to investigate evolutionary lineages, both ancient and recent. Because of the relative similarity of sequence mtDNA is also used in species identification in ecology studies. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.02%3A_Sex_Chromosomes_and_Sex_Determination/13.2.02%3A_Inferring_the_Mode_of_Inheritance.txt |
Learning Objectives
• Explain why genetic information in organelles is passed independently of nuclear DNA.
In eukaryotes, DNA and genes also exist outside of the nuclear chromosomes. Both the chloroplast and mitochondrion have circular chromosomes (Figure \(1\)). These organellar genomes are often present in multiple copies within each organelle. In most sexually-reproducing species, organellar chromosomes are inherited from only one parent, usually the one that produces the largest gamete. Thus, in mammals, angiosperms, and many other organisms, mitochondria and chloroplasts are inherited only through the oocyte.
These organelles are likely the remnants of prokaryotic endosymbionts that entered the cytoplasm of ancient progenitors of today’s eukaryotes (endosymbiont theory). These endosymbionts had their own, circular chromosomes, like most bacteria that exist today. Chloroplasts and mitochondria typically have circular chromosomes that behave more like bacterial chromosomes than eukaryotic chromosomes, i.e. these organellar genomes do not undergo mitosis or meiosis.
Implications of mitochondrial inheritance
As with nuclear DNA, organellar DNA can be mutated. Cells can have a mixture of hundreds to thousands of organelles with different alleles for genes. Because there are not simply one or two organelles in a cell, terms like heterozygous and homozygous do not apply to this situation and patterns of inheritance can be unpredictable. Some patterns of inheritance that are usually observed for mitochondrial inheritance are:
• Traits can be passed via egg to offspring
• Traits are not passed via sperm to offspring
• Variable penetrance and expressivity are often observed are often due to different proportions of wild type and mutant organelles in the organism of even differing proportions between different tissues in the same organism.
Mitochondrial DNA polymorphisms are also used to investigate evolutionary lineages, both ancient and recent.
Current Research in Plant Genetics:
Although organelles are most often inherited through oocytes, exceptions have been identified. Recent studies of cucumber plants (Cucumis sativus var. sativus) identified SNPs in true-breeding lines and performed reciprocal crosses. The results showed that the chloroplasts were inherited from the maternal parent but that mitochondria are inherited from the male parent.
Reference: Park, HS., Lee, W.K., Lee, SC. et al. Inheritance of chloroplast and mitochondrial genomes in cucumber revealed by four reciprocal F1 hybrid combinations. Sci Rep 11, 2506 (2021). https://doi.org/10.1038/s41598-021-81988-w
13.4.01: Classification and Detection of Molecular Markers
Regardless of their origins, molecular markers can be classified as polymorphisms that either vary in the length of a DNA sequence, or vary only in the identity of nucleotides at a particular position on a chromosome (Figure \(1\)). In both cases, because two or more alternative versions of the DNA sequence exist, we can treat each variant as a different allele of a single locus. Each allele gives a different molecular phenotype. For example, polymorphisms of SSRs (short sequence repeats) can be distinguished based on the length of PCR products: one allele of a particular SSR locus might produce a 100bp band, while the same primers used with a different allele as a template might produce a 120bp band (Figure \(2\)). A different type of marker, called a SNP (single nucleotide polymorphism), is an example of polymorphism that varies in nucleotide identity, but not length. SNPs are the most common of any molecular markers, and the genotypes of thousands of SNP loci can be determined in parallel, using new, hybridization based instruments. Note that the alleles of most molecular markers are co-dominant, since it is possible to distinguish the molecular phenotype of a heterozygote from either homozygote.
Mutations that do not affect the function of protein sequences or gene expression are likely to persist in a population as polymorphisms, since there will be no selection either in favor or against them (i.e. they are neutral). Note that the although the rate of spontaneous mutation in natural populations is sufficiently high so as to generate millions of polymorphisms that accumulate over thousands of generations, the rate of mutation is on the other hand sufficiently low that existing polymorphisms are stable throughout the few generations we study in a typical genetic experiment. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.03%3A_Exceptions_to_Chromosomal_Theory_of_Inheritance/13.3.01%3A_Organellar_Inheritance.txt |
Learning Objectives
• Discuss how linked genes can be inherited separately
Genetic Linkage and Distances
Mendel’s work suggested that traits are inherited independently of each other. Morgan identified a 1:1 ratio between a segregating trait and the X chromosome, suggesting that the random segregation of chromosomes was the physical basis of Mendel’s model. This also demonstrated that linked genes disrupt Mendel’s predicted outcomes. The fact that each chromosome can carry many linked genes explains how individuals can have many more traits than they have chromosomes. However, observations by researchers in Morgan’s laboratory suggested that alleles positioned on the same chromosome were not always inherited together. During meiosis, linked genes somehow became unlinked.
Homologous Recombination
In 1909, Frans Janssen observed chiasmata (the point at which chromatids are in contact with each other and may exchange segments) prior to the first division of meiosis. He suggested that alleles become unlinked when chromosomes physically exchange segments. As chromosomes condensed and paired with their homologs, they appeared to interact at distinct points. Janssen suggested that these points corresponded to regions in which chromosome segments were exchanged. It is now known that the pairing and interaction between homologous chromosomes, known as synapsis, does more than simply organize the homologs for migration to separate daughter cells. When synapsed, homologous chromosomes undergo reciprocal physical exchanges of DNA at their arms in a process called homologous recombination, or more simply, “crossing over.”
Genetic Maps
In 1913, Alfred Sturtevant, a student in Morgan’s laboratory, created the first “chromosome map,” a linear representation of gene order and relative distance on a chromosome.To construct a chromosome map, Sturtevant assumed that genes were ordered serially on threadlike chromosomes. He also assumed that the incidence of recombination between two homologous chromosomes could occur with equal likelihood anywhere along the length of the chromosome. Operating under these assumptions, Sturtevant hypothesized alleles that were far apart on a chromosome were more likely to dissociate during meiosis simply because there was a larger region over which recombination could occur. Conversely, alleles that were close to each other on the chromosome were likely to be inherited together. The average number of crossovers between two alleles, or their recombination frequency, correlated with their genetic distance from each other, relative to the locations of other genes on that chromosome. Sturtevant divided his genetic map into map units, or centimorgans (cM), in which a recombination frequency of 0.01 corresponds to 1 cM.
By representing alleles in a linear map, Sturtevant suggested that genes can range from being perfectly linked (recombination frequency = 0) to being perfectly unlinked (recombination frequency = 0.5) when genes are on different chromosomes or genes are separated very far apart on the same chromosome. Perfectly unlinked genes correspond to the frequencies predicted by Mendel to assort independently in a dihybrid cross. A recombination frequency of 0.5 indicates that 50 percent of offspring are recombinants and the other 50 percent are parental types. That is, every type of allele combination is represented with equal frequency. This allowed Sturtevant to calculate distances between several genes on the same chromosome.
Key Points
• Alleles positioned on the same chromosome are not always inherited together because during meiosis linked genes can became unlinked.
• Frans Janssen suggested chromosomes become unlinked during homologous recombination, a process where homologous chromosomes exchange segments of DNA.
• Alfred Sturtevant hypothesized that alleles that were closer together on a gene were more likely to be inherited together rather than alleles that were farther apart and used measurements of recombination between genes to create the first genetic map.
• When genes are perfectly linked, they have a recombination frequency of 0.
• When genes are unlinked, they have a recombination frequency of 0.5, which means 50 percent of offspring are recombinants and the other 50 percent are parental types.
Key Terms
• homologous recombination: a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA
• linkage: the property of genes of being inherited together
• synapsis: the association of homologous maternal and paternal chromosomes during the initial part of meiosis | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.04%3A_Genetic_Mapping/13.4B%3A_Genetic_Linkage_and_Distances.txt |
Is being short-statured inherited?
It can be. Achondroplasia is the most common form of dwarfism in humans, and it is caused by a dominant mutation. The mutation can be passed from one generation to the next.
Genetic Disorders
Many genetic disorders are caused by mutations in one or a few genes. Other genetic disorders are caused by abnormal numbers of chromosomes.
Genetic Disorders Caused by Mutations
The Table below lists several genetic disorders caused by mutations in just one gene. Some of the disorders are caused by mutations in autosomal genes, others by mutations in X-linked genes. Which disorder would you expect to be more common in males than females? You can watch a video about the human genome, genetic disorders, and mutations at this link:http://www.pbs.org/wgbh/nova/programs/ht/rv/2809_03.html.
You can click on any human chromosome at this link to see the genetic disorders associated with it:http://www.ornl.gov/sci/techresource.../chooser.shtml.
Genetic Disorder Direct Effect of Mutation Signs and Symptoms of the Disorder Mode of Inheritance
Marfan syndrome defective protein in connective tissue heart and bone defects and unusually long, slender limbs and fingers autosomal dominant
Sickle cell anemia abnormal hemoglobin protein in red blood cells sickle-shaped red blood cells that clog tinyblood vessels, causing pain and damaging organs and joints autosomal recessive
Vitamin D-resistant rickets lack of a substance needed for bones to absorb minerals soft bones that easily become deformed, leading to bowed legs and other skeletal deformities X-linked dominant
Hemophilia A reduced activity of a protein needed for blood clotting internal and external bleeding that occurs easily and is difficult to control X-linked recessive
Few genetic disorders are controlled by dominant alleles. A mutant dominant allele is expressed in every individual who inherits even one copy of it. If it causes a serious disorder, affected people may die young and fail to reproduce. Therefore, the mutant dominant allele is likely to die out of the population.
A mutant recessive allele, such as the allele that causes sickle cell anemia (see Figure belowand the link that follows), is not expressed in people who inherit just one copy of it. These people are called carriers. They do not have the disorder themselves, but they carry the mutant allele and can pass it to their offspring. Thus, the allele is likely to pass on to the next generation rather than die out. http://www.dnalc.org/resources/3d/17-sickle-cell.html
Sickle-Shaped and Normal Red Blood Cells. Sickle cell anemia is an autosomal recessive disorder. The mutation that causes the disorder affects just one amino acid in a single protein, but it has serious consequences for the affected person. This photo shows the sickle shape of red blood cells in people with sickle cell anemia.
Cystic Fibrosis and Tay-Sachs disease are two additional severe genetic disorders. They are discussed in the following video: http://www.youtube.com/watch?v=8s4he3wLgkM (9:31). Tay-Sachs is further discussed at http://www.youtube.com/watch?v=1RO0LOgHbIo (3:13) andhttp://www.youtube.com/watch?v=6zNj5LdDuTA (2:01).
Chromosomal Disorders
Mistakes may occur during meiosis that result in nondisjunction. This is the failure of replicated chromosomes to separate during meiosis (the animation at the link below shows how this happens). Some of the resulting gametes will be missing a chromosome, while others will have an extra copy of the chromosome. If such gametes are fertilized and form zygotes, they usually do not survive. If they do survive, the individuals are likely to have serious genetic disorders. Table below lists several genetic disorders that are caused by abnormal numbers of chromosomes. Most chromosomal disorders involve the X chromosome. Look back at the X and Y chromosomes and you will see why. The X and Y chromosomes are very different in size, so nondisjunction of the sex chromosomes occurs relatively often. learn.genetics.utah.edu/conte...der/index.html
Genetic Disorder Genotype Phenotypic Effects
Down syndrome extra copy (complete or partial) of chromosome 21 (see Figure below) developmental delays, distinctive facial appearance, and other abnormalities (see Figurebelow)
Turner’s syndrome one X chromosome but no other sex chromosome (XO) female with short height and infertility (inability to reproduce)
Triple X syndrome three X chromosomes (XXX) female with mild developmental delays and menstrual irregularities
Klinefelter’s syndrome one Y chromosome and two or more X chromosomes (XXY, XXXY) male with problems in sexual development and reduced levels of the male hormone testosterone
(left) Trisomy 21 (Down Syndrome) Karyotype. A karyotype is a picture of a cell's chromosomes. Note the extra chromosome 21. (right) Child with Down syndrome, exhibiting characteristic facial appearance.
Diagnosing Genetic Disorders
A genetic disorder that is caused by a mutation can be inherited. Therefore, people with a genetic disorder in their family may be concerned about having children with the disorder. Professionals known as genetic counselors can help them understand the risks of their children being affected. If they decide to have children, they may be advised to have prenatal(“before birth”) testing to see if the fetus has any genetic abnormalities. One method of prenatal testing is amniocentesis. In this procedure, a few fetal cells are extracted from the fluid surrounding the fetus, and the fetal chromosomes are examined.
Treating Genetic Disorders
The symptoms of genetic disorders can sometimes be treated, but cures for genetic disorders are still in the early stages of development. One potential cure that has already been used with some success is gene therapy. This involves inserting normal genes into cells with mutant genes. At the following link, you can watch the video ‘‘Sickle Cell Anemia: Hope fromGene Therapy’’, to learn how scientists are trying to cure sickle-cell anemia with gene therapy. www.pubinfo.vcu.edu/secretsof...list_frame.asp
If you could learn your risk of getting cancer or another genetic disease, would you? Though this is a personal decision, it is a possibility. A number of companies now makes it easy to order medical genetic tests through the Web. See Genetic Testing through the Web athttp://www.kqed.org/quest/televisi...hrough-the-web.
Summary
• Many genetic disorders are caused by mutations in one or a few genes.
• Other genetic disorders are caused by abnormal numbers of chromosomes.
Explore More
Use this resource to answer the questions that follow.
1. How do mutations affect proteins?
2. What is a single-gene disorder?
3. What is a chromosomal disorder?
4. What is a complex disorder?
5. Give an example of a chromosomal disorder.
Review
1. Describe a genetic disorder caused by a mutation in a single gene.
2. What causes Down syndrome?
3. What is nondisjunction?
4. What is gene therapy?
5. Explain why genetic disorders caused by abnormal numbers of chromosomes most often involve the X chromosome.
13.05: Human Genetic Disorders
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/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.05%3A_Human_Genetic_Disorders/13.5.01%3A_Imprinted_Genes.txt |
Many tests are now available to detect genetic diseases such as sickle cell disease, cystic fibrosis and phenylketonuria (PKU). Most of these tests can not only be performed on cells removed from adults but also on cells removed from the fetus and even from a pre-implantation embryo.
Amniocentesis
During its development, the fetus sheds cells into the amniotic fluid. After 14–22 weeks of pregnancy, a small volume of this fluid can be removed (using a needle inserted through the abdominal wall).
Using ultrasound to locate the position of the placenta prior to amniocentesis. These sonograms are made by recording the echoes received from structures within the abdomen. A, amniotic cavity; B, urinary bladder; F, part of the fetus; P, placenta. Both longitudinal (left) and transverse (right) scans are needed for accurate localization of the placenta. (Courtesy of the Downstate Medical Center of the State University of New York.)
Separating the cells and culturing them enables the clinician to look for
• chromosome abnormalities (e.g., the three number 21 chromosomes of Down syndrome)
• certain enzymatic defects (e.g., an inability to metabolize galactose, hence milk)
• the sex of the fetus
Over 100 genetic abnormalities can be diagnosed by amniocentesis and the pregnancy deliberately ended if the parents wish it.
Chorionic Villus Sampling (CVS)
This is an alternate method of prenatal diagnosis. A small amount of placental tissue is sucked out by a tube inserted through the abdominal wall or through the vagina (the latter avoiding the need for an incision). For some tests the fetal cells can be examined immediately without the need to culture them. Another advantage of CVS is that it can be performed earlier in pregnancy (after only 10–12 weeks) than amniocentesis. If an abortion is to be performed, it is a simpler process early in pregnancy.
Non-Invasive Prenatal Genetic Testing (NIPT)
Although the blood vessels of the placenta are in close contact with the mother's blood vessels in the uterus, intermingling of their blood does not normally occur. However some of cells of the fetus do manage to get into the mother's circulation where they may represent 1 in a million of her white blood cells (so only some 2–6 cells per ml of blood). Fragments of fetal DNA (~ 300 bp long) from apoptotic cells of the placenta are also found in the mother's plasma as early as 5 weeks after implantation. This raises the possibility of using genetic tests (e.g., PCR) to identify mutations or chromosomal abnormalities in the fetus using a small (~10 ml) sample of blood drawn from the mother.
Two home blood test kits for determining the sex of the fetus are already on the market. The collected drops of blood are sent to a laboratory to determine whether any Y-chromosome-specific DNA (e.g., SRY) is present. Tests of fetal DNA for Down syndrome (trisomy 21), as well as for trisomy 13 and 18, have both higher sensitivity (false negatives <0.1%) and specificity (false positives <0.2%) than amniocentesis and CVS.
In several European countries, Rh-negative mothers can now have their blood screened for the presence of an Rh-positive fetus. The time will surely come when such NIPT screening will become available for many genetic disorders. The procedure is also called non-invasive prenatal diagnosis - NIPD.
The level of fetal DNA in the mother's blood rises to a peak at the time of birth and some evidence suggests that this rise may be a trigger to start the birth process.
Preimplantation Genetic Diagnosis (PGD)
Genetic Analysis of Blastomeres
One of the remarkable facts about mammalian development is that all the cells in the early (e.g., 8-cell) embryo are not needed to produce a healthy fetus (which is why a single fertilized egg can on occasions produce identical twins, triplets, etc.). So couples using in vitro fertilization (IVF) also can take advantage of genetic screening. While the embryo is in culture, one or two cells can safely be removed and tested for their genotype. For example:
• The sex of the embryo can be determined with a probe for Y-specific DNA. This permits prospective mothers carrying a severe X-linked trait like hemophilia A to choose a female rather than a male embryo for attempted implantation.
• Fluorescent probes specific for the DNA of particular chromosomes can detect (by FISH) if there is an abnormal number (aneuploidy) such as the three #21 chromosomes of Down syndrome.
• In fact the entire karyotype of the embryo can be determined. Random fragments of DNA prepared by the polymerase chain reaction (PCR) of all the DNA of a cell from the embryo can be
• given a fluorescent label
• applied to the metaphase chromosomes of a standard reference cell that has a normal karyotype along with
• DNA fragments from the reference cell labelled with a different color.
Comparing the intensity of the two colors from each chromosome shows whether the embryo has the normal amount of DNA for that chromosome or is aneuploid containing either:
• too much (e.g. 3 copies of #21 — trisomy)
• too little (only a single copy of #14 — monosomy)
Genetic Analysis of Polar Bodies
As meiosis I is completed, one set of duplicated chromosomes (dyads) is extruded into the first polar body. The DNA of the polar body can be amplified by the polymerase chain reaction (PCR) and tested.
If the mother is heterozygous for a trait, and no crossover has occurred, and the polar body contains the mutant alleles (see figure), the egg can be safely fertilized. (For simplicity, the figure shows only the pair of homologues carrying the locus of concern.)
However, if crossing over has occurred, the first polar body would contain one mutant and one healthy allele. In that case, there is a 50:50 chance that, after fertilization, the other copy of the mutant allele will end up in the egg (instead of in the second polar body). So the second polar body should also be tested to see if it also contains the mutant allele. Only if it does can the egg be safely used. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/13%3A_Chromosomes_Mapping_and_the_Meiosis-Inheritance_Connection/13.05%3A_Human_Genetic_Disorders/13.5D%3A_Prenatal_Screening.txt |
• 14.1: The Nature of Genetic Material
• 14.2: DNA Structure
The DNA molecule is a polymer of nucleotides. Each nucleotide is composed of a nitrogenous base, a five-carbon sugar (deoxyribose), and a phosphate group. There are four nitrogenous bases in DNA, two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). A DNA molecule is composed of two strands. Each strand is composed of nucleotides bonded together covalently between the phosphate group of one and the deoxyribose sugar of the next. From this backbone extend the bases.
• 14.3: Basic Characteristics of DNA Replication
The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive.
• 14.4: Prokaryotic Replication
In both prokaryotic and eukaryotic cell division, the genomic DNA is replicated and each copy is allocated into a daughter cell. The cytoplasmic contents are also divided evenly to the new cells. However, there are many differences between prokaryotic and eukaryotic cell division. Bacteria have a single, circular DNA chromosome and no nucleus. Therefore, mitosis is not necessary in bacterial cell division. Bacterial cytokinesis is directed by a ring composed of a protein called FtsZ.
• 14.5: Eukaryotic Replication
Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on the eukaryotic chromosome; humans can have up to 100,000 origins of replication
• 14.6: DNA Repair
DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.
14: DNA- The Genetic Material
The spiral structure in the picture is a large organic molecule. What type of organic molecule is it?
Here’s a hint: molecules like this one determine who you are. They contain genetic information that controls your characteristics. They determine your eye color, facial features, and other physical attributes. What molecule is it?
You probably answered "DNA." Today, it is commonly known that DNA is the genetic material. For a long time, scientists knew such molecules existed. They were aware that genetic information was contained within organic molecules. However, they didn’t know which type of molecules play this role. In fact, for many decades, scientists thought that proteins were the molecules that carry genetic information. In this section, you will learn how scientists discovered that DNA carries the code of life.
DNA, the Genetic Material
DNA, deoxyribonucleic acid, is the genetic material in your cells. It was passed on to you from your parents and determines your characteristics. The discovery that DNA is the genetic material was another important milestone in molecular biology.
Griffith Searches for the Genetic Material
Many scientists contributed to the identification of DNA as the genetic material. In the 1920s, Frederick Griffith made an important discovery. He was studying two different strains of a bacterium, called R (rough) strain and S (smooth) strain. He injected the two strains into mice. The S strain killed (virulent) the mice, but the R strain did not (non-virulent) (see Figure below). Griffith also injected mice with S-strain bacteria that had been killed by heat. As expected, the killed bacteria did not harm the mice. However, when the dead S-strain bacteria were mixed with live R-strain bacteria and injected, the mice died.
Griffith’s Experimental Results. Griffith showed that a substance could be transferred to harmless bacteria and make them deadly.
Based on his observations, Griffith deduced that something in the killed S strain was transferred to the previously harmless R strain, making the R strain deadly. He called this process transformation, as something was "transforming" the bacteria from one strain into another strain. What was that something? What type of substance could change the characteristics of the organism that received it?
Avery’s Team Makes a Major Contribution
In the early 1940s, a team of scientists led by Oswald Avery tried to answer the question raised by Griffith’s results. They inactivated various substances in the S-strain bacteria. They then killed the S-strain bacteria and mixed the remains with live R-strain bacteria. (Keep in mind, the R-strain bacteria usually did not harm the mice.) When they inactivated proteins, the R-strain was deadly to the injected mice. This ruled out proteins as the genetic material. Why? Even without the S-strain proteins, the R strain was changed, or transformed, into the deadly strain. However, when the researchers inactivated DNA in the S strain, the R strain remained harmless. This led to the conclusion that DNA is the substance that controls the characteristics of organisms. In other words, DNA is the genetic material. You can watch an animation about the research of both Griffith and Avery at this link:http://www.dnalc.org/view/16375-Animation-17-A-gene-is-made-of-DNA-.html.
Hershey and Chase Seal the Deal
The conclusion that DNA is the genetic material was not widely accepted at first. It had to be confirmed by other research. In the 1950s, Alfred Hershey and Martha Chase did experiments with viruses and bacteria. Viruses are not made of cells. They are basically DNA inside a protein coat. To reproduce, a virus must insert its own genetic material into a cell (such as a bacterium). Then it uses the cell’s machinery to make more viruses. The researchers used different radioactive elements to label the DNA and proteins in viruses. This allowed them to identify which molecule the viruses inserted into bacteria. DNA was the molecule they identified. This confirmed that DNA is the genetic material.
Summary
• The work of several researchers led to the discovery that DNA is the genetic material.
• Along the way, Griffith discovered the process of transformation.
Explore More
I) Bacteria and viruses have DNA too at
www.dnalc.org/resources/nobel/hershey.html
1. What is a bacteriophage?
2. How do phages reproduce?
3. Why was DNA labeled with radioactive phosphorus?
4. After the experiment, where was the radioactive phosphorus found?
5. What is the genetic material? Why?
Review
1. List the research that determined that DNA is the genetic material.
2. What is transformation?
3. What happened to the R-strain bacteria when Avery and his colleagues inactivated DNA in the S strain bacteria? | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.01%3A_The_Nature_of_Genetic_Material.txt |
In the 1950s, Francis Crick and James Watson worked together at the University of Cambridge, England, to determine the structure of DNA. Other scientists, such as Linus Pauling and Maurice Wilkins, were also actively exploring this field. Pauling had discovered the secondary structure of proteins using X-ray crystallography. X-ray crystallography is a method for investigating molecular structure by observing the patterns formed by X-rays shot through a crystal of the substance. The patterns give important information about the structure of the molecule of interest. In Wilkins’ lab, researcher Rosalind Franklin was using X-ray crystallography to understand the structure of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule using Franklin's data (Figure \(1\)). Watson and Crick also had key pieces of information available from other researchers such as Chargaff’s rules. Chargaff had shown that of the four kinds of monomers (nucleotides) present in a DNA molecule, two types were always present in equal amounts and the remaining two types were also always present in equal amounts. This meant they were always paired in some way. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine for their work in determining the structure of DNA.
Now let’s consider the structure of the two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The building blocks of DNA are nucleotides, which are made up of three parts: a deoxyribose (5-carbon sugar), a phosphate group, and a nitrogenous base (Figure \(2\)). There are four types of nitrogenous bases in DNA. Adenine (A) and guanine (G) are double-ringed purines, and cytosine (C) and thymine (T) are smaller, single-ringed pyrimidines. The nucleotide is named according to the nitrogenous base it contains.
The phosphate group of one nucleotide bonds covalently with the sugar molecule of the next nucleotide, and so on, forming a long polymer of nucleotide monomers. The sugar–phosphate groups line up in a “backbone” for each single strand of DNA, and the nucleotide bases stick out from this backbone. The carbon atoms of the five-carbon sugar are numbered clockwise from the oxygen as 1', 2', 3', 4', and 5' (1' is read as “one prime”). The phosphate group is attached to the 5' carbon of one nucleotide and the 3' carbon of the next nucleotide. In its natural state, each DNA molecule is actually composed of two single strands held together along their length with hydrogen bonds between the bases.
Watson and Crick proposed that the DNA is made up of two strands that are twisted around each other to form a right-handed helix, called a double helix. Base-pairing takes place between a purine and pyrimidine: namely, A pairs with T, and G pairs with C. In other words, adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. This is the basis for Chargaff’s rule; because of their complementarity, there is as much adenine as thymine in a DNA molecule and as much guanine as cytosine. Adenine and thymine are connected by two hydrogen bonds, and cytosine and guanine are connected by three hydrogen bonds. The two strands are anti-parallel in nature; that is, one strand will have the 3' carbon of the sugar in the “upward” position, whereas the other strand will have the 5' carbon in the upward position. The diameter of the DNA double helix is uniform throughout because a purine (two rings) always pairs with a pyrimidine (one ring) and their combined lengths are always equal (Figure \(3\)).
The Structure of RNA
There is a second nucleic acid in all cells called ribonucleic acid, or RNA. Like DNA, RNA is a polymer of nucleotides. Each of the nucleotides in RNA is made up of a nitrogenous base, a five-carbon sugar, and a phosphate group. In the case of RNA, the five-carbon sugar is ribose, not deoxyribose. Ribose has a hydroxyl group at the 2' carbon, unlike deoxyribose, which has only a hydrogen atom (Figure \(4\)).
RNA nucleotides contain the nitrogenous bases adenine, cytosine, and guanine. However, they do not contain thymine, which is instead replaced by uracil, symbolized by a “U.” RNA exists as a single-stranded molecule rather than a double-stranded helix. Molecular biologists have named several kinds of RNA on the basis of their function. These include messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)—molecules that are involved in the production of proteins from the DNA code.
How DNA Is Arranged in the Cell
DNA is a working molecule; it must be replicated when a cell is ready to divide, and it must be “read” to produce the molecules, such as proteins, to carry out the functions of the cell. For this reason, the DNA is protected and packaged in very specific ways. In addition, DNA molecules can be very long. Stretched end-to-end, the DNA molecules in a single human cell would come to a length of about 2 meters. Thus, the DNA for a cell must be packaged in a very ordered way to fit and function within a structure (the cell) that is not visible to the naked eye. The chromosomes of prokaryotes are much simpler than those of eukaryotes in many of their features (Figure \(5\)). Most prokaryotes contain a single, circular chromosome that is found in an area in the cytoplasm called the nucleoid.
The size of the genome in one of the most well-studied prokaryotes, Escherichia coli, is 4.6 million base pairs, which would extend a distance of about 1.6 mm if stretched out. So how does this fit inside a small bacterial cell? The DNA is twisted beyond the double helix in what is known as supercoiling. Some proteins are known to be involved in the supercoiling; other proteins and enzymes help in maintaining the supercoiled structure.
Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a different type of packing strategy to fit their DNA inside the nucleus (Figure \(6\)). At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The DNA is wrapped tightly around the histone core. This nucleosome is linked to the next one by a short strand of DNA that is free of histones. This is also known as the “beads on a string” structure; the nucleosomes are the “beads” and the short lengths of DNA between them are the “string.” The nucleosomes, with their DNA coiled around them, stack compactly onto each other to form a 30-nm–wide fiber. This fiber is further coiled into a thicker and more compact structure. At the metaphase stage of mitosis, when the chromosomes are lined up in the center of the cell, the chromosomes are at their most compacted. They are approximately 700 nm in width, and are found in association with scaffold proteins.
In interphase, the phase of the cell cycle between mitoses at which the chromosomes are decondensed, eukaryotic chromosomes have two distinct regions that can be distinguished by staining. There is a tightly packaged region that stains darkly, and a less dense region. The darkly staining regions usually contain genes that are not active, and are found in the regions of the centromere and telomeres. The lightly staining regions usually contain genes that are active, with DNA packaged around nucleosomes but not further compacted.
CONCEPT IN ACTION
Watch this animation of DNA packaging.
Summary
The model of the double-helix structure of DNA was proposed by Watson and Crick. The DNA molecule is a polymer of nucleotides. Each nucleotide is composed of a nitrogenous base, a five-carbon sugar (deoxyribose), and a phosphate group. There are four nitrogenous bases in DNA, two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). A DNA molecule is composed of two strands. Each strand is composed of nucleotides bonded together covalently between the phosphate group of one and the deoxyribose sugar of the next. From this backbone extend the bases. The bases of one strand bond to the bases of the second strand with hydrogen bonds. Adenine always bonds with thymine, and cytosine always bonds with guanine. The bonding causes the two strands to spiral around each other in a shape called a double helix. Ribonucleic acid (RNA) is a second nucleic acid found in cells. RNA is a single-stranded polymer of nucleotides. It also differs from DNA in that it contains the sugar ribose, rather than deoxyribose, and the nucleotide uracil rather than thymine. Various RNA molecules function in the process of forming proteins from the genetic code in DNA.
Prokaryotes contain a single, double-stranded circular chromosome. Eukaryotes contain double-stranded linear DNA molecules packaged into chromosomes. The DNA helix is wrapped around proteins to form nucleosomes. The protein coils are further coiled, and during mitosis and meiosis, the chromosomes become even more greatly coiled to facilitate their movement. Chromosomes have two distinct regions which can be distinguished by staining, reflecting different degrees of packaging and determined by whether the DNA in a region is being expressed (euchromatin) or not (heterochromatin).
Glossary
deoxyribose
a five-carbon sugar molecule with a hydrogen atom rather than a hydroxyl group in the 2' position; the sugar component of DNA nucleotides
double helix
the molecular shape of DNA in which two strands of nucleotides wind around each other in a spiral shape
nitrogenous base
a nitrogen-containing molecule that acts as a base; often referring to one of the purine or pyrimidine components of nucleic acids
phosphate group
a molecular group consisting of a central phosphorus atom bound to four oxygen atoms | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.02%3A_DNA_Structure.txt |
Skills to Develop
• Explain how the structure of DNA reveals the replication process
• Describe the Meselson and Stahl experiments
The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested (Figure \(1\)): conservative, semi-conservative, and dispersive.
In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.
Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure \(2\)).
The E. coli culture was then shifted into medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15N will band at a higher density position than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N after they had been shifted from 15N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N and 14N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.
During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.
Link to Learning
Click through this tutorial on DNA replication.
Summary
The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In conservative replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative method suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. The dispersive mode suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA.
Contributors and Attributions
• Connie Rye (East Mississippi Community College), Robert Wise (University of Wisconsin, Oshkosh), Vladimir Jurukovski (Suffolk County Community College), Jean DeSaix (University of North Carolina at Chapel Hill), Jung Choi (Georgia Institute of Technology), Yael Avissar (Rhode Island College) among other contributing authors. Original content by OpenStax (CC BY 4.0; Download for free at http://cnx.org/contents/[email protected]). | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.03%3A_Basic_Characteristics_of_DNA_Replication.txt |
Prokaryotes such as bacteria propagate by binary fission. For unicellular organisms, cell division is the only method to produce new individuals. In both prokaryotic and eukaryotic cells, the outcome of cell reproduction is a pair of daughter cells that are genetically identical to the parent cell. In unicellular organisms, daughter cells are individuals.
To achieve the outcome of identical daughter cells, some steps are essential. The genomic DNA must be replicated and then allocated into the daughter cells; the cytoplasmic contents must also be divided to give both new cells the machinery to sustain life. In bacterial cells, the genome consists of a single, circular DNA chromosome; therefore, the process of cell division is simplified. Mitosis is unnecessary because there is no nucleus or multiple chromosomes. This type of cell division is called binary fission.
Binary Fission
The cell division process of prokaryotes, called binary fission, is a less complicated and much quicker process than cell division in eukaryotes. Because of the speed of bacterial cell division, populations of bacteria can grow very rapidly. The single, circular DNA chromosome of bacteria is not enclosed in a nucleus, but instead occupies a specific location, the nucleoid, within the cell. As in eukaryotes, the DNA of the nucleoid is associated with proteins that aid in packaging the molecule into a compact size. The packing proteins of bacteria are, however, related to some of the proteins involved in the chromosome compaction of eukaryotes.
The starting point of replication, the origin, is close to the binding site of the chromosome to the plasma membrane (Figure \(1\)). Replication of the DNA is bidirectional—moving away from the origin on both strands of the DNA loop simultaneously. As the new double strands are formed, each origin point moves away from the cell-wall attachment toward opposite ends of the cell. As the cell elongates, the growing membrane aids in the transport of the chromosomes. After the chromosomes have cleared the midpoint of the elongated cell, cytoplasmic separation begins. A septum is formed between the nucleoids from the periphery toward the center of the cell. When the new cell walls are in place, the daughter cells separate.
EVOLUTION IN ACTION: Mitotic Spindle Apparatus
The precise timing and formation of the mitotic spindle is critical to the success of eukaryotic cell division. Prokaryotic cells, on the other hand, do not undergo mitosis and therefore have no need for a mitotic spindle. However, the FtsZ protein that plays such a vital role in prokaryotic cytokinesis is structurally and functionally very similar to tubulin, the building block of the microtubules that make up the mitotic spindle fibers that are necessary for eukaryotes. The formation of a ring composed of repeating units of a protein called FtsZ directs the partition between the nucleoids in prokaryotes. Formation of the FtsZ ring triggers the accumulation of other proteins that work together to recruit new membrane and cell-wall materials to the site. FtsZ proteins can form filaments, rings, and other three-dimensional structures resembling the way tubulin forms microtubules, centrioles, and various cytoskeleton components. In addition, both FtsZ and tubulin employ the same energy source, GTP (guanosine triphosphate), to rapidly assemble and disassemble complex structures.
FtsZ and tubulin are an example of homology, structures derived from the same evolutionary origins. In this example, FtsZ is presumed to be similar to the ancestor protein to both the modern FtsZ and tubulin. While both proteins are found in extant organisms, tubulin function has evolved and diversified tremendously since the evolution from its FtsZ-like prokaryotic origin. A survey of cell-division machinery in present-day unicellular eukaryotes reveals crucial intermediary steps to the complex mitotic machinery of multicellular eukaryotes (Table \(1\)).
Table \(1\): Mitotic Spindle Evolution
Structure of genetic material Division of nuclear material Separation of daughter cells
Prokaryotes There is no nucleus. The single, circular chromosome exists in a region of cytoplasm called the nucleoid. Occurs through binary fission. As the chromosome is replicated, the two copies move to opposite ends of the cell by an unknown mechanism. FtsZ proteins assemble into a ring that pinches the cell in two.
Some protists Linear chromosomes exist in the nucleus. Chromosomes attach to the nuclear envelope, which remains intact. The mitotic spindle passes through the envelope and elongates the cell. No centrioles exist. Microfilaments form a cleavage furrow that pinches the cell in two.
Other protists Linear chromosomes exist in the nucleus. A mitotic spindle forms from the centrioles and passes through the nuclear membrane, which remains intact. Chromosomes attach to the mitotic spindle. The mitotic spindle separates the chromosomes and elongates the cell. Microfilaments form a cleavage furrow that pinches the cell in two.
Animal cells Linear chromosomes exist in the nucleus. A mitotic spindle forms from the centrioles. The nuclear envelope dissolves. Chromosomes attach to the mitotic spindle, which separates them and elongates the cell. Microfilaments form a cleavage furrow that pinches the cell in two.
Summary
In both prokaryotic and eukaryotic cell division, the genomic DNA is replicated and each copy is allocated into a daughter cell. The cytoplasmic contents are also divided evenly to the new cells. However, there are many differences between prokaryotic and eukaryotic cell division. Bacteria have a single, circular DNA chromosome and no nucleus. Therefore, mitosis is not necessary in bacterial cell division. Bacterial cytokinesis is directed by a ring composed of a protein called FtsZ. Ingrowth of membrane and cell-wall material from the periphery of the cells results in a septum that eventually forms the separate cell walls of the daughter cells.
Glossary
binary fission
the process of prokaryotic cell division
FtsZ
a tubulin-like protein component of the prokaryotic cytoskeleton that is important in prokaryotic cytokinesis (name origin: Filamenting temperature-sensitive mutant Z)
origin
the region of the prokaryotic chromosome at which replication begins
septum
a wall formed between bacterial daughter cells as a precursor to cell separation
14.04: Prokaryotic Replication
Skills to Develop
• Explain the process of DNA replication in prokaryotes
• Discuss the role of different enzymes and proteins in supporting this process
DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistakes.
DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.
How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand (Figure \(1\)).
Exercise \(1\)
You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?
Answer
DNA ligase, as this enzyme joins together Okazaki fragments.
The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5' to 3' direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction. One strand, which is complementary to the 3' to 5' parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand.
The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in by deoxyribonucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment.
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows.
DNA replication steps
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase starts adding nucleotides to the 3'-OH end of the primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed by exonuclease activity.
9. Gaps are filled by DNA pol by adding dNTPs.
10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Table \(1\) summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.
Table \(1\): Prokaryotic DNA Replication: Enzymes and Their Function
Enzyme/protein Specific Function
DNA pol I Exonuclease activity removes RNA primer and replaces with newly synthesized DNA
DNA pol II Repair function
DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA
Single-strand binding proteins (SSB) Binds to single-stranded DNA to avoid DNA rewinding back.
Link to Learning
Review the full process of DNA replication here.
Summary
Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication fork; this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand.
Glossary
helicase
during replication, this enzyme helps to open up the DNA helix by breaking the hydrogen bonds
lagging strand
during replication, the strand that is replicated in short fragments and away from the replication fork
leading strand
strand that is synthesized continuously in the 5'-3' direction which is synthesized in the direction of the replication fork
ligase
enzyme that catalyzes the formation of a phosphodiester linkage between the 3' OH and 5' phosphate ends of the DNA
Okazaki fragment
DNA fragment that is synthesized in short stretches on the lagging strand
primase
enzyme that synthesizes the RNA primer; the primer is needed for DNA pol to start synthesis of a new DNA strand
primer
short stretch of nucleotides that is required to initiate replication; in the case of replication, the primer has RNA nucleotides
replication fork
Y-shaped structure formed during initiation of replication
single-strand binding protein
during replication, protein that binds to the single-stranded DNA; this helps in keeping the two strands of DNA apart so that they may serve as templates
sliding clamp
ring-shaped protein that holds the DNA pol on the DNA strand
topoisomerase
enzyme that causes underwinding or overwinding of DNA when DNA replication is taking place | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.04%3A_Prokaryotic_Replication/14.4.01%3A_DNA_Replication_in_Prokaryotes.txt |
Skills to Develop
• Discuss the similarities and differences between DNA replication in eukaryotes and prokaryotes
• State the role of telomerase in DNA replication
Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on the eukaryotic chromosome; humans can have up to 100,000 origins of replication. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as Autonomously Replicating Sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E. coli.
The number of DNA polymerases in eukaryotes is much more than prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as pol α, pol β, pol γ, pol δ, and pol ε.
The essential steps of replication are the same as in prokaryotes. Before replication can start, the DNA has to be made available as template. Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Other proteins are then recruited to start the replication process (Table \(1\)).
A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis. While the leading strand is continuously synthesized by the enzyme pol δ, the lagging strand is synthesized by pol ε. A sliding clamp protein known as PCNA (Proliferating Cell Nuclear Antigen) holds the DNA pol in place so that it does not slide off the DNA. RNase H removes the RNA primer, which is then replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined together after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond.
Telomere replication
Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5' to 3' direction. In the leading strand, synthesis continues until the end of the chromosome is reached. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and over time these ends may get progressively shorter as cells continue to divide.
The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that code for no particular gene. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure \(1\)) helped in the understanding of how chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3' end of the DNA strand. Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.
Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure \(2\)) received the Nobel Prize for Medicine and Physiology in 2009.
Telomerase and Aging
Cells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older.
In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine.1 Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.
Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.
Table \(1\): Difference between Prokaryotic and Eukaryotic Replication
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
DNA polymerase types 5 14
Telomerase Not present Present
RNA primer removal DNA pol I RNase H
Strand elongation DNA pol III Pol δ, pol ε
Sliding clamp Sliding clamp PCNA
Summary
Replication in eukaryotes starts at multiple origins of replication. The mechanism is quite similar to prokaryotes. A primer is required to initiate synthesis, which is then extended by DNA polymerase as it adds nucleotides one by one to the growing chain. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short stretches called Okazaki fragments. The RNA primers are replaced with DNA nucleotides; the DNA remains one continuous strand by linking the DNA fragments with DNA ligase. The ends of the chromosomes pose a problem as polymerase is unable to extend them without a primer. Telomerase, an enzyme with an inbuilt RNA template, extends the ends by copying the RNA template and extending one end of the chromosome. DNA polymerase can then extend the DNA using the primer. In this way, the ends of the chromosomes are protected.
Footnotes
1. 1 Jaskelioff et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature 469 (2011): 102-7.
Glossary
telomerase
enzyme that contains a catalytic part and an inbuilt RNA template; it functions to maintain telomeres at chromosome ends
telomere
DNA at the end of linear chromosomes | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.05%3A_Eukaryotic_Replication.txt |
Skills to Develop
• Discuss the different types of mutations in DNA
• Explain DNA repair mechanisms
DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.
Most of the mistakes during DNA replication are promptly corrected by DNA polymerase by proofreading the base that has been just added (Figure \(1\)). In proofreading, the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.
Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair (Figure \(2\)). The enzymes recognize the incorrectly added nucleotide and excise it; this is then replaced by the correct base. If this remains uncorrected, it may lead to more permanent damage. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli, after replication, the nitrogenous base adenine acquires a methyl group; the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA polymerase is able to remove the wrongly incorporated bases from the newly synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.
In another type of repair mechanism, nucleotide excision repair, enzymes replace incorrect bases by making a cut on both the 3' and 5' ends of the incorrect base (Figure \(3\)). The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.
A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa (Figure \(4\)). Affected individuals have skin that is highly sensitive to UV rays from the sun. When individuals are exposed to UV, pyrimidine dimers, especially those of thymine, are formed; people with xeroderma pigmentosa are not able to repair the damage. These are not repaired because of a defect in the nucleotide excision repair enzymes, whereas in normal individuals, the thymine dimers are excised and the defect is corrected. The thymine dimers distort the structure of the DNA double helix, and this may cause problems during DNA replication. People with xeroderma pigmentosa may have a higher risk of contracting skin cancer than those who dont have the condition.
Errors during DNA replication are not the only reason why mutations arise in DNA. Mutations, variations in the nucleotide sequence of a genome, can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.
Mutations may have a wide range of effects. Some mutations are not expressed; these are known as silent mutations. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These can be of two types, either transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa; for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, also known as deletion. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome; this is also known as translocation. These mutation types are shown in Figure \(5\).
Exercise \(1\)
A frameshift mutation that results in the insertion of three nucleotides is often less deleterious than a mutation that results in the insertion of one nucleotide. Why?
Answer
If three nucleotides are added, one additional amino acid will be incorporated into the protein chain, but the reading frame won't shift.
Mutations in repair genes have been known to cause cancer. Many mutated repair genes have been implicated in certain forms of pancreatic cancer, colon cancer, and colorectal cancer. Mutations can affect either somatic cells or germ cells. If many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. If a mutation takes place in germ cells, the mutation will be passed on to the next generation, as in the case of hemophilia and xeroderma pigmentosa.
Summary
DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then a new base is added. Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base. In yet another type of repair, nucleotide excision repair, the incorrect base is removed along with a few bases on the 5' and 3' end, and these are replaced by copying the template with the help of DNA polymerase. The ends of the newly synthesized fragment are attached to the rest of the DNA using DNA ligase, which creates a phosphodiester bond.
Most mistakes are corrected, and if they are not, they may result in a mutation defined as a permanent change in the DNA sequence. Mutations can be of many types, such as substitution, deletion, insertion, and translocation. Mutations in repair genes may lead to serious consequences such as cancer. Mutations can be induced or may occur spontaneously.
Glossary
induced mutation
mutation that results from exposure to chemicals or environmental agents
mutation
variation in the nucleotide sequence of a genome
mismatch repair
type of repair mechanism in which mismatched bases are removed after replication
nucleotide excision repair
type of DNA repair mechanism in which the wrong base, along with a few nucleotides upstream or downstream, are removed
proofreading
function of DNA pol in which it reads the newly added base before adding the next one
point mutation
mutation that affects a single base
silent mutation
mutation that is not expressed
spontaneous mutation
mutation that takes place in the cells as a result of chemical reactions taking place naturally without exposure to any external agent
transition substitution
when a purine is replaced with a purine or a pyrimidine is replaced with another pyrimidine
transversion substitution
when a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/14%3A_DNA-_The_Genetic_Material/14.06%3A_DNA_Repair.txt |
Beadle and Tatum: one gene, one enzyme hypothesis
Life depends on (bio)chemistry to supply energy and to produce the molecules to construct and regulate cells. In 1908, A. Garrod described “in born errors of metabolism” in humans using the congenital disorder, alkaptonuria (black urine disease), as an example of how “genetic defects” led to the lack of an enzyme in a biochemical pathway and caused a disease (phenotype). Over 40 years later, in 1941, Beadle and Tatum built on this connection between genes and metabolic pathways. Their research led to the “one gene, one enzyme (or protein)” hypothesis, which states that each of the enzymes that act in a biochemical pathway is encoded by a different gene. Although we now know of many exceptions to the “one gene, one enzyme (or protein)” principle, it is generally true that each different gene produces a protein that has a distinct catalytic, regulatory, or structural function.
Beadle and Tatum used the fungus Neurospora crassa (a mold) for their studies because it had practical advantages as a laboratory organism. They knew that Neurospora was prototrophic, meaning that it could synthesize its own amino acids when grown on minimal medium, which lacked most nutrients except for a few minerals, simple sugars, and one vitamin (biotin). They also knew that by exposing Neurospora spores to X-rays, they could randomly damage its DNA to create mutations in genes. Each different spore exposed to X-rays potentially contained a mutation in a different gene. After genetically screening many, many spores for growth, most appeared to still be prototrophic and still able to grow on minimal medium. However, some spores had mutations that changed them into auxotrophic strains that could no longer grow on minimal medium, but did grow on complete medium supplemented with nutrients (Figure \(12\)). In fact, some auxotrophic mutations could grow on minimal medium with only one, single nutrient supplied, such as arginine.
B&T’s 1 gene: 1 enzyme hypothesis led to Biochemical Pathway dissection using genetic screens and mutations
Beadle and Tatum’s experiments are important not only for its conceptual advances in understanding genes, but also because they demonstrate the utility of screening for genetic mutants to investigate a biological process – genetic analysis. Beadle and Tatum’s results were useful to investigate biological processes, specifically the metabolic pathways that produce amino acids. For example, Srb and Horowitz in 1944 tested the ability of the amino acids to rescue auxotrophic strains. They added one of each of the amino acids to minimal medium and recorded which of these restored growth to independent mutants. For example, if the progeny of a mutagenized spore could grow on minimal medium only when it was supplemented with arginine (Arg), then the auxotroph must bear a mutation in the Arg biosynthetic pathway and was called an “arginineless” strain (arg-).
Synthesis of even a relatively simple molecule such as arginine requires many steps, each with a different enzyme. Each enzyme works sequentially on a different intermediate in the pathway (Figure \(13\)). For arginine (Arg), two of the intermediates are ornithine (Orn) and citrulline (Cit). Thus, mutation of any one of the enzymes in this pathway could turn Neurospora into an Arg auxotroph (arg-). Srb and Horowitz extended their analysis of Arg auxotrophs by testing the intermediates of amino acid biosynthesis for the ability to restore growth of the mutants (Figure \(14\)).
They found that some of the Arg auxotrophs could be rescued only by Arg, while others could be rescued by either Arg or Cit, and still other mutants could be rescued by Arg, Cit, or Orn (Table \(1\)). Based on these results, they deduced the location of each mutation in the Arg biochemical pathway, (i.e. which gene was responsible for the metabolism of which intermediate).
Table \(1\): Ability of auxotrophic mutants of each of the three enzymes of the Arg biosynthetic pathways to grow on minimal medium (MM) supplemented with Arg or either of its precursors, Orn and Cit. Gene names refer to the labels used in Figure \(14\)
MM + Orn
MM + Cit
MM + Arg
gene A mutants
Yes
Yes
Yes
gene B mutants
No
Yes
Yes
gene C mutants
No
No
Yes | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.01%3A_The_Nature_of_Genes/15.1.01%3A_The_Function_of_Genes.txt |
A few years after he and James Watson had proposed the double helical structure for DNA, Francis Crick (with other collaborators) proposed that a less stable nucleic acid, RNA, served as a messenger RNA that provided a transient copy of the genetic material that could be translated into the protein product encoded by the gene. Such mRNAs were indeed found. These and other studies led Francis Crick to formulate this “central dogma” of molecular biology (Figure 1.21).
This model states that DNA serves as the repository of genetic information. It can be replicated accurately and indefinitely. The genetic information is expressed by the DNA first serving as a template for the synthesis of (messenger) RNA; this occurs in a process called transcription. The mRNA then serves as a template, which is read by ribosomes and translatedinto protein. The protein products can be enzymes that catalyze the many metabolic transformations in the cell, or they can be structural proteins.
Although there have been some additional steps added since its formulation, the central dogma has stood the test of time and myriad experiments. It provides a strong unifying theme to molecular genetics and information flow in cell biology and biochemistry.
Although in many cases a gene encodes one polypeptide, other genes encode a functional RNA. Some genes encode tRNAs and rRNAs needed for translation, others encode other structural and catalytic RNAs. Genes encode some product that is used in the cell, i.e. that when altered generates an identifiable phenotype. More generally, genes encode RNAs, some of which are functional as transcribed (or with minor alterations via processing) such as tRNAs and rRNAs, and others are messengers that are then translated into proteins. These proteins can provide structural, catalytic and regulatory roles in the cell.
Note the static role of DNA in this process. Implicit in this model is the idea that DNA does not provide an active cellular function, but rather it encodes macromolecules that are functional. However, the expression of virtually all genes is highly regulated. The sites on the DNA where this control is exerted are indeed functional entities, such as promoters and enhancers. In this case, the DNA is directly functional (cis‑regulatory sites), but the genes being regulated by these sites still encode some functional product (RNA or protein).
Studies of retroviruses lead Dulbecco to argue that the flow of information is not unidirectional, but in fact RNA can be converted into DNA (some viral RNA genomes are converted into DNA proviruses integrated into the genome). Subsequently Temin and Baltimore discovered the enzyme that can make a DNA copy of RNA, i.e. reverse transcriptase.
15.1.03: Transcription and mRNA structure
Several aspects of the structure of genes can be illustrated by examining the general features of a bacterial gene as now understood.
A gene is a string of nucleotides in the duplex DNA that encodes a mRNA, which itself codes for protein. Only one strand of the duplex DNA is copied into mRNA (Figure 1.22). Sometimes genes overlap, and in some of those cases each strand of DNA is copied, but each for a different mRNA. The strand of DNA that reads the same as the sequence of mRNA is the nontemplate strand. The strand that reads as the reverse complement of the mRNA is the template strand.
Note
The term "sense strand" has two opposite uses (unfortunately). Sidney Brenner first used it to designate the strand that served as the template to make RNA (bottom strand above), and this is still used in many genetics texts. However, now many authors use the term to refer to the strand that reads the same as the mRNA (top strand above). The same confusion applies to the term "coding strand" which can refer to the strand encoding mRNA (bottom strand) or the strand "encoding" the protein (top strand). Interestingly, "antisense" is used exclusively to refer to the strand that is the reverse complement of the mRNA (bottom strand).
Figure 1.22 helps illustrate the origin of terms used in gene expression. Copying the information of DNA into RNA stays in the same "language" in that both of these polymers are nucleic acids, hence the process is called transcription. An analogy would be writing exercises where you had to copy, e.g. a poem, from a book onto your paper - you transcribed the poem, but it is still in English. Converting the information from RNA into DNA is equivalent to converting from one "language" to another, in this case from one type of polymer (the nucleic acid RNA) to a different one (a polypeptide or protein). Hence the process is called translation. This is analogous to translating a poem written in French into English.
Figure 1.23 illustrates the point that a gene may be longer than the region coding for the protein because of 5' and/or 3' untranslated regions.
Eukaryotic mRNAs have covalent attachment of nucleotides at the 5' and 3' ends, and in some cases nucleotides are added internally (a process called RNA editing). Recent work shows that additional nucleotides are added post‑transcriptionally to some bacterial mRNAs as well.
Regulatory signals can be considered parts of genes
In order to express a gene at the correct time, the DNA also carries signals to start transcription (e.g. promoters), signals for regulating the efficiency of starting transcription (e.g. operators, enhancers or silencers), and signals to stop transcription (e.g. terminators). Minimally, a gene includes the transcription unit, which is the segment of DNA that is copied into RNA in the primary transcript. The signals directing RNA polymerase to start at the correct site, and other DNA segments that influence the efficiency of this process are regulatory elements for the gene. One can also consider them to be part of the gene, along with the transcription unit. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.01%3A_The_Nature_of_Genes/15.1.02%3A_Central_Dogma-_DNA_to_RNA_to_protein.txt |
• 15.1: The Nature of Genes
• 15.2: The Genetic Code
The cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon.
• 15.3: Prokaryotic Transcription
The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. Prokaryotes often have abundant plasmids that are shorter circular DNA molecules that may only contain one or a few genes.
• 15.4: Eukaryotic Transcription
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated.
• 15.5: Eukaryotic pre-mRNA Splicing
After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated. Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein synthesis machinery.
• 15.6: The Structure of tRNA and Ribosomes
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other component of living organisms (other than water), and proteins perform virtually every function of a cell. The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product. Amino acids are covalently bonded by interlinking peptide bonds in lengths ranging from ~50 amino acid residues to >1,000.
• 15.7: The Process of Translation
The synthesis of proteins is one of a cell’s most energy-consuming metabolic processes. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform a wide variety of the functions of a cell. The process of translation, or protein synthesis, involves decoding an mRNA message into a polypeptide product. Amino acids are covalently strung together in lengths ranging from approximately 50 amino acids to more than 1,000.
• 15.8: Summarizing Gene Expression
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the
• 15.9: Mutation- Altered Genes
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.
15: Genes and How They Work
Skills to Develop
• Explain the “central dogma” of protein synthesis
• Describe the genetic code and how the nucleotide sequence prescribes the amino acid and the protein sequence
The cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters (Figure $1$). Each amino acid is defined by a three-nucleotide sequence called the triplet codon. Different amino acids have different chemistries (such as acidic versus basic, or polar and nonpolar) and different structural constraints. Variation in amino acid sequence gives rise to enormous variation in protein structure and function.
The Central Dogma: DNA Encodes RNA; RNA Encodes Protein
The flow of genetic information in cells from DNA to mRNA to protein is described by the Central Dogma (Figure $2$), which states that genes specify the sequence of mRNAs, which in turn specify the sequence of proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected. The copying of DNA to RNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand. The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear, such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.
The Genetic Code Is Degenerate and Universal
Given the different numbers of “letters” in the mRNA and protein “alphabets,” scientists theorized that combinations of nucleotides corresponded to single amino acids. Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (42). In contrast, there are 64 possible nucleotide triplets (43), which is far more than the number of amino acids. Scientists theorized that amino acids were encoded by nucleotide triplets and that the genetic code was degenerate. In other words, a given amino acid could be encoded by more than one nucleotide triplet. This was later confirmed experimentally; Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, protein synthesis was completely abolished. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that three nucleotides specify each amino acid. These nucleotide triplets are called codons. The insertion of one or two nucleotides completely changed the triplet reading frame, thereby altering the message for every subsequent amino acid (Figure $4$). Though insertion of three nucleotides caused an extra amino acid to be inserted during translation, the integrity of the rest of the protein was maintained.
Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure $3$).
In addition to instructing the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons, or stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA.
The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 1084 possible combinations of 20 amino acids and 64 triplet codons.
Link to Learning
Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.
Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might either specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.
Scientific Method Connection: Which Has More DNA: A Kiwi or a Strawberry?
Question: Would a kiwifruit and strawberry that are approximately the same size (Figure $5$) also have approximately the same amount of DNA?
Background: Genes are carried on chromosomes and are made of DNA. All mammals are diploid, meaning they have two copies of each chromosome. However, not all plants are diploid. The common strawberry is octoploid (8n) and the cultivated kiwi is hexaploid (6n). Research the total number of chromosomes in the cells of each of these fruits and think about how this might correspond to the amount of DNA in these fruits’ cell nuclei. Read about the technique of DNA isolation to understand how each step in the isolation protocol helps liberate and precipitate DNA.
Hypothesis: Hypothesize whether you would be able to detect a difference in DNA quantity from similarly sized strawberries and kiwis. Which fruit do you think would yield more DNA?
Test your hypothesis: Isolate the DNA from a strawberry and a kiwi that are similarly sized. Perform the experiment in at least triplicate for each fruit.
1. Prepare a bottle of DNA extraction buffer from 900 mL water, 50 mL dish detergent, and two teaspoons of table salt. Mix by inversion (cap it and turn it upside down a few times).
2. Grind a strawberry and a kiwifruit by hand in a plastic bag, or using a mortar and pestle, or with a metal bowl and the end of a blunt instrument. Grind for at least two minutes per fruit.
3. Add 10 mL of the DNA extraction buffer to each fruit, and mix well for at least one minute.
4. Remove cellular debris by filtering each fruit mixture through cheesecloth or porous cloth and into a funnel placed in a test tube or an appropriate container.
5. Pour ice-cold ethanol or isopropanol (rubbing alcohol) into the test tube. You should observe white, precipitated DNA.
6. Gather the DNA from each fruit by winding it around separate glass rods.
Record your observations: Because you are not quantitatively measuring DNA volume, you can record for each trial whether the two fruits produced the same or different amounts of DNA as observed by eye. If one or the other fruit produced noticeably more DNA, record this as well. Determine whether your observations are consistent with several pieces of each fruit.
Analyze your data: Did you notice an obvious difference in the amount of DNA produced by each fruit? Were your results reproducible?
Draw a conclusion: Given what you know about the number of chromosomes in each fruit, can you conclude that chromosome number necessarily correlates to DNA amount? Can you identify any drawbacks to this procedure? If you had access to a laboratory, how could you standardize your comparison and make it more quantitative?
Summary
The genetic code refers to the DNA alphabet (A, T, C, G), the RNA alphabet (A, U, C, G), and the polypeptide alphabet (20 amino acids). The Central Dogma describes the flow of genetic information in the cell from genes to mRNA to proteins. Genes are used to make mRNA by the process of transcription; mRNA is used to synthesize proteins by the process of translation. The genetic code is degenerate because 64 triplet codons in mRNA specify only 20 amino acids and three nonsense codons. Almost every species on the planet uses the same genetic code.
Glossary
Central Dogma
states that genes specify the sequence of mRNAs, which in turn specify the sequence of proteins
codon
three consecutive nucleotides in mRNA that specify the insertion of an amino acid or the release of a polypeptide chain during translation
colinear
in terms of RNA and protein, three “units” of RNA (nucleotides) specify one “unit” of protein (amino acid) in a consecutive fashion
degeneracy
(of the genetic code) describes that a given amino acid can be encoded by more than one nucleotide triplet; the code is degenerate, but not ambiguous
nonsense codon
one of the three mRNA codons that specifies termination of translation
reading frame
sequence of triplet codons in mRNA that specify a particular protein; a ribosome shift of one or two nucleotides in either direction completely abolishes synthesis of that protein | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.02%3A_The_Genetic_Code.txt |
Skills to Develop
• List the different steps in prokaryotic transcription
• Discuss the role of promoters in prokaryotic transcription
• Describe how and when transcription is terminated
The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids, which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance.
Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially unwind in the region of mRNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The mRNA product is complementary to the template strand and is almost identical to the other DNA strand, called the nontemplate strand. The only difference is that in mRNA, all of the T nucleotides are replaced with U nucleotides. In an RNA double helix, A can bind U via two hydrogen bonds, just as in A–T pairing in a DNA double helix.
The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' mRNA nucleotide is transcribed is called the +1 site, or the initiation site. Nucleotides preceding the initiation site are given negative numbers and are designated upstream. Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called downstream nucleotides.
Initiation of Transcription in Prokaryotes
Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein.
Our discussion here will exemplify transcription by describing this process in Escherichia coli, a well-studied bacterial species. Although some differences exist between transcription in E. coli and transcription in archaea, an understanding of E. coli transcription can be applied to virtually all bacterial species.
Prokaryotic RNA Polymerase
Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β' comprise the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two α-subunits are necessary to assemble the polymerase on the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent “recently born” mRNA molecule; and the β' binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme.
Prokaryotic Promoters
A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species (Figure \(1\)). The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template, and several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released.
Link to Learning
View this MolecularMovies animation to see the first part of transcription and the base sequence repetition of the TATA box.
Elongation and Termination in Prokaryotes
The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5' to 3' direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it (Figure \(2\)). The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.
Prokaryotic Termination Signals
Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.
Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides. The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.
Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently. The unification of transcription, translation, and even mRNA degradation is possible because all of these processes occur in the same 5' to 3' direction, and because there is no membranous compartmentalization in the prokaryotic cell (Figure \(3\)). In contrast, the presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation.
Link to Learning
Visit this BioStudio animation to see the process of prokaryotic transcription.
Summary
In prokaryotes, mRNA synthesis is initiated at a promoter sequence on the DNA template comprising two consensus sequences that recruit RNA polymerase. The prokaryotic polymerase consists of a core enzyme of four protein subunits and a σ protein that assists only with initiation. Elongation synthesizes mRNA in the 5' to 3' direction at a rate of 40 nucleotides per second. Termination liberates the mRNA and occurs either by rho protein interaction or by the formation of an mRNA hairpin.
Glossary
consensus
DNA sequence that is used by many species to perform the same or similar functions
core enzyme
prokaryotic RNA polymerase consisting of α, α, β, and β' but missing σ; this complex performs elongation
downstream
nucleotides following the initiation site in the direction of mRNA transcription; in general, sequences that are toward the 3' end relative to a site on the mRNA
hairpin
structure of RNA when it folds back on itself and forms intramolecular hydrogen bonds between complementary nucleotides
holoenzyme
prokaryotic RNA polymerase consisting of α, α, β, β', and σ; this complex is responsible for transcription initiation
initiation site
nucleotide from which mRNA synthesis proceeds in the 5' to 3' direction; denoted with a “+1”
nontemplate strand
strand of DNA that is not used to transcribe mRNA; this strand is identical to the mRNA except that T nucleotides in the DNA are replaced by U nucleotides in the mRNA
plasmid
extrachromosomal, covalently closed, circular DNA molecule that may only contain one or a few genes; common in prokaryotes
promoter
DNA sequence to which RNA polymerase and associated factors bind and initiate transcription
Rho-dependent termination
in prokaryotes, termination of transcription by an interaction between RNA polymerase and the rho protein at a run of G nucleotides on the DNA template
Rho-independent
termination sequence-dependent termination of prokaryotic mRNA synthesis; caused by hairpin formation in the mRNA that stalls the polymerase
TATA box
conserved promoter sequence in eukaryotes and prokaryotes that helps to establish the initiation site for transcription
template strand
strand of DNA that specifies the complementary mRNA molecule
transcription bubble
region of locally unwound DNA that allows for transcription of mRNA
upstream
nucleotides preceding the initiation site; in general, sequences toward the 5' end relative to a site on the mRNA | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.03%3A_Prokaryotic_Transcription.txt |
Skills to Develop
• List the steps in eukaryotic transcription
• Discuss the role of RNA polymerases in transcription
• Compare and contrast the three RNA polymerases
• Explain the significance of transcription factors
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes. Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein.
Initiation of Transcription in Eukaryotes
Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase.
The Three Eukaryotic RNA Polymerases
The features of eukaryotic mRNA synthesis are markedly more complex those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more. Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template.
RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes (Table \(1\)). The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation. RNA polymerase I synthesizes all of the rRNAs except for the 5S rRNA molecule. The “S” designation applies to “Svedberg” units, a nonadditive value that characterizes the speed at which a particle sediments during centrifugation.
Table \(1\): Locations, Products, and Sensitivities of the Three Eukaryotic RNA Polymerases
RNA Polymerase Cellular Compartment Product of Transcription α-Amanitin Sensitivity
I Nucleolus All rRNAs except 5S rRNA Insensitive
II Nucleus All protein-coding nuclear pre-mRNAs Extremely sensitive
III Nucleus 5S rRNA, tRNAs, and small nuclear RNAs Moderately sensitive
RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. For clarity, this module’s discussion of transcription and translation in eukaryotes will use the term “mRNAs” to describe only the mature, processed molecules that are ready to be translated. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.
RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation; they serve as the adaptor molecules between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.
A scientist characterizing a new gene can determine which polymerase transcribes it by testing whether the gene is expressed in the presence of a particular mushroom poison, α-amanitin (table above). Interestingly, α-amanitin produced by Amanita phalloides, the Death Cap mushroom, affects the three polymerases very differently. RNA polymerase I is completely insensitive to α-amanitin, meaning that the polymerase can transcribe DNA in vitro in the presence of this poison. In contrast, RNA polymerase II is extremely sensitive to α-amanitin, and RNA polymerase III is moderately sensitive. Knowing the transcribing polymerase can clue a researcher into the general function of the gene being studied. Because RNA polymerase II transcribes the vast majority of genes, we will focus on this polymerase in our subsequent discussions about eukaryotic transcription factors and promoters.
Structure of an RNA Polymerase II Promoter
Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but both have a TATA box. For example, in the mouse thymidine kinase gene, the TATA box is located at approximately -30 relative to the initiation (+1) site (Figure \(1\)). For this gene, the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on the nontemplate strand. This sequence is not identical to the E. coli TATA box, but it conserves the A–T rich element. The thermostability of A–T bonds is low and this helps the DNA template to locally unwind in preparation for transcription.
Art Connection
A scientist splices a eukaryotic promoter in front of a bacterial gene and inserts the gene in a bacterial chromosome. Would you expect the bacteria to transcribe the gene?
The mouse genome includes one gene and two pseudogenes for cytoplasmic thymidine kinase. Pseudogenes are genes that have lost their protein-coding ability or are no longer expressed by the cell. These pseudogenes are copied from mRNA and incorporated into the chromosome. For example, the mouse thymidine kinase promoter also has a conserved CAAT box (GGCCAATCT) at approximately -80. This sequence is essential and is involved in binding transcription factors. Further upstream of the TATA box, eukaryotic promoters may also contain one or more GC-rich boxes (GGCG) or octamer boxes (ATTTGCAT). These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more “active” genes that are constantly being expressed by the cell.
Transcription Factors for RNA Polymerase II
The complexity of eukaryotic transcription does not end with the polymerases and promoters. An army of basal transcription factors, enhancers, and silencers also help to regulate the frequency with which pre-mRNA is synthesized from a gene. Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed. Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation.
The names of the basal transcription factors begin with “TFII” (this is the transcription factor for RNA polymerase II) and are specified with the letters A–J. The transcription factors systematically fall into place on the DNA template, with each one further stabilizing the preinitiation complex and contributing to the recruitment of RNA polymerase II.
The processes of bringing RNA polymerases I and III to the DNA template involve slightly less complex collections of transcription factors, but the general theme is the same. Eukaryotic transcription is a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand. Although the process of transcription in eukaryotes involves a greater metabolic investment than in prokaryotes, it ensures that the cell transcribes precisely the pre-mRNAs that it needs for protein synthesis.
Evolution Connection: The Evolution of Promoters
The evolution of genes may be a familiar concept. Mutations can occur in genes during DNA replication, and the result may or may not be beneficial to the cell. By altering an enzyme, structural protein, or some other factor, the process of mutation can transform functions or physical features. However, eukaryotic promoters and other gene regulatory sequences may evolve as well. For instance, consider a gene that, over many generations, becomes more valuable to the cell. Maybe the gene encodes a structural protein that the cell needs to synthesize in abundance for a certain function. If this is the case, it would be beneficial to the cell for that gene’s promoter to recruit transcription factors more efficiently and increase gene expression.
Scientists examining the evolution of promoter sequences have reported varying results. In part, this is because it is difficult to infer exactly where a eukaryotic promoter begins and ends. Some promoters occur within genes; others are located very far upstream, or even downstream, of the genes they are regulating. However, when researchers limited their examination to human core promoter sequences that were defined experimentally as sequences that bind the preinitiation complex, they found that promoters evolve even faster than protein-coding genes.
It is still unclear how promoter evolution might correspond to the evolution of humans or other higher organisms. However, the evolution of a promoter to effectively make more or less of a given gene product is an intriguing alternative to the evolution of the genes themselves.1
Promoter Structures for RNA Polymerases I and III
In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves.
Eukaryotic Elongation and Termination
Following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with the polymerase synthesizing pre-mRNA in the 5' to 3' direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus on how this polymerase accomplishes elongation and termination.
Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool.
For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein complex called FACT, which stands for “facilitates chromatin transcription.” This complex pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.
The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.
Summary
Transcription in eukaryotes involves one of three types of polymerases, depending on the gene being transcribed. RNA polymerase II transcribes all of the protein-coding genes, whereas RNA polymerase I transcribes rRNA genes, and RNA polymerase III transcribes rRNA, tRNA, and small nuclear RNA genes. The initiation of transcription in eukaryotes involves the binding of several transcription factors to complex promoter sequences that are usually located upstream of the gene being copied. The mRNA is synthesized in the 5' to 3' direction, and the FACT complex moves and reassembles nucleosomes as the polymerase passes by. Whereas RNA polymerases I and III terminate transcription by protein- or RNA hairpin-dependent methods, RNA polymerase II transcribes for 1,000 or more nucleotides beyond the gene template and cleaves the excess during pre-mRNA processing.
Art Connections
Figure \(2\): A scientist splices a eukaryotic promoter in front of a bacterial gene and inserts the gene in a bacterial chromosome. Would you expect the bacteria to transcribe the gene?
Answer
No. Prokaryotes use different promoters than eukaryotes.
Footnotes
1. 1 H Liang et al., “Fast evolution of core promoters in primate genomes,” Molecular Biology and Evolution 25 (2008): 1239–44.
Glossary
CAAT box
(GGCCAATCT) essential eukaryotic promoter sequence involved in binding transcription factors
FACT
complex that “facilitates chromatin transcription” by disassembling nucleosomes ahead of a transcribing RNA polymerase II and reassembling them after the polymerase passes by
GC-rich box
(GGCG) nonessential eukaryotic promoter sequence that binds cellular factors to increase the efficiency of transcription; may be present several times in a promoter
Octamer box
(ATTTGCAT) nonessential eukaryotic promoter sequence that binds cellular factors to increase the efficiency of transcription; may be present several times in a promoter
preinitiation complex
cluster of transcription factors and other proteins that recruit RNA polymerase II for transcription of a DNA template
small nuclear RNA
molecules synthesized by RNA polymerase III that have a variety of functions, including splicing pre-mRNAs and regulating transcription factors | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.04%3A_Eukaryotic_Transcription.txt |
Skills to Develop
• Describe the different steps in RNA processing
• Understand the significance of exons, introns, and splicing
• Explain how tRNAs and rRNAs are processed
After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated. Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein synthesis machinery.
mRNA Processing
The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.
Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5' and 3' ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.
Evolution Connection: RNA Editing in Trypanosomes
The trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes sleeping sickness in humans (Figure \(1\)). Trypanosomes, and virtually all other eukaryotes, have organelles called mitochondria that supply the cell with chemical energy. Mitochondria are organelles that express their own DNA and are believed to be the remnants of a symbiotic relationship between a eukaryote and an engulfed prokaryote. The mitochondrial DNA of trypanosomes exhibit an interesting exception to The Central Dogma: their pre-mRNAs do not have the correct information to specify a functional protein. Usually, this is because the mRNA is missing several U nucleotides. The cell performs an additional RNA processing step called RNA editing to remedy this.
Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs. One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript. However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides to bind with. In these regions, the guide RNA loops out. The 3' ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped. This process is entirely mediated by RNA molecules. That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing.
RNA editing is not just a phenomenon of trypanosomes. In the mitochondria of some plants, almost all pre-mRNAs are edited. RNA editing has also been identified in mammals such as rats, rabbits, and even humans. What could be the evolutionary reason for this additional step in pre-mRNA processing? One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNA-based method for regulating gene expression. In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions. Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions.
5' Capping
While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5' end of the growing transcript by a phosphate linkage. This moiety (functional group) protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.
3' Poly-A Tail
Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm.
Pre-mRNA Splicing
Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (int-ron denotes their intervening role), which may be involved in gene regulation but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.
The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. These regions may correspond to regulatory sequences; however, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.
All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing (Figure \(2\)). Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.
Art Connection
Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors? Think of different possible outcomes if splicing errors occur.
Note that more than 70 individual introns can be present, and each has to undergo the process of splicing—in addition to 5' capping and the addition of a poly-A tail—just to generate a single, translatable mRNA molecule.
Link to Learning
See how introns are removed during RNA splicing at this website.
Processing of tRNAs and rRNAs
The tRNAs and rRNAs are structural molecules that have roles in protein synthesis; however, these RNAs are not themselves translated. Pre-rRNAs are transcribed, processed, and assembled into ribosomes in the nucleolus. Pre-tRNAs are transcribed and processed in the nucleus and then released into the cytoplasm where they are linked to free amino acids for protein synthesis.
Most of the tRNAs and rRNAs in eukaryotes and prokaryotes are first transcribed as a long precursor molecule that spans multiple rRNAs or tRNAs. Enzymes then cleave the precursors into subunits corresponding to each structural RNA. Some of the bases of pre-rRNAs are methylated; that is, a –CH3 moiety (methyl functional group) is added for stability. Pre-tRNA molecules also undergo methylation. As with pre-mRNAs, subunit excision occurs in eukaryotic pre-RNAs destined to become tRNAs or rRNAs.
Mature rRNAs make up approximately 50 percent of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities. Mature tRNAs take on a three-dimensional structure through intramolecular hydrogen bonding to position the amino acid binding site at one end and the anticodon at the other end (Figure \(3\)). The anticodon is a three-nucleotide sequence in a tRNA that interacts with an mRNA codon through complementary base pairing.
Summary
Eukaryotic pre-mRNAs are modified with a 5' methylguanosine cap and a poly-A tail. These structures protect the mature mRNA from degradation and help export it from the nucleus. Pre-mRNAs also undergo splicing, in which introns are removed and exons are reconnected with single-nucleotide accuracy. Only finished mRNAs that have undergone 5' capping, 3' polyadenylation, and intron splicing are exported from the nucleus to the cytoplasm. Pre-rRNAs and pre-tRNAs may be processed by intramolecular cleavage, splicing, methylation, and chemical conversion of nucleotides. Rarely, RNA editing is also performed to insert missing bases after an mRNA has been synthesized.
Art Connections
Figure \(2\): Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors? Think of different possible outcomes if splicing errors occur.
Answer
Mutations in the spliceosome recognition sequence at each end of the intron, or in the proteins and RNAs that make up the spliceosome, may impair splicing. Mutations may also add new spliceosome recognition sites. Splicing errors could lead to introns being retained in spliced RNA, exons being excised, or changes in the location of the splice site.
Glossary
7-methylguanosine cap
modification added to the 5' end of pre-mRNAs to protect mRNA from degradation and assist translation
anticodon
three-nucleotide sequence in a tRNA molecule that corresponds to an mRNA codon
exon
sequence present in protein-coding mRNA after completion of pre-mRNA splicing
intron
non–protein-coding intervening sequences that are spliced from mRNA during processing
poly-A tail
modification added to the 3' end of pre-mRNAs to protect mRNA from degradation and assist mRNA export from the nucleus
RNA editing
direct alteration of one or more nucleotides in an mRNA that has already been synthesized
splicing
process of removing introns and reconnecting exons in a pre-mRNA | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.05%3A_Eukaryotic_pre-mRNA_Splicing.txt |
Skills to Develop
• Describe the different steps in protein synthesis
• Discuss the role of ribosomes in protein synthesis
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform virtually every function of a cell. The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product. Amino acids are covalently strung together by interlinking peptide bonds in lengths ranging from approximately 50 amino acid residues to more than 1,000. Each individual amino acid has an amino group (NH2) and a carboxyl (COOH) group. Polypeptides are formed when the amino group of one amino acid forms an amide (i.e., peptide) bond with the carboxyl group of another amino acid (Figure $1$). This reaction is catalyzed by ribosomes and generates one water molecule.
The Protein Synthesis Machinery
In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.
Link to Learning
Click through the steps of this PBS interactive to see protein synthesis in action.
Ribosomes
Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes. In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.
Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and rough endoplasmic reticulum in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.
tRNAs
The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C—three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of these 61, one codon (AUG) also encodes the initiation of translation. Each tRNA anticodon can base pair with one of the mRNA codons and add an amino acid or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a tRNA expressing the complementary sequence, GAU, which would be linked to the amino acid leucine.
As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. Consider that tRNAs need to interact with three factors: 1) they must be recognized by the correct aminoacyl synthetase (see below); 2) they must be recognized by ribosomes; and 3) they must bind to the correct sequence in mRNA.
Aminoacyl tRNA Synthetases
The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released.
The Mechanism of Protein Synthesis
As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.
Initiation of Translation
Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called $\text{tRNA}_\text{f}^\text{Met}$. The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by $\text{fMet} - \text{tRNA}_\text{f}^\text{Met}$ at the beginning of every polypeptide chain synthesized by E. coli, but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNAMet.
In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.
In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNAi, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.
Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.
Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.
Translation, Elongation, and Termination
In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one exception to this assembly line of tRNAs: in E. coli, $\text{fMet} - \text{tRNA}_\text{f}^\text{Met}$ is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNAi, with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.
Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled (Figure $2$). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.
Art Connection
Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?
Tetracycline would directly affect:
1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain
Chloramphenicol would directly affect
1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain
Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.
Protein Folding, Modification, and Targeting
During and after translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment. These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. For instance, a specific sequence at the amino terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.
Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.
Summary
The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit forms on the mRNA template either at the Shine-Dalgarno sequence (prokaryotes) or the 5' cap (eukaryotes). Translation begins at the initiating AUG on the mRNA, specifying methionine. The formation of peptide bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. Charged tRNAs enter the ribosomal A site, and their amino acid bonds with the amino acid at the P site. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a nonsense codon is encountered, a release factor binds and dissociates the components and frees the new protein. Folding of the protein occurs during and after translation.
Art Connections
Figure $2$: Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?
Tetracycline would directly affect:
1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain
Chloramphenicol would directly affect
1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain
Answer
Tetracycline: a; Chloramphenicol: c.
Glossary
aminoacyl tRNA synthetase
enzyme that “charges” tRNA molecules by catalyzing a bond between the tRNA and a corresponding amino acid
initiator tRNA
in prokaryotes, called $\text{tRNA}_\text{f}^\text{Met}$; in eukaryotes, called tRNAi; a tRNA that interacts with a start codon, binds directly to the ribosome P site, and links to a special methionine to begin a polypeptide chain
Kozak’s rules
determines the correct initiation AUG in a eukaryotic mRNA; the following consensus sequence must appear around the AUG: 5’-GCC(purine)CCAUGG-3’; the bolded bases are most important
peptidyl transferase
RNA-based enzyme that is integrated into the 50S ribosomal subunit and catalyzes the formation of peptide bonds
polysome
mRNA molecule simultaneously being translated by many ribosomes all going in the same direction
Shine-Dalgarno sequence
(AGGAGG); initiates prokaryotic translation by interacting with rRNA molecules comprising the 30S ribosome
signal sequence
short tail of amino acids that directs a protein to a specific cellular compartment
start codon
AUG (or rarely, GUG) on an mRNA from which translation begins; always specifies methionine | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.06%3A_The_Structure_of_tRNA_and_Ribosomes.txt |
The synthesis of proteins is one of a cell’s most energy-consuming metabolic processes. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform a wide variety of the functions of a cell. The process of translation, or protein synthesis, involves decoding an mRNA message into a polypeptide product. Amino acids are covalently strung together in lengths ranging from approximately 50 amino acids to more than 1,000.
The Protein Synthesis Machinery
In addition to the mRNA template, many other molecules contribute to the process of translation. The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNA) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors (Figure \(1\)).
In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.
Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction.
Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is bonded to its correct amino acid.
The Genetic Code
To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code.
Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations; therefore, a given amino acid is encoded by more than one nucleotide triplet (Figure \(2\)).
Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.
The Mechanism of Protein Synthesis
Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.
Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small ribosome subunit, the mRNA template, three initiation factors, and a special initiator tRNA. The initiator tRNA interacts with the AUG start codon, and links to a special form of the amino acid methionine that is typically removed from the polypeptide after translation is complete.
In prokaryotes and eukaryotes, the basics of polypeptide elongation are the same, so we will review elongation from the perspective of E. coli. The large ribosomal subunit of E. coli consists of three compartments: the A site binds incoming charged tRNAs (tRNAs with their attached specific amino acids). The P site binds charged tRNAs carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E site releases dissociated tRNAs so they can be recharged with free amino acids. The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP (Figure \(3\)). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.
Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released and the ribosome subunits dissociate and leave the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.
CONCEPT IN ACTION
Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.
Summary
The central dogma describes the flow of genetic information in the cell from genes to mRNA to proteins. Genes are used to make mRNA by the process of transcription; mRNA is used to synthesize proteins by the process of translation. The genetic code is the correspondence between the three-nucleotide mRNA codon and an amino acid. The genetic code is “translated” by the tRNA molecules, which associate a specific codon with a specific amino acid. The genetic code is degenerate because 64 triplet codons in mRNA specify only 20 amino acids and three stop codons. This means that more than one codon corresponds to an amino acid. Almost every species on the planet uses the same genetic code.
The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit binds to the mRNA template. Translation begins at the initiating AUG on the mRNA. The formation of bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. The ribosome accepts charged tRNAs, and as it steps along the mRNA, it catalyzes bonding between the new amino acid and the end of the growing polypeptide. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a stop codon is encountered, a release factor binds and dissociates the components and frees the new protein.
Glossary
codon
three consecutive nucleotides in mRNA that specify the addition of a specific amino acid or the release of a polypeptide chain during translation
genetic code
the amino acids that correspond to three-nucleotide codons of mRNA
rRNA
ribosomal RNA; molecules of RNA that combine to form part of the ribosome
stop codon
one of the three mRNA codons that specifies termination of translation
start codon
the AUG (or, rarely GUG) on an mRNA from which translation begins; always specifies methionine
tRNA
transfer RNA; an RNA molecule that contains a specific three-nucleotide anticodon sequence to pair with the mRNA codon and also binds to a specific amino acid
15.07: The Process of Translation
The synthesis of proteins occurs in the cytoplasm, where mature ribosomes are located. Generally, if no information is added, a newly synthesized polypeptide will remain in the cytoplasm. Yet even in the structurally simplest of cells, those of the bacteria and archaea, there is more than one place that a protein may need to be to function correctly: it can remain in the cytoplasm, it can be inserted into the plasma membrane or it may be secreted from the cell. Both membrane and secreted polypeptides must be inserted into, or pass through, the plasma membrane.
Polypeptides destined for the membrane or for secretion are generally marked by a specific tag, known as a signal sequence. The signal sequence consists of a stretch of hydrophobic amino acids, often located at the N-terminus of the polypeptide. As the signal sequence emerges from the ribosomal tunnel it interacts with a signal recognition particle (SRP) - a complex of polypeptides and a structural RNA. The binding of SRP to the signal sequence causes translation to pause. SRP acts as a chaperone for a subset of membrane proteins. The nascent mRNA/ribosome/nascent polypeptide/SRP complex will find (by diffusion), and attach to, a ribosome/SRP receptor complex on the cytoplasmic surface of the plasma membrane (in bacteria and archaea) or a cytoplasmic facing membrane (in eukaryotes). This ribosome/SRP receptor is associated with a polypeptide pore. When the ribosome/SRP complex docks with the receptor, translation resumes and the nascent polypeptide passes through the protein pore and so enters into or passes through the membrane. As the polypeptide emerges on the external, non-cytoplasmic face of the membrane, the signal sequence is generally removed by an enzyme, signal sequence peptidase. If the polypeptide is a membrane protein, it will fold and remain within the membrane. If it is a secreted polypeptide, it will be released into the periplasmic space, that is the region topologically outside of the cytoplasm (either within a vesicle or other side of the plasma membrane. Other mechanisms can lead to the release of the protein from the cell.
Because eukaryotic cells are structurally and topologically more complex than bacterial and archaeal cells there are more places for a newly synthesized protein to end up. While we will not discuss the details of those processes, one rule of thumb is worth keeping in mind. Generally, in the absence of added information, a newly synthesized polypeptide will end up in the cytoplasm. As in bacteria and archaea, a eukaryotic polypeptides destined for secretion or insertion into the cell’s plasma membrane or internal membrane systems (that is the endoplasmic reticulum and Golgi apparatus) are directed to their final location by a signal sequence/SRP system. Proteins that must function in the nucleus generally get there because they have a nuclear localization sequence, other proteins are actively excluded from the nucleus using a nuclear exclusion sequence (see above). Likewise, other localization signals and receptors are used to direct proteins to other intracellular compartments, including mitochondria and chloroplasts. While details of these targeting systems are beyond the scope of this course, you can assume that each specific targeting event requires signals, receptors, and various mechanisms that drive what are often thermodynamically unfavorable reactions.
Contributors and Attributions
• Michael W. Klymkowsky (University of Colorado Boulder) and Melanie M. Cooper (Michigan State University) with significant contributions by Emina Begovic & some editorial assistance of Rebecca Klymkowsky. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.07%3A_The_Process_of_Translation/15.7.01%3A_Regulating_protein_localization.txt |
Skills to Develop
• Discuss why every cell does not express all of its genes
• Describe how prokaryotic gene regulation occurs at the transcriptional level
• Discuss how eukaryotic gene regulation occurs at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels
For a cell to function properly, necessary proteins must be synthesized at the proper time. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.
Prokaryotic versus Eukaryotic Gene Expression
To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners.
Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesize a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription. All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.
Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA. The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process (Figure \(1\)). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when the RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level).
The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized below. The regulation of gene expression is discussed in detail in subsequent modules.
Table \(1\): Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms
Prokaryotic organisms Eukaryotic organisms
Lack nucleus Contain nucleus
DNA is found in the cytoplasm DNA is confined to the nuclear compartment
RNA transcription and protein formation occur almost simultaneously RNA transcription occurs prior to protein formation, and it takes place in the nucleus. Translation of RNA to protein occurs in the cytoplasm.
Gene expression is regulated primarily at the transcriptional level Gene expression is regulated at many levels (epigenetic, transcriptional, nuclear shuttling, post-transcriptional, translational, and post-translational)
Evolution Connection: Evolution of Gene Regulation
Prokaryotic cells can only regulate gene expression by controlling the amount of transcription. As eukaryotic cells evolved, the complexity of the control of gene expression increased. For example, with the evolution of eukaryotic cells came compartmentalization of important cellular components and cellular processes. A nuclear region that contains the DNA was formed. Transcription and translation were physically separated into two different cellular compartments. It therefore became possible to control gene expression by regulating transcription in the nucleus, and also by controlling the RNA levels and protein translation present outside the nucleus.
Some cellular processes arose from the need of the organism to defend itself. Cellular processes such as gene silencing developed to protect the cell from viral or parasitic infections. If the cell could quickly shut off gene expression for a short period of time, it would be able to survive an infection when other organisms could not. Therefore, the organism evolved a new process that helped it survive, and it was able to pass this new development to offspring.
Summary
While all somatic cells within an organism contain the same DNA, not all cells within that organism express the same proteins. Prokaryotic organisms express the entire DNA they encode in every cell, but not necessarily all at the same time. Proteins are expressed only when they are needed. Eukaryotic organisms express a subset of the DNA that is encoded in any given cell. In each cell type, the type and amount of protein is regulated by controlling gene expression. To express a protein, the DNA is first transcribed into RNA, which is then translated into proteins. In prokaryotic cells, these processes occur almost simultaneously. In eukaryotic cells, transcription occurs in the nucleus and is separate from the translation that occurs in the cytoplasm. Gene expression in prokaryotes is regulated only at the transcriptional level, whereas in eukaryotic cells, gene expression is regulated at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels.
Glossary
epigenetic
heritable changes that do not involve changes in the DNA sequence
gene expression
processes that control the turning on or turning off of a gene
post-transcriptional
control of gene expression after the RNA molecule has been created but before it is translated into protein
post-translational
control of gene expression after a protein has been created | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.08%3A_Summarizing_Gene_Expression.txt |
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/Map%3A_Raven_Biology_12th_Edition/15%3A_Genes_and_How_They_Work/15.09%3A_Mutation-_Altered_Genes.txt |
For a cell to function properly, necessary proteins must be synthesized at the proper time. All organisms and cells control or regulate the transcription and translation of their DNA into protein. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or in a complex multicellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.
Cells in multicellular organisms are specialized; cells in different tissues look very different and perform different functions. For example, a muscle cell is very different from a liver cell, which is very different from a skin cell. These differences are a consequence of the expression of different sets of genes in each of these cells. All cells have certain basic functions they must perform for themselves, such as converting the energy in sugar molecules into energy in ATP. Each cell also has many genes that are not expressed, and expresses many that are not expressed by other cells, such that it can carry out its specialized functions. In addition, cells will turn on or off certain genes at different times in response to changes in the environment or at different times during the development of the organism. Unicellular organisms, both eukaryotic and prokaryotic, also turn on and off genes in response to the demands of their environment so that they can respond to special conditions.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.
Prokaryotic versus Eukaryotic Gene Expression
To understand how gene expression is regulated, we must first understand how a gene becomes a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different fashions.
Because prokaryotic organisms lack a cell nucleus, the processes of transcription and translation occur almost simultaneously. When the protein is no longer needed, transcription stops. As a result, the primary method to control what type and how much protein is expressed in a prokaryotic cell is through the regulation of DNA transcription into RNA. All the subsequent steps happen automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is almost entirely at the transcriptional level.
The first example of such control was discovered using E. coli in the 1950s and 1960s by French researchers and is called the lac operon. The lac operon is a stretch of DNA with three adjacent genes that code for proteins that participate in the absorption and metabolism of lactose, a food source for E. coli. When lactose is not present in the bacterium’s environment, the lac genes are transcribed in small amounts. When lactose is present, the genes are transcribed and the bacterium is able to use the lactose as a food source. The operon also contains a promoter sequence to which the RNA polymerase binds to begin transcription; between the promoter and the three genes is a region called the operator. When there is no lactose present, a protein known as a repressor binds to the operator and prevents RNA polymerase from binding to the promoter, except in rare cases. Thus very little of the protein products of the three genes is made. When lactose is present, an end product of lactose metabolism binds to the repressor protein and prevents it from binding to the operator. This allows RNA polymerase to bind to the promoter and freely transcribe the three genes, allowing the organism to metabolize the lactose.
Eukaryotic cells, in contrast, have intracellular organelles and are much more complex. Recall that in eukaryotic cells, the DNA is contained inside the cell’s nucleus and it is transcribed into mRNA there. The newly synthesized mRNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the mRNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation only occurs outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process (Figure \(1\)). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level).
The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in Table \(1\).
Table \(1\): Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms
Prokaryotic organisms Eukaryotic organisms
Lack nucleus Contain nucleus
RNA transcription and protein translation occur almost simultaneously
• RNA transcription occurs prior to protein translation, and it takes place in the nucleus. RNA translation to protein occurs in the cytoplasm.
• RNA post-processing includes addition of a 5' cap, poly-A tail, and excision of introns and splicing of exons.
Gene expression is regulated primarily at the transcriptional level Gene expression is regulated at many levels (epigenetic, transcriptional, post-transcriptional, translational, and post-translational)
EVOLUTION IN ACTION: Alternative RNA Splicing
In the 1970s, genes were first observed that exhibited alternative RNA splicing. Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of introns (and sometimes exons) are removed from the transcript (Figure \(2\)). This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene regulation, with the frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells, or at different stages of development. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; according to one estimate, 70% of genes in humans are expressed as multiple proteins through alternative splicing.
How could alternative splicing evolve? Introns have a beginning and ending recognition sequence, and it is easy to imagine the failure of the splicing mechanism to identify the end of an intron and find the end of the next intron, thus removing two introns and the intervening exon. In fact, there are mechanisms in place to prevent such exon skipping, but mutations are likely to lead to their failure. Such “mistakes” would more than likely produce a nonfunctional protein. Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a sequence. However, alternative splicing would create a protein variant without the loss of the original protein, opening up possibilities for adaptation of the new variant to new functions. Gene duplication has played an important role in the evolution of new functions in a similar way—by providing genes that may evolve without eliminating the original functional protein.
Summary
While all somatic cells within an organism contain the same DNA, not all cells within that organism express the same proteins. Prokaryotic organisms express the entire DNA they encode in every cell, but not necessarily all at the same time. Proteins are expressed only when they are needed. Eukaryotic organisms express a subset of the DNA that is encoded in any given cell. In each cell type, the type and amount of protein is regulated by controlling gene expression. To express a protein, the DNA is first transcribed into RNA, which is then translated into proteins. In prokaryotic cells, these processes occur almost simultaneously. In eukaryotic cells, transcription occurs in the nucleus and is separate from the translation that occurs in the cytoplasm. Gene expression in prokaryotes is regulated only at the transcriptional level, whereas in eukaryotic cells, gene expression is regulated at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels.
Glossary
alternative RNA splicing
a post-transcriptional gene regulation mechanism in eukaryotes in which multiple protein products are produced by a single gene through alternative splicing combinations of the RNA transcript
epigenetic
describing non-genetic regulatory factors, such as changes in modifications to histone proteins and DNA that control accessibility to genes in chromosomes
gene expression
processes that control whether a gene is expressed
post-transcriptional
control of gene expression after the RNA molecule has been created but before it is translated into protein
post-translational
control of gene expression after a protein has been created | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.01%3A_Control_of_Gene_Expression.txt |
Given the relative structural simplicity and repetitiveness of DNA, it would follow that proteins that bind specifically to it might have common DNA binding domain motifs but with specific amino acids side chains allowing for specific binding interactions.
• helix-turn-helix: found in prokaryotic DNA binding proteins.
Figure: helix-turn-helix
The figures shows two such proteins, the cro repressor from bacteriophage 434 and the lambda repressor from the bacteriophage lambda. (Bacteriophages are viruses that infect bacteia.) Notice how specificity is achieved, in part, by the formation of specific H-bonds between the protein and the major grove of the operator DNA.
Figure: Lambda Repressor/DNA Complex
Figure: H Bond interactions betweenλ repressor and DNA
Jmol: Updated Lambda Repressor/DNA complex Jmol14 (Java) | JSMol (HTML5)
• zinc finger: (eukaryotes) These proteins have a common sequence motif of X3-Cys-X2-4-Cys-X12-His-X3-4-His-X4- in which X is any amino acid. Zn2+ is tetrahedrally coordinated with the Cys and His side chains, which are on one of two antiparallel beta strands, and an alpha helix, respectively. The zinc finger, stabilized by the zinc, binds to the major groove of DNA. ]
Figure: zinc finger
Jmol: Updated Zif268:DNA Complex Jmol14 (Java) | JSMol (HTML5)
Zn finger proteins, of which 900 are encoded in the human genome (including the eukaryotic insulators binding protein CTCF described above) can be mobilized to actual repair specific mutations in cells, which if carried out in a high enough percentage of mutant cells could cure specific genetic diseases such as some forms of severe combined immunodeficiency disease. In this new technique (Urnov et al, 2005), multiple linked Zn finger binding domains, (one of the natural-occurring ones or mutant forms produced in the lab), each one specific for a certain nucleotide sequence, is linked to a nonspeciifc endonuclease, derived from the enzyme FokI. The nuclease is active in dimeric form so the active complex requires two endonuclease domains, each bound to four different Zn finger domains, to assemble at the target site. Specificity of binding is achieved by selection by the Zn finger domains. A nick is then made by the DNA by the nuclease, and host cell repair mechanisms ensue. This process involves strand separation, homologous recombination of the nicked region with complementary DNA within the cell, and repair of the nick. If excess wild type (non-mutated) DNA is added to the cells and uses as the template, the normal DNA repair mutation would fix the mutation. Urnov et al have shown the up to 20% of cultured cells containing a mutation can be repair in the lab. If these cells gain a selective growth advantage, the mutated cells would eventually be replaced with wild type cells.
• steroid hormone receptors: (eukaryotes) In contrast to most hormones, which bind to cell surface receptors, steroid hormones (derivatives of cholesterol) pass through the cell membrane and bind to cytoplasmic receptors through a hormone binding domain. This changes the shape of the receptor which then binds to a specific site on the DNA (hormone response element) though a DNA binding domain. In a structure analogous to the zinc finger, Zn 2+ is tetrahedrally coordinated to 4 Cys, in a globular-like structure which binds as a dimer to two identical, but reversed sequences of DNA (palindrome) within the major grove. (Examples of palindromes: Able was I ere I saw Elba; Dennis and Edna dine, said I, as Enid and Edna sinned.
Consider the glucocorticoid receptor (GR) as a specific example. It binds DNA as a dimer. The two DNA binding domains of the dimer associate with two adjacent major grooves of the DNA in the GR binding sequence (GBS), a short sequence of DNA within the promoter. Meijsing, et al. have found that not only does the GBS act as a binding site for GR, allowing transcription of genes, but it also affects the conformation of the receptor, causing gene transcription to be regulated in another way. The group constructed luciferase "reporters genes" which have GBS linked to the gene for the protein luciferase, that would express the protein luciferase (which fluoresces) if they were being transcribed, with the GBS. They found that relative transcriptional activity did not correlate to relative binding affinity of GR to the GBS. GBSs which were much more active than others bound comparably with those of lower activity, while GBSs with similar transcriptional activity bound with different affinities. This shows that the GBS is conferring unique function to the GR associated with it (i.e. transcription is not simply affected by whether or not the GR is bound to the GBS). A �lever arm� of the receptor was found to undergo conformational changes when bound to DNA, with changes specific to the sequence to which it was bound. A mutant protein, GR-γ, was made to be identical to the wild-type protein, GR-α, except in the lever arm was found to have different transcriptional activity even though they were binding to the same site on the DNA, showing that the lever arm and its conformation affects transcription.
• leucine zippers (or scissors): (eukaryotes) These proteins contain stretches of 35 amino acids in which Leu is found repeatedly at 7 amino acid intervals. These regions of the protein form amphiphilic helices, with Leu on one face, one Leu after two turns of a helix. Two of these proteins can form a dimer, stabilized by the binding of these nonpolar, leucine-rich amphiphilic helices to one another, forming a coiled-coil, much as in the muscle protein myosin. The leucine zipper represents the protein binding domain of the protein. The DNA binding domain is found in the first 30 N-terminal amino acids, which are basic and form an alpha helix when the protein binds to DNA. The leucine zipper then functions to bring two DNA binding proteins together, allowing the N-terminal bases helices to interact with the major grove of DNA in a base-specific fashion. Valine and isoleucine, along with leucine, are often found in stretches of amino acids that can interact to form other types of coiled coils.
Figure: leucine zippers (made with VMD)
Jmol: Updated Leucine Zipper Jmol14 (Java) | JSMol (HTML5)
Just as Zinc fingers nucleases have been used to induce repair of mutations, another study of the rat genome used specially designed ZFNs to cause breaks in ds-DNA that contain mutations from inaccurate DNA repair mechanism (by NHEJ) and hence contained specific mutations (Geurts, et al. 2009). This process, �knockout of the gene,� prevents the production of the protein normally transcribed by the target gene. Five- and six-finger ZFNs were used to achieve a high level of specificity in the targeted binding to the gene for three different proteins: green fluorescent protein (GFP), Immunoglobulin M (IgM) and Rab38. The knockout was successful in 12% of the rats tested; these animals had no wild-type protein and no expression. The ZFNs were sufficiently specific that no mutations were observed at any of 20 predicted non-target sites. This study supports the viability of control of transcription and expression for the treatment of disease and the importance of specific binding.
We have seen that two main factors contribute to the specific recognition of DNA by proteins; the formation of hydrogen bonds to specific nucleotide donors and acceptors in the major groove, and sequence-dependent deformations of the DNA helix to altered shapes with increased affinity of protein ligands. For example the Tata Binding Protein (TBP) can interact with a widened minor grove in the TATA box. New findings support that in addition proteins are able to use information in minor grooves that have become "narrowed" depending on the nucleotide sequence.
Tracks of DNA enriched in A can lead to twisting conformations that cause inter-base-pair hydrogen bonding in the major grooves, results in the narrowing of minor grooves. High amounts of AT base pairs are concentrated in narrow minor grooves (width <5.0 �) and CG base pairs are found more frequently in wide minor grooves.
How does minor groove narrowing affect DNA recognition? Narrow minor groves enhance the negative electrostatic potential of the DNA, making it a more specific and recognizable site. The backbone phosphates of the DNA are closer to the middle of the groove when it is narrow, thus correlating narrow minor grooves with a more negative electrostatic potential.
The minor grove-interacting parts of proteins contain arginine whose side chain can be accommodated into the more narrow and negative minor groove. Arginines can bind and in some cases insert themselves as short sequence motifs which enhance the specificity of the DNA shape recognition. Arg is preferred over Lys since the effective radii of the charge in Arg is greater than of the charge carrier in Lys. This would lead to a decreased desolvation energy for Arg which would promote its binding to the narrowed major grove. This discovery shows that "the role of DNA shape must be taken into consideration when annotating the entire genome and predicting transcription-factor-binding sites".
Figure: Arg in T3c Transposase binding in Narrowed Minor Grove of T3c Transposon
• Sliding Model for protein/DNA Interactions | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.02%3A_Regulatory_Proteins/16.2.01%3A_D5._DNA_Binding_Proteins.txt |
Skills to Develop
• Describe the steps involved in prokaryotic gene regulation
• Explain the roles of activators, inducers, and repressors in gene regulation
The DNA of prokaryotes is organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon.
In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors are proteins that suppress transcription of a gene in response to an external stimulus, whereas activators are proteins that increase the transcription of a gene in response to an external stimulus. Finally, inducers are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate.
The trp Operon: A Repressor Operon
Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure \(1\)). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.
A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site; each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription.
A DNA sequence called the operator sequence is encoded between the promoter region and the first trp coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.
Link to Learning
Watch this video to learn more about the trp operon.
Catabolite Activator Protein (CAP): An Activator Regulator
Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources (Figure \(2\)). In these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes.
The lac Operon: An Inducer Operon
The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. To do so, the cAMP–CAP protein complex serves as a positive regulator to induce transcription. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. CAP binds to the operator sequence upstream of the promoter that initiates transcription of the lac operon. However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed (Figure \(3\)). This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available.
Art Connection
In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think this is the case?
If glucose is absent, then CAP can bind to the operator sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is met, then transcription remains off. Only when both conditions are satisfied is the lac operon transcribed (Table \(1\)).
Table \(1\): Signals that Induce or Repress Transcription of the lac Operon
Glucose CAP binds Lactose Repressor binds Transcription
+ - - + No
+ - + - Some
- + - + No
- + + - Yes
Link to Learning
Watch an animated tutorial about the workings of lac operon here.
Summary
The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are three ways to control the transcription of an operon: repressive control, activator control, and inducible control. Repressive control, typified by the trp operon, uses proteins bound to the operator sequence to physically prevent the binding of RNA polymerase and the activation of transcription. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains off. Activator control, typified by the action of CAP, increases the binding ability of RNA polymerase to the promoter when CAP is bound. In this case, low levels of glucose result in the binding of cAMP to CAP. CAP then binds the promoter, which allows RNA polymerase to bind to the promoter better. In the last example—the lac operon—two conditions must be met to initiate transcription. Glucose must not be present, and lactose must be available for the lac operon to be transcribed. If glucose is absent, CAP binds to the operator. If lactose is present, the repressor protein does not bind to its operator. Only when both conditions are met will RNA polymerase bind to the promoter to induce transcription.
Art Connections
Figure \(3\): In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think that this is the case?
Answer
Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present.
Glossary
activator
protein that binds to prokaryotic operators to increase transcription
catabolite activator protein (CAP)
protein that complexes with cAMP to bind to the promoter sequences of operons that control sugar processing when glucose is not available
inducible operon
operon that can be activated or repressed depending on cellular needs and the surrounding environment
lac operon
operon in prokaryotic cells that encodes genes required for processing and intake of lactose
negative regulator
protein that prevents transcription
operator
region of DNA outside of the promoter region that binds activators or repressors that control gene expression in prokaryotic cells
operon
collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells
positive regulator
protein that increases transcription
repressor
protein that binds to the operator of prokaryotic genes to prevent transcription
transcriptional start site
site at which transcription begins
trp operon
series of genes necessary to synthesize tryptophan in prokaryotic cells
tryptophan
amino acid that can be synthesized by prokaryotic cells when necessary | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.03%3A_Prokaryotic_Regulation.txt |
Learning Objectives
• Describe the role of promoters in RNA transcription
Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long). The longer the promoter, the more available space for proteins to bind. This also adds more control to the transcription process. The length of the promoter is gene-specific and can differ dramatically between genes. Consequently, the level of control of gene expression can also differ quite dramatically between genes. The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
Within the promoter region, just upstream of the transcriptional start site, resides the TATA box. This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA repeats). RNA polymerase binds to the transcription initiation complex, allowing transcription to occur. To initiate transcription, a transcription factor (TFIID) is the first to bind to the TATA box. Binding of TFIID recruits other transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH to the TATA box. Once this transcription initiation complex is assembled, RNA polymerase can bind to its upstream sequence. When bound along with the transcription factors, RNA polymerase is phosphorylated. This releases part of the protein from the DNA to activate the transcription initiation complex and places RNA polymerase in the correct orientation to begin transcription; DNA-bending protein brings the enhancer, which can be quite a distance from the gene, in contact with transcription factors and mediator proteins.
In addition to the general transcription factors, other transcription factors can bind to the promoter to regulate gene transcription. These transcription factors bind to the promoters of a specific set of genes. They are not general transcription factors that bind to every promoter complex, but are recruited to a specific sequence on the promoter of a specific gene. There are hundreds of transcription factors in a cell that each bind specifically to a particular DNA sequence motif. When transcription factors bind to the promoter just upstream of the encoded gene, they are referred to as cis-acting elements because they are on the same chromosome, just next to the gene. The region that a particular transcription factor binds to is called the transcription factor binding site. Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed.
Key Points
• The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
• The promoter region can be short or quite long; the longer the promoter is, the more available space for proteins to bind.
• To initiate transcription, a transcription factor (TFIID) binds to the TATA box, which causes other transcription factors to subsequently bind to the TATA box.
• Once the transcription initiation complex is assembled, RNA polymerase can bind to its upstream sequence and is then phosphorylated.
• Phosphorylation of RNA polymerase releases part of the protein from the DNA to activate the transcription initiation complex and places RNA polymerase in the correct orientation to begin transcription.
• Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed.
Key Terms
• TATA box: a DNA sequence (cis-regulatory element) found in the promoter region of genes in archaea and eukaryotes
• transcription factor: a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA
• promoter: the section of DNA that controls the initiation of RNA transcription
16.4B: Transcriptional Enhancers and Repressors
Learning Objectives
• Explain how enhancers and repressors regulate gene expression
Enhancers and Transcription
In some eukaryotic genes, there are regions that help increase or enhance transcription. These regions, called enhancers, are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away.
Enhancer regions are binding sequences, or sites, for transcription factors. When a DNA-bending protein binds to an enhancer, the shape of the DNA changes. This shape change allows the interaction between the activators bound to the enhancers and the transcription factors bound to the promoter region and the RNA polymerase to occur. Whereas DNA is generally depicted as a straight line in two dimensions, it is actually a three-dimensional object. Therefore, a nucleotide sequence thousands of nucleotides away can fold over and interact with a specific promoter.
Turning Genes Off: Transcriptional Repressors
Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Like the transcriptional activators, repressors respond to external stimuli to prevent the binding of activating transcription factors.
A corepressor is a protein that decreases gene expression by binding to a transcription factor that contains a DNA-binding domain. The corepressor is unable to bind DNA by itself. The corepressor can repress transcriptional initiation by recruiting histone deacetylase, which catalyzes the removal of acetyl groups from lysine residues. This increases the positive charge on histones, which strengthens the interaction between the histones and DNA, making the DNA less accessible to the process of transcription.
Key Points
• Enhancers can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or thousands of nucleotides away.
• When a DNA -bending protein binds to the enhancer, the shape of the DNA changes, which allows interactions between the activators and transcription factors to occur.
• Repressors respond to external stimuli to prevent the binding of activating transcription factors.
• Corepressors can repress transcriptional initiation by recruiting histone deacetylase.
• Histone deactylation increases the positive charge on histones, which strengthens the interaction between the histones and DNA, making the DNA less accessible to transcription.
Key Terms
• enhancer: a short region of DNA that can increase transcription of genes
• repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription
• activator: any chemical or agent which regulates one or more genes by increasing the rate of transcription | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.04%3A_Eukaryotic_Regulation/16.4A%3A_The_Promoter_and_the_Transcription_Machinery.txt |
Learning Objectives
• Discuss how eukaryotic gene regulation occurs at the epigenetic level and the various epigenetic changes that can be made to DNA
Epigenetic Control: Regulating Access to Genes within the Chromosome
The human genome encodes over 20,000 genes; each of the 23 pairs of human chromosomes encodes thousands of genes. The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments can be accessed as needed by a specific cell type.
The first level of organization, or packing, is the winding of DNA strands around histone proteins. Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions. Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string. These beads (histone proteins) can move along the string (DNA) and change the structure of the molecule.
If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery ( RNA polymerase ) to initiate transcription. Nucleosomes can move to open the chromosome structure to expose a segment of DNA, but do so in a very controlled manner.
How the histone proteins move is dependent on signals found on both the histone proteins and on the DNA. These signals are tags, or modifications, added to histone proteins and DNA that tell the histones if a chromosomal region should be open or closed. These tags are not permanent, but may be added or removed as needed. They are chemical modifications (phosphate, methyl, or acetyl groups) that are attached to specific amino acids in the protein or to the nucleotides of the DNA. The tags do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins. DNA is a negatively-charged molecule; therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a large positive charge; by adding chemical modifications, such as acetyl groups, the charge becomes less positive.
The DNA molecule itself can also be modified. This occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes. When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added). This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region. Highly-methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive. These changes to DNA are inherited from parent to offspring, such that while the DNA sequence is not altered, the pattern of gene expression is passed to the next generation.
This type of gene regulation is called epigenetic regulation. Epigenetics means “above genetics.” The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent. Instead, these changes are temporary (although they often persist through multiple rounds of cell division) and alter the chromosomal structure (open or closed) as needed. A gene can be turned on or off depending upon the location and modifications to the histone proteins and DNA. If a gene is to be transcribed, the histone proteins and DNA are modified surrounding the chromosomal region encoding that gene. This opens the chromosomal region to allow access for RNA polymerase and other proteins, called transcription factors, to bind to the promoter region, located just upstream of the gene, and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur.
Key Points
• DNA is packaged by wrapping around histone proteins into structures called nucleosomes, which resemble beads on a string.
• When DNA is to be transcribed, the nucleosomes can slide away from that region of DNA, opening it up to the transcription machinery of the cell.
• Chemical modifications to either the histone proteins or the DNA itself signals whether or not a particular region of the genome should be “open” or “closed” to the transcription machinery.
• Modifications such as acetylation or methylation of the histones can alter how tightly DNA is wrapped around them, while methylation of DNA changes how the DNA interacts with proteins, including the histone proteins that control access to the region.
• This type of genetic regulation is called epigenetic regulation (“above genetics”) as it does not change the nucleotide sequence of the DNA.
Key Terms
• nucleosome: any of the subunits that repeat in chromatin; a coil of DNA surrounding a histone core
• epigenetics: the study of heritable changes caused by the activation and deactivation of genes without any change in DNA sequence
• histone: any of various simple water-soluble proteins that are rich in the basic amino acids lysine and arginine and are complexed with DNA in the nucleosomes of eukaryotic chromatin | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.05%3A_Chromatin_Structure_Affects_Gene_Expression/16.5C%3A_Epigenetic_Control-_Regulating_Access_to_Genes_within_the_Chr.txt |
Learning Objectives
• Gene expression can be regulated at various stages after an RNA transcript has been produced.
• Some transcripts can undergo alternative splicing. This regulated process makes different mRNAs and proteins from the same initial RNA transcript.
• Some mRNAs are targeted by small regulatory RNAs, including miRNAs, which can cause mRNA degradation or block translation.
• A protein’s activity may be regulated after translation by mechanisms such as proteolysis (“snipping out” of pieces) and addition of chemical groups.
The genes that a eukaryotic cell turns “on” largely determine its identity and properties. For instance, a photoreceptor cell in your eye can detect light because it expresses genes for light-sensitive proteins, as well as as genes for neurotransmitters that allow signals to be relayed to the brain.
In eukaryotic cells like photoreceptors, gene expression is often controlled primarily at the level of transcription. However, that doesn’t mean transcription is the last chance for regulation. Later stages of gene expression can also be regulated, including the following:
• RNA processing, such as splicing, capping, and addition of a poly-A tail
• Messenger RNA (mRNA) translation and lifetime in the cytosol
• Protein modifications, such as addition of chemical groups or removal of amino acids
In the sections below, we’ll discuss some common types of gene regulation that occur after an RNA transcript has been made.
Regulation of RNA processing
When a eukaryotic gene is transcribed in the nucleus, the primary transcript (freshly made RNA molecule) isn’t yet considered a messenger RNA. Instead, it’s an “immature” molecule called a pre-mRNA.
The pre-mRNA has to go through some modifications to become a mature mRNA molecule that can leave the nucleus and be translated. These include splicing, capping, and addition of a poly-A tail, all of which can potentially be regulated – sped up, slowed down, or altered to result in a different product.
Alternative splicing
Most pre-mRNA molecules have sections that are removed from the molecule, called introns, and sections that are linked or together to make the final mRNA, called exons. This process is called splicing.
In the process of alternative splicing, different portions of an mRNA can be selected for use as exons. This allows either of two (or more) mRNA molecules to be made from one pre-mRNA.
Alternative splicing is not a random process. Instead, it’s typically controlled by regulatory proteins. The proteins bind to specific sites on the pre-mRNA and “tell” the splicing factors which exons should be used. Different cell types may express different regulatory proteins, so different exon combinations can be used in each cell type, leading to the production of different proteins.
Small regulatory RNA
Once an mRNA has left the nucleus, it may or may not be translated many times to make proteins. Two key determinants of how much protein is made from an mRNA are its “lifespan” (how long it floats around in the cytosol) and how readily the translation machinery, such as the ribosome, can attach to it.
A recently discovered class of regulators, called small regulatory RNAs, can control mRNA lifespan and translation. Let’s see how this works.
microRNAs
microRNAs (miRNAs) were among the first small regulatory RNAs to be discovered. A miRNA is first transcribed as a long RNA molecule, which forms base pairs with itself and folds over to make a hairpin. Next, the hairpin is chopped up by enzymes, releasing a small double-stranded fragment of about 20 nucleotides. One of the strands in this fragment is the mature miRNA, which binds to a specific protein to make an RNA-protein complex.
The miRNA directs the protein complex to “matching” mRNA molecules (ones that form base pairs with the miRNA). When the RNA-protein complex binds:
• If the miRNA and its target match perfectly, an enzyme in the RNA-protein complex will typically chop the mRNA in half, leading to its breakdown.
• If the miRNA and its target have some mismatches, the RNA-protein complex may instead bind to the mRNA and keep it from being translated.
These are not the only ways that miRNAs inhibit expression of their targets, and scientists are still investigating their many modes of action.[1]
1. Carthew, R. W. and Sontheimer, E. J. (2009). Origins and mechanisms of miRNAs and siRNAs. Cell, 136(4), 642–655. [1]http://dx.doi.org/10.1016/j.cell.2009.01.035. ↵
Contributors and Attributions
CC licensed content, Shared previously | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.06%3A_Eukaryotic_Posttranscriptional_Regulation/16.6.01%3A_miRNA.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/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.06%3A_Eukaryotic_Posttranscriptional_Regulation/16.6.02%3A_The_Translation_of_RNA_into_Proteins.txt |
Learning Objectives
• Explain the role of RNA splicing in regulating gene expression
RNA splicing, the first stage of post-transcriptional control
Gene expression is the process that transfers genetic information from a gene made of DNA to a functional gene product made of RNA or protein. Genetic Information flows from DNA to RNA by the process of transcription and then from RNA to protein by the process of translation. In order to ensure that the proper products are produced, gene expression is regulated at many different stages during and in between transcription and translation. In eukaryotes, the gene contains extra sequences that do not code for protein. In these organisms, transcription of DNA produces pre-mRNA. These pre-mRNA transcripts often contain regions, called introns, that are intervening sequences which must be removed prior to translation by the process of splicing. The regions of RNA that code for protein are called exons. Splicing can be regulated so that different mRNAs can contain or lack exons, in a process called alternative splicing. Alternative splicing allows more than one protein to be produced from a gene and is an important regulatory step in determining which functional proteins are produced from gene expression. Thus, splicing is the first stage of post-transcriptional control.
Alternative Splicing
Alternative splicing is a process that occurs during gene expression and allows for the production of multiple proteins (protein isoforms) from a single gene coding. Alternative splicing can occur due to the different ways in which an exon can be excluded from or included in the messenger RNA. It can also occur if portions on an exon are excluded/included or if there is an inclusion of introns. For example, if a pre-mRNA has four exons (A, B, C, and D), these can be spliced and translated in a number of different combinations. Exons A, B, and C can be translated together or Exons A, C, and D can be translated. This results in what is called alternative splicing. The pattern of splicing and production of alternatively-spliced messenger RNA is controlled by the binding of regulatory proteins (trans-acting proteins that contain the genes) to cis-acting sites that are found on the pre-RNA. Some of these regulatory proteins include splicing activators (proteins that promote certain splicing sites) and splicing repressors (proteins that reduce the use of certain sites). Some common splicing repressors include: heterogeneous nuclear ribonucleoprotein (hnRNP) and polypyrimidine tract binding protein (PTB). Proteins that are translated from alternatively-spliced messenger RNAs differ in the sequence of their amino acids which results in altered function of the protein. This is one reason why the human genome can encode a wide diversity of proteins. Alternative splicing is a common process that occurs in eukaryotes; most of the multi-exonic genes in humans are spliced alternatively. Unfortunately, abnormal variations in splicing are also the reason why there are many genetic diseases and disorders.
Spliceosome
The splicing of messenger RNA is accomplished and catalyzed by a macro-molecule complex known as the spliceosome. The areas for ligation and cleavage are determined by the many sub-units of the spliceosome which include the branch site (A) and the 5′ and 3′ splice sites. Interactions between these sub-units and the small nuclear ribonucleoproteins (snRNP) found in the spliceosome create a spliceosome A complex which helps determine which introns to leave out and which exons to keep and bind together. Once the introns are cleaved and removed, the exons are joined together by a phosphodiester bond.
Regulatory Proteins
As noted above, splicing is regulated by repressor proteins and activator proteins, which are are also known as trans-acting proteins. Equally as important are the silencers and enhancers that are found on the messenger RNAs, also known as cis-acting sites. These regulatory functions work together in order to create splicing code that determines alternative splicing.
Key Points
• Introns are intervening sequences within a pre-mRNA molecule that do not code for proteins and are removed during RNA processing by a spliceosome.
• Exons are expressing sequences within a pre-mRNA molecule that are spliced together once introns are removed to form mature mRNA molecules that are translated into proteins.
• Alternative splicing allows for the production of various protein isoforms from one single gene coding.
• A spliceosome is a complex comprised of both RNA molecules and proteins which determine which introns to leave out and which exons to keep and bind together.
Key Terms
• intron: a portion of a split gene that is included in pre-RNA transcripts but is removed during RNA processing and rapidly degraded
• exon: a region of a transcribed gene present in the final functional RNA molecule
• spliceosome: a dynamic complex of RNA and protein subunits that removes introns from precursor mRNA | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.06%3A_Eukaryotic_Posttranscriptional_Regulation/16.6D%3A_RNA_Splicing.txt |
Learning Objectives
• Discuss how eukaryotes assemble ribosomes on the mRNA to begin translation
Ribosome Assembly and Translation Rate
Like transcription, translation is controlled by proteins that bind and initiate the process. In translation, before protein synthesis can begin, ribosome assembly has to be completed. This is a multi-step process.
In ribosome assembly, the large and small ribosomal subunits and an initiator tRNA (tRNAi) containing the first amino acid of the final polypeptide chain all come together at the translation start codon on an mRNA to allow translation to begin. First, the small ribosomal subunit binds to the tRNAi which carries methionine in eukaryotes and archaea and carries N-formyl-methionine in bacteria. (Because the tRNAi is carrying an amino acid, it is said to be charged.) Next, the small ribosomal subunit with the charged tRNAi still bound scans along the mRNA strand until it reaches the start codon AUG, which indicates where translation will begin. The start codon also establishes the reading frame for the mRNA strand, which is crucial to synthesizing the correct sequence of amino acids. A shift in the reading frame results in mistranslation of the mRNA. The anticodon on the tRNAi then binds to the start codon via basepairing. The complex consisting of mRNA, charged tRNAi, and the small ribosomal subunit attaches to the large ribosomal subunit, which completes ribosome assembly. These components are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit during initiation and are found in all three domains of life. In addition, the cell spends GTP energy to help form the initiation complex. Once ribosome assembly is complete, the charged tRNAi is positioned in the P site of the ribosome and the empty A site is ready for the next aminoacyl-tRNA. The polypeptide synthesis begins and always proceeds from the N-terminus to the C-terminus, called the N-to-C direction.
In eukaryotes, several eukaryotic initiation factor proteins (eIFs) assist in ribosome assembly. The eukaryotic initiation factor-2 (eIF-2) is active when it binds to guanosine triphosphate (GTP). With GTP bound to it, eIF-2 protein binds to the small 40S ribosomal subunit. Next, the initiatior tRNA charged with methionine (Met-tRNAi) associates with the GTP-eIF-2/40S ribosome complex, and once all these components are bound to each other, they are collectively called the 43S complex.
Eukaryotic initiation factors eIF1, eIF3, eIF4, and eIF5 help bring the 43S complex to the 5′-m7G cap of an mRNA be translated. Once bound to the mRNA’s 5′ m7G cap, the 43S complex starts travelling down the mRNA until it reaches the initiation AUG codon at the start of the mRNA’s reading frame. Sequences around the AUG may help ensure the correct AUG is used as the initiation codon in the mRNA.
Once the 43S complex is at the initiation AUG, the tRNAi-Met is positioned over the AUG. The anticodon on tRNAi-Met basepairs with the AUG codon. At this point, the GTP bound to eIF2 in the 43S complexx is hydrolyzed to GDP + phosphate, and energy is released. This energy is used to release the eIF2 (with GDP bound to it) from the 43S complex, leaving the 40S ribosomal subunit and the tRNAi-Met at the translation start site of the mRNA.
Next, eIF5 with GTP bound binds to the 40S ribosomal subunit complexed to the mRNA and the tRNAi-Met. The eIF5-GTP allows the 60S large ribosomal subunit to bind. Once the 60S ribosomal subunit arrives, eIF5 hydrolyzes its bound GTP to GDP + phosphate, and energy is released. This energy powers assembly of the two ribosomal subunits into the intact 80S ribosome, with tRNAi-Met in its P site while also basepaired to the initiation AUG codon on the mRNA. Translation is ready to begin.
The binding of eIF-2 to the 40S ribosomal subunit is controlled by phosphorylation. If eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP. Therefore, the 43S complex cannot form properly and translation is impeded. When eIF-2 remains unphosphorylated, it binds the 40S ribosomal subunit and actively translates the protein.
The ability to fully assemble the ribosome directly affects the rate at which translation occurs. But protein synthesis is regulated at various other levels as well, including mRNA synthesis, tRNA synthesis, rRNA synthesis, and eukaryotic initiation factor synthesis. Alteration in any of these components affects the rate at which translation can occur.
Key Points
• The components involved in ribosome assembly are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit.
• Initiator tRNA is used to locate the start codon AUG (the amino acid methionine) which establishes the reading frame for the mRNA strand.
• GTP carried by eIF2 is the energy source used for loading the initiator tRNA carried by the small ribosomal subunit on the correct start codon in the mRNA.
• GTP carried by eIF5 is the energy source for assembling the large and small ribosomal subunits together.
Key Terms
• reading frame: either of three possible triplets of codons in which a DNA sequence could be transcribed
• phosphorylation: the addition of a phosphate group to a compound; often catalyzed by enzymes | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.06%3A_Eukaryotic_Posttranscriptional_Regulation/16.6E%3A_The_Initiation_Complex_and_Translation_Rate.txt |
Learning Objectives
• Explain how chemical modifications affect protein activity and longevity
Chemical Modifications, Protein Activity, and Longevity
Proteins can be chemically modified with the addition of methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell; for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.
Chemical modifications occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. These changes can alter protein function, epigenetic accessibility, transcription, mRNA stability, or translation; all resulting in changes in expression of various genes. This is an efficient way for the cell to rapidly change the abundance levels of specific proteins in response to the environment. Because proteins are involved in every stage of gene regulation, the phosphorylation of a protein (depending on the protein that is modified) can alter accessibility to the chromosome, can alter translation (by altering transcription factor binding or function), can change nuclear shuttling (by influencing modifications to the nuclear pore complex), can alter RNA stability (by binding or not binding to the RNA to regulate its stability), can modify translation (increase or decrease), or can change post-translational modifications (add or remove phosphates or other chemical modifications). All of these protein activities are affected by the phosphorylation process. The enzymes which are responsible for phosphorylation are known as protein kinases. The addition of a phosphate group to a protein can result in either activation or deactivation; it is protein dependent.
Another example of chemical modifications affecting protein activity include the addition or removal of methyl groups. Methyl groups are added to proteins via the process of methylation; this is the most common form of post-translational modification. The addition of methyl groups to a protein can result in protein-protein interactions that allows for transcriptional regulation, response to stress, protein repair, nuclear transport, and even differentiation processes. Methylation on side chain nitrogens is considered largely irreversible while methylation of the carboxyl groups is potentially reversible. Methylation in the proteins negates the negative charge on it and increases the hydrophobicity of the protein. Methylation on carboxylate side chains covers up a negative charge and adds hydrophobicity. The addition of this chemical group changes the property of the protein and, thus, affects it activity.
The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete. These proteins are moved to the proteasome, an organelle that functions to remove proteins to be degraded. One way to control gene expression is to alter the longevity of the protein: ubiquitination shortens a protein’s lifespan.
Key Points
• Proteins can be chemically modified by adding methyl, phosphate, acetyl, and ubiquitin groups.
• Protein longevity can be affected by altering stages of gene regulation, including but not limited to altering: accessibility to chromosomal DNA for transcription, rate of translation, nuclear shuttling, RNA stability, and post-translational modifications.
• Ubiquitin is added to a protein to mark it for degradation by the proteasome.
Key Terms
• ubiquitin: a small polypeptide present in the cells of all eukaryotes; it plays a part in modifying and degrading proteins
• proteasome: a complex protein, found in bacterial, archaeal and eukaryotic cells, that breaks down other proteins via proteolysis | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.06%3A_Eukaryotic_Posttranscriptional_Regulation/16.6F%3A_Regulating_Protein_Activity_and_Longevity.txt |
Learning Objectives
• Explain how chemical modifications affect protein activity and longevity
Chemical Modifications, Protein Activity, and Longevity
Proteins can be chemically modified with the addition of methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell; for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.
Chemical modifications occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. These changes can alter protein function, epigenetic accessibility, transcription, mRNA stability, or translation; all resulting in changes in expression of various genes. This is an efficient way for the cell to rapidly change the abundance levels of specific proteins in response to the environment. Because proteins are involved in every stage of gene regulation, the phosphorylation of a protein (depending on the protein that is modified) can alter accessibility to the chromosome, can alter translation (by altering transcription factor binding or function), can change nuclear shuttling (by influencing modifications to the nuclear pore complex), can alter RNA stability (by binding or not binding to the RNA to regulate its stability), can modify translation (increase or decrease), or can change post-translational modifications (add or remove phosphates or other chemical modifications). All of these protein activities are affected by the phosphorylation process. The enzymes which are responsible for phosphorylation are known as protein kinases. The addition of a phosphate group to a protein can result in either activation or deactivation; it is protein dependent.
Another example of chemical modifications affecting protein activity include the addition or removal of methyl groups. Methyl groups are added to proteins via the process of methylation; this is the most common form of post-translational modification. The addition of methyl groups to a protein can result in protein-protein interactions that allows for transcriptional regulation, response to stress, protein repair, nuclear transport, and even differentiation processes. Methylation on side chain nitrogens is considered largely irreversible while methylation of the carboxyl groups is potentially reversible. Methylation in the proteins negates the negative charge on it and increases the hydrophobicity of the protein. Methylation on carboxylate side chains covers up a negative charge and adds hydrophobicity. The addition of this chemical group changes the property of the protein and, thus, affects it activity.
The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete. These proteins are moved to the proteasome, an organelle that functions to remove proteins to be degraded. One way to control gene expression is to alter the longevity of the protein: ubiquitination shortens a protein’s lifespan.
Key Points
• Proteins can be chemically modified by adding methyl, phosphate, acetyl, and ubiquitin groups.
• Protein longevity can be affected by altering stages of gene regulation, including but not limited to altering: accessibility to chromosomal DNA for transcription, rate of translation, nuclear shuttling, RNA stability, and post-translational modifications.
• Ubiquitin is added to a protein to mark it for degradation by the proteasome.
Key Terms
• ubiquitin: a small polypeptide present in the cells of all eukaryotes; it plays a part in modifying and degrading proteins
• proteasome: a complex protein, found in bacterial, archaeal and eukaryotic cells, that breaks down other proteins via proteolysis | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/16%3A_Control_of_Gene_Expression/16.07%3A_Protein_Degradation/16.7F%3A_Regulating_Protein_Activity_and_Longevity.txt |
Recombinant DNA technology utilizes the power of microbiological selection and screening procedures to allow investigators to isolate a gene that represents as little as 1 part in a million of the genetic material in an organism. The DNA from the organism of interest is divided into small pieces that are then placed into individual cells (usually bacterial). These can then be separated as individual colonies on plates, and they can be screened through rapidly to find the gene of interest. This process is called molecular cloning.
Joining DNA in vitro to form recombinant molecules
Restriction endonucleasescut at defined sequences of (usually) 4 or 6 bp. This allows the DNA of interest to be cut at specific locations. The physiological function of restriction endonucleases is to serve as part of system to protect bacteria from invasion by viruses or other organisms. (See Chapter 7)
Table 3.1. List of restriction endonucleases and their cleavage sites. A ' means that the nuclease cuts between these 2 nucleotides to generate a 3' hydroxyl and a 5' phosphate.
Enzyme Site Enzyme Site
AluI AG'CT NotI GC'GGCCGC
BamHI G'GATCC PstI CTGCA'G
BglII A'GATCT PvuII CAG'CTG
EcoRI G'AATTC SalI G'TCGAC
HaeIII GG'CC Sau3AI 'GATC
HhaI GCG'C SmaI CCC'GGG
HincII GTY'RAC SpeI A'CTAGT
HindIII A'AGCTT TaqI T'CGA
HinfI G'ANTC XbaI T'CTAGA
HpaII C'CGG XhoI C'TCGAG
KpnI GGTAC'C XmaI C'CCGGG
MboI 'GATC
N = A,G,C or T
R = A or G
Y = C or T
S = G or C
W = A or T
a. Sticky ends
(1) Since the recognition sequences for restriction endonucleases are pseudopalindromes, an off-center cleavage in the recognition site will generate either a 5' overhang or a 3' overhang with self-complementary (or "sticky") ends.
e.g. 5' overhang EcoRI G'AATTC
BamHI G'GATCC
3' overhangPstI CTGCA'G
(2) When the ends of the restriction fragments are complementary,
e.g. for EcoRI 5'‑‑‑G AATTC‑‑‑3'
3'‑‑‑CTTAA G‑‑‑5'
the ends can anneal to each other. Any two fragments, regardless of their origin (animal, plant, fungal, bacterial) can be joined in vitro to form recombinant molecules (Figure 3.3).
Figure 3.3.
b. Blunt ends
(1) The restriction endonuclease cleaves in the center of the pseudopalindromic recognition site to generate blunt (or flush) ends.
(2) E.g. HaeIII GG'CC
HincII GTY'RAC
T4 DNA ligase is used to tie together fragments of DNA (Figure 3.4). Note that the annealed "sticky" ends of restriction fragments have nicks(usually 4 bp apart). Nicks are breaks in the phosphodiester backbone, but all nucleotides are present. Gapsin one strand are missing a string of nucleotides.
T4 DNA ligase uses ATP as source of adenylyl group attached to 5' end of the nick, which is a good leaving group after attack by the 3' OH. (See Chapter 5 on Replication).
At high concentration of DNA ends and of ligase, the enzyme can also ligate together blunt‑ended DNA fragments. Thus any two blunt‑ended fragments can be ligated together. Note: Any fragment with a 5' overhang can be readily converted to a blunt‑ended molecule by fill‑in synthesis catalyzed by a DNA polymerase (often the Klenow fragment of DNA polymerase I). Then it can be ligated to another blunt‑ended fragment.
Linkers are short duplex oligonucleotides that contain a restriction endonuclease cleavage site. They can be ligated onto any blunt‑ended molecule, thereby generating a new restriction cleavage site on the ends of the molecule. Ligation of a linker on a restriction fragment followed by cleavage with the restriction endonuclease is one of several ways to generate an end that is easy to ligate to another DNA fragment.
Annealing of homopolymer tails are another way to joint two different DNA molecules.
The enzyme terminal deoxynucleotidyl transferasewill catalyze the addition of a string of nucleotides to the 3' end of a DNA fragment. Thus by incubating each DNA fragment with the appropriate dNTP and terminal deoxynucleotidyl transferase, one can add complementary homopolymers to the ends of the DNAs that one wants to combine. E.g., one can add a string of G's to the 3' ends of one fragment and a string of C's to the 3' ends of the other fragment. Now the two fragments will join together via the homopolymer tails. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.01%3A_Recombinant_DNA/17.1.01%3A_Overview_of_Recombinant_DNA_Technology.txt |
Vectors used to move DNA between species, or from the lab bench into a living cell, must meet three requirements (Figure \(1\)):
1. They must be autonomously replicating DNA molecules in the host cell. The most common vectors are designed for replicating in bacteria or yeast, but there are vectors for plants, animals and other species.
2. They must contain a selectable marker so cells containing the recombinant DNA can be distinguished from those that do not. An example is drug resistance in bacteria.
3. They must have an insertion site to accommodate foreign DNA. Usually a unique restriction cleavage site in a nonessential region of the vector DNA. Later generation vectors have a set of about 15 or more unique restriction cleavage sites.
Plasmid Vectors
Plasmids are autonomously replicating circular DNA molecules found in bacteria. They have their own origin of replication, and they replicate independently of the origins on the "host" chromosome. Replication is usually dependent on host functions, such as DNA polymerases, but regulation of plasmid replication is distinct from that of the host chromosome. Plamsids, such as the sex-factor F, can be very large (94 kb), but others can be small (2‑4 kb). Plasmids do not encode an essential function to the bacterium, which distinguishes them from chromosomes. Plasmids can be present in a single copy, such as F, or in multiple copies, like those used as most cloning vectors, such as pBR322, pUC, and pBluescript.
In nature, plasmids provide carry some useful function, such as transfer (F), or antibiotic resistance. This is what keeps the plasmids in a population. In the absence of selection, plasmids are lost from bacteria. The antibiotic resistance genes on plasmids are often carried within, or are derived from, transposons, a types of transposable element. These are DNA segments that are capable of "jumping" or moving to new locations (Chapter 9).
A plasmid that was widely used in many recombinant DNA projects is pBR322 (Figure \(2\)). It replicates from an origin derived from a colicin-resistance plasmid (ColE1). This origin allows a fairly high copy number, about 100 copies of the plasmid per cell. Plasmid pBR322 carries two antibiotic resistance genes, each derived from different transposons. These transposons were initially found in R-factors, which are larger plasmids that confer antibiotic resistance.
Use of the TcR and ApR genes allows for easy screening for recombinants carrying inserts of foreign DNA. For instance, insertion of a restriction fragment in the BamHI site of the TcR gene inactivates that gene. One can still select for ApR colonies, and then screen to see which ones have lost TcR .
Exercise \(1\)
What effects on drug resistance are seen when you use the EcoRI or PstI sites in pBR322 for inserting foreign DNA?
A generation of vectors developed after pBR322 are designed for even more efficient screening for recombinant plasmids, i.e. those that have foreign DNA inserted. The pUC plasmids (named for plasmid universal cloning) and plasmids derived from them use a rapid screen for inactivation of the b-galactosidase gene to identify recombinants (Figure \(3\)).
One can screen for production of functional b‑galactosidase in a cell by using the chromogenic substrate X‑gal (a halogenated indoyl b‑galactoside). When cleaved by b‑galactosidase, the halogenated indoyl compound is liberated and forms a blue precipitate. The pUC vector has the b‑galactosidase gene {actually only part of it, but enough to form a functional enzyme with the rest of the gene that is encoded either on the E. coli chromosome or an F' factor}. When introduced into E. coli, the colonies are blue on plates containing X‑gal.
The multiple cloning sites (unique restriction sites) are in the b‑galactosidase gene (lacZ). When a restriction fragment is introduced into one or more of these sites, the b‑galactosidase activity is lost by this insertional mutation. Thus cells containing recombinant plasmids form white(not blue) colonies on plates containing X‑gal.
The replication origin is a modified ColE1 origin of replication that has been mutated to eliminate a negative control region. Hence the copy number is very high(several hundred or more plasmid molecules per cell), and one obtains an very high yield of plasmid DNA from cultures of transformed bacteria. The plasmid has ApR as a selectable marker. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.01%3A_Recombinant_DNA/17.1.02%3A_Introduction_of_recombinant_DNA_into_cell_and_replication-_Vectors.txt |
Transformation in E. coli
E. coli does not have a natural system for taking up DNA, but when treated with \(\ce{CaCl2}\), the cells will take up the added DNA (Figure \(1\)). The recombinant vectors will give a new phenotype to the cells (usually drug resistance), so this process can be considered DNA-mediated transformation. An average efficiency is about 106 transformants per mg of DNA, although some more elaborate transformation cocktails procedures can give up to about 108 transformants per mg of DNA.
Usually one will transform with a mixture of recombinant vector molecules, most of which carry a different restriction fragment. Each transformed E. coli cell will pick up only one plasmid molecule, so the complex mixture of plasmids in the ligation mix has been separated into a population of transformed bacteria (Figre \(1\)). The bacterial cells are then plated at a sufficiently low density that individual colonies can be identified. Each colony (or transformant) carries a single plasmid, so as one screens the colonies, one is actually screening through individual DNA molecules. A colony is a visible group of bacterial cells on a plate, all of which are derived from a single bacterial cell. A group of identical cells derived from a single cell is called a clone. Since each clone carries a single type of recombinant DNA molecule, the process is called molecular cloning.
Phage Vectors
Phase vectors are a more efficient introduction of DNA into bacteria. Phage vectors such as those derived from bacteriophage l can carry larger inserts and can be introduced into bacteria more efficiently. l phage has a duplex DNA genome of about 50 kb. The internal 20 kb can be replaced with foreign DNA and still retain the lytic functions. Hence restriction fragments up to 20 kb can replace the l sequences, allowing larger genomic DNA fragments to be cloned (Figure \(2\)).
Recombinant bacteriophage can be introduction into E. coli by infection. DNA that has the cohesive ends of l can be packaged in vitro into infective phage particles. Being in a viral particle brings the efficiency of infection reliably over 108 plaque forming units per mg of recombinant DNA.
Some other bacteriphage vectors for cloning are derived from the virus M13. One can obtain single stranded DNA from M13 vectors and recombinants. M13 is a virus with a genome of single stranded DNA. It has a nonessential region into which foreign genes can be inserted. It has been modified to carry a gene for b‑galactosidase as a way to screen for recombinants. Introduction of recombinant M13 DNA into E. coli will lead to an infection of the host, and the progeny viral particles will contain single‑stranded DNA. The replicative form is duplex, allowing one to cleave with restriction enzymes and insert foreign DNA.
Some vectors are hybrids between plasmids and single‑strand phage; these are called phagemids. One example is pBluescript. Phagemids are plasmids (with the modified, high-copy number ColE1 origin) that also have an M13 origin of replication. Infection of transformed bacteria (containing the phagemid) with a helper virus (e.g. derived from M13) will cause the M13 origin to be activated, and progeny viruses carrying single‑stranded copies of the phagemid can be obtained. Hence one can easily obtain either double‑ or single‑stranded forms of thes plasmids. {The "blue" comes from the blue‑white screening for recombinants that can be done when the multiple cloning sites are in the b‑galactosidase gene. The "script" refers to the ability to make RNA copies of either strand in vitro with phage RNA polymerases.}
Vectors Designed to Carry Larger Inserts
Fragments even larger than those carried in l vectors are useful for studies of longer segments of chromosomes or whole genomes. Several vectors have been designed for cloning these very large fragments, 50 to 400 kb.
• Cosmids are plasmids that have the cohesive ends of l phage. They can be packaged in vitro into infective phage particles to give a more efficient delivery of the DNA into the cells. They can carry about 35 to 45 kb inserts.
• Yeast artificial chromosomes (YACs) are yeast vectors with centromeres and telomeres. They can carry about 200 kb or larger fragments (in principle up to 1000 kb = 1 Mb). Thus very large fragments of DNA can be cloned in yeast (Figure \(3\)). In practice, chimeric clones with fragments from different regions of the genome are obtained fairly often, and some of the inserts are unstable.
Vectors derived from bacteriophage P1 can carry fragments of about 100 kb. Fragments in a similar size range are also cloned into bacterial artificial chromosomes (BACs), which are derived from the F-factor (Figure \(4\)). These have a lower copy number (like F) but they are stable and relatively easy to work with in the laboratory. BACs have become one of the most frequently used vectors for large inserts in genome projects.
Shuttle vectors for testing functions of isolated genes
Shuttle vectors can replicate in two different organisms, e.g. bacteria and yeast, or mammalian cells and bacteria. They have the appropriate origins of replication. Hence one can, e.g. clone a gene in bacteria, maybe modify it or mutate it in bacteria, and test its function by introducing it into yeast or animal cells. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.01%3A_Recombinant_DNA/17.1.03%3A_Introducting_Recombinant_DNA_into_Host_Cells.txt |
The first step in making a cDNA library is to isolate cellular mRNA. This mRNA extract should represent all of the transcripts in the cells at the time of isolation, or the cell’s transcriptome. This term is used by analogy to genome. However, a genome is all of the genetic information of an organism. In contrast, a transcriptome (usually eukaryotic) reflects all of the genes expressed in a given cell type at a moment in time. Reversetranscribed cDNAs from an mRNA extract are also referred to as a transcriptome…, and likewise, a cDNA library. A cDNA library is a tube full of bacterial cells that have taken up (i.e., been transformed with) plasmids recombined with cDNAs. cDNA libraries made from mRNAs taken from different cell types or the same cells grown under different conditions are in effect, different transcriptomes. Each reflects mRNAs transcribed in cells at the moment of their extraction. When cells in a cDNA library are spread out on a nutrient agar petri dish, each cell grows into a colony of cells; each cell in the colony is a clone of a starting cell. cDNA libraries can be used isolate and sequence the DNA encoding a polypeptide that you are studying.
Recall that the mature mRNA in eukaryotic cells has been spliced. This means that cDNAs from eukaryotic cells do not include introns. Introns, as well as sequences of enhancers and other regulatory elements in and surrounding a gene must be studied in genomic libraries, to be discussed later. Here we look at how to make a cDNA library.
A. cDNA Construction
mRNA is only a few percent of a eukaryotic cell; most is rRNA. But that small amount of mRNA can be separated from other cellular RNAs by virtue of their 3’ poly(A) tails. Simply pass a total RNA extract over an oligo-d(T) column (illustrated below).
The strings of thymidine (T) can H-bond with the poly(A) tails of mRNAs, tethering them to the column. All RNAs without a 3’ poly(A) tail will flow through the column as waste. A second buffer is passed over the column to destabilize the A-T H-bonds to allow elution of an mRNA fraction. When free’ oligo d(T) is added to the eluted mRNA, it forms H-bonds with the poly(A) tails of the mRNAs, serving as a primer for the synthesis of cDNA copies of the poly(A) mRNAs originally in the cells. Finally, four deoxynucleotide DNA precursors and reverse transcriptase (originally isolated from chicken retrovirus-infected cells) are added to start reverse transcription. The synthesis of a cDNA strand complementary to an mRNA is shown below.
After heating to separate the cDNAs from the mRNAs, the cDNA is replicated to produce double-stranded, or (ds)cDNA, as illustrated below.
Synthesis of the second cDNA strand is also catalyzed by reverse transcriptase! The enzyme recognizes DNA as well as RNA templates, and has the same 5’-to-3’ DNA polymerizing activity as DNA polymerases. After 2nd cDNA strand synthesis, S1 nuclease (a single-stranded endonuclease originally isolated from an East Asian fungus!) is added to open the loop of the (ds) cDNA structure and trim the rest of the single-stranded DNA. What remains is the (ds) cDNA.
258 Isolate mRNA and Make cDNA
259 Reverse Transcriptase
B. Cloning cDNAs into Plasmid Vectors
To understand cDNA cloning and other aspects of making recombinant DNA, we need to talk a bit more about the recombinant DNA tool kit. In addition to reverse transcriptase and S1 nuclease, other necessary enzymes in the ‘kit’ include restriction endonucleases (restriction enzymes) and DNA ligase. The natural function of restriction enzymes in bacteria is to recognize specific restriction site sequences in phage DNA (most often palindromic DNA sequences), hydrolyze it and thus avoid infection.
Restriction enzymes that make a scissors cut through the two strands of the double helix leaves blunt ends. Restriction enzymes that make a staggered cut on each strand at their restriction site leave behind complementary (‘sticky’) ends (below).
If you mix two of double-stranded DNA fragments with the same sticky ends from different sources (e.g., different species), they will form H-bonds at their complementary ends, making it easy to recombine plasmid DNA with (ds)cDNA, that have the same complementary ‘sticky ends’. Using the language of recombinant DNA technologies, let’s look at how plasmid vectors and cDNAs can be made to recombine.
1. Preparing Recombinant Plasmid Vectors Containing cDNA Inserts
Vectors are carrier DNAs engineered to recombine with foreign DNAs of interest. When a recombinant vector with its foreign DNA insert gets into a host cell, it can replicate many copies of itself, enough in fact for easy isolation and study. cDNAs are typically inserted into plasmid vectors that are usually purchased “off-the-shelf”. They can be cut with a restriction enzyme at a suitable location, leaving those sticky ends. On the other hand, it would not do to digest (ds)cDNA with restriction endonucleases since the goal is not to clone cDNA fragments, but entire cDNA molecules. Therefore, it will be necessary to attach linkers to either end of the (ds)cDNAs. Plasmid DNAs and cDNA-linker constructs can then be digested with the same restriction enzyme to produce compatible ‘sticky ends’. Steps in the preparation of vector and (ds)cDNA for recombination are shown below.
To prepare for recombination, a plasmid vector is digested with a restriction enzyme to open the DNA circle. To have compatible sticky ends, double-stranded cDNAs to be inserted are mixed with linkers and DNA ligase to put a linker DNA at both ends of the (ds) cDNA. DNA ligase is another tool in the recombinant DNA toolkit. Linkers are short, synthetic double-stranded DNA oligomers containing restriction sites recognized and cut by the same restriction enzyme as the plasmid. Once the linkers are attached to the ends of the plasmid DNAs, they are digested with the appropriate restriction enzyme. This leaves both the (ds)cDNAs and the plasmid vectors with complementary sticky ends.
260 Restriction Enzymes and Recombinant DNA
2. Recombining Plasmids and cDNA Inserts and Transforming Host Cells
The next step is to mix the cut plasmids with the digested linker-cDNAs in just the right proportions so that the most of the cDNA (linker) ends will anneal (form Hbonds) with the most of the sticky plasmid ends. Adding DNA ligase to the plasmid/linker-cDNA mixture forms phosphodiester bonds between plasmid and cDNA insert, completing the recombinant circle of DNA, as shown below.
In early cloning experiments, an important consideration was to generate plasmids with only one copy of a given cDNA insert, rather than lots of re-ligated plasmids with no inserts or lots of plasmids with multiple inserts. Using betterengineered vector and linker combinations, this issue became less important.
261 Recombine a cDNA Insert with a Plasmid Vector
3. Transforming Host Cells with Recombinant Plasmids
The recombinant DNA molecules are now ready for ‘cloning’. They are added to E. coli (sometimes other host cells) made permeable so that they can be easily transformed. Recall that transformation as defined by Griffith is bacterial uptake of foreign DNA leading to a genetic change. The transforming principle in cloning is the recombinant plasmid! The transformation step is shown below.
The tube full of transformed cells is the cDNA Library.
262 Making the cDNA Library.
After all these treatments, not all plasmid molecules in the mix are recombinant; some cells in the mix haven’t even taken up a plasmid. So when the recombinant cells are plated on agar, how do you tell which of the colonies that grow came from cells that took up a recombinant plasmid? Both the host strain of E. coli and plasmid vectors used these days were further engineered to solve this problem. One such plasmid vector carries an antibiotic resistance gene. In this case, ampicillin-sensitive cells would be transformed with recombinant plasmids containing the resistance gene. When these cells are plated on media containing ampicillin (a form of penicillin), they grow, as illustrated below.
Untransformed cells (cells that failed to take up a plasmid) lack the ampicillin resistance gene and thus, do not grow on ampicillin-medium. But, there is still a question. How can you tell whether the cells that grew were transformed by a recombinant plasmid containing a cDNA insert? It is possible that some of the transformants contain only non-recombinant plasmids that still have the ampicillin resistance gene!
To address this question, plasmids were further engineered with a streptomycin resistance gene. But this antibiotic resistance gene was also engineered to contain restriction enzyme sites in the middle of the gene. Thus, inserting a cDNA in this plasmid would disrupt and inactivate the gene. Here is how this second bit of genetic engineering enabled growth only of cells transformed with a recombinant plasmid containing a cDNA insert. We can tell transformants containing recombinant plasmids apart from those containing non-recombinant plasmids by the technique of replica plating shown (illustrated below).
After colonies grow on the ampicillin agar plate, lay a filter over the plate. The filter will pick up a few cells from each colony, in effect becoming a replica (mirror image) of the colonies on the plate. Place the replica filter on a new agar plate containing streptomycin; the new colonies that grow on the filter must be streptomycin-resistant, containing only non-recombinant plasmids. Colonies containing recombinant plasmids, those that did not grow in streptomycin are easily identified on the original ampicillin agar plate. In practice, highly efficient recombination and transformation procedures typically reveal very streptomycinresistant cells (i.e., colonies) after replica plating. In this case, ampicillin-resistant cells constitute a good cDNA library, ready for screening.
263 Making a Replica Plate Filter
4. Identifying Colonies Containing Plasmids with Inserts of Interest
The next step is to screen the colonies from the cDNA library for those containing the specific cDNA that you’re after. This typically begins preparing multiple replica filters like the one above. Remember, these filters are replicas of bacterial cells containing recombinant plasmids that grow on ampicillin but not streptomycin. The number of replica filters that must be screened can be calculated from assumptions and formulas for estimating how many colonies must be screened to represent an entire transcriptome (i.e., the number of different mRNAs in the original cellular mRNA source). Once the requisite number of replica filters are made, they are subjected to in situ lysis to disrupt cell walls and membranes. The result is that the cell contents are released and the DNA is denatured (i.e., becomes single-stranded). The DNA then adheres to the filter in place (in situ, where the colonies were). The result of in situ lysis is a filter with faint traces of the original colony (below).
Next, a molecular probe is used to identify DNA containing the sequence of interest. The probe is often a synthetic oligonucleotide whose sequence was inferred from known amino acid sequences. These oligonucleotides are made radioactive and placed in a bag with the filter(s). DNA from cells that contained recombinant plasmids with a cDNA of interest will bind the complementary probe. The results of in situ lysis and hybridization of a radioactive probe to a replica filter are shown below.
264 Probing a Replica Plate Filter
The filters are rinsed to remove un-bound radioactive oligomer probe, and then placed on X-ray film. After a period of exposure, the film is developed. Black spots will form on the film from radioactive exposure, creating an autoradiograph of the filter. The black spots in the autoradiograph correspond to colonies on a filter that contain a recombinant plasmid with your target cDNA sequence (below).
Once a positive clone is identified on the film, the corresponding recombinant colony is located on the original plate. This colony is grown up in a liquid culture and the plasmid DNA is isolated. At that point, the cloned plasmid DNA can be sequenced and the amino acid sequence encoded by its cDNA can be inferred from the genetic code dictionary to verify that the cDNA insert in fact encodes the protein of interest. Once verified as the sequence of interest, a cloned plasmid cDNA can be made radioactive or fluorescent, and used to
• probe for the genes from which they originated.
• identify and quantitate the mRNA even locate the transcripts in the cells.
• quantitatively measure amounts of specific mRNAs.
Isolated plasmid cDNAs can even be expressed in suitable cells to make the encoded protein. These days, diabetics no longer receive pig insulin, but get synthetic human insulin human made from expressed human cDNAs. Moreover, while the introduction of the polymerase chain reaction (PCR, see below) has superseded some uses of cDNAs, they still play a role in genome-level and transcriptome-level studies.
265 Pick a Clone From a Replica Filter and Play With It! | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.01%3A_Recombinant_DNA/17.1.04%3A_Make_and_Screen_a_cDNA_Library.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.
17.02: Amplifying DNA Using the Polymerase Chain Reaction
Quantitative PCR (qPCR)
Measurements can be made of individual genes of interest through PCR of those specific genes. A process known as Real-Time PCR or quantitative PCR (qPCR) is used to measure individual genes using fluorescence measurements. An intercalating agent that binds only to double-stranded DNA called Sybr Green is used in a qPCR machine that is measuring fluorescence after each cycle of PCR indirectly indicates the amount of amplified product. However, non-specific products of amplification may also be measured and not discriminated from the authentic amplicon.
An alternative to Sybr Green is exemplified by the TaqMan technology. With TaqMan, a third primer (TaqMan probe) is designed in the middle of the area to be amplified. This middle primer is designed with a hairpin self-complementarity so that the 5′ and 3′ ends are in close proximity. At one end, a fluorescent reporter is attached while the other terminus has a quencher that absorbs any fluorescence signal. Under normal circumstances, measurements of fluorescence will be very low. When PCR extension occurs, the Polymerase hydrolyzes this middle primer, thereby separating the quencher and reporter. The name TaqMan is a play on words since it is imagined that the polymerase is chewing up the probe like Pacman. With increased distance between quencher/reporter, the fluorescence signal from this probe can now be measured. This method is much more specific than Sybr Green. However, the use of specific probes increases the cost considerably.
Threshold Cycles (Ct)
Credit: Zuzanna K. Filutowska (CC-BY-SA 3.0)
Fluorescence measurement early during the PCR process will be very low due to the small number of dsDNA molecules (Sybr Green) or most TaqMan primers being quenched. During this exponential DNA production, a threshold will be reached in which the fluorescence will linearly increase. A specific point where the fluorescence is clearly measurable is called the Threshold Cycle (Ct) is used as a reference point to compare expression values.
Looking at the example of Sybr Green qPCR above, it can be observed that samples exponentially increasing at a lower cycle number (Ct) has a higher level of mRNA expression (towards the left) of that gene than samples with higher cycle number (towards the right). Notice that the fluorescence eventually plateaus and stops increasing. This is due to the depletion of raw materials for DNA production like dNTPs.
Since the PCR reactions theoretically represent a doubling of DNA after each cycle, the Ct values can be interpreted on a base 2 system. If there is a difference in Ct between two samples (ΔCt) of 5 cycles, this corresponds to 25 or 32 fold difference. We can control for variations in the RNA preparation through comparing the fluorescence values of our gene of interest to a housekeeping gene like actin. The use of a house-keeping gene to normalize the initial input to the reactions and comparison between samples is referred to as Relative Quantification.
Melt Curves for Sybr Green
The top panel illustrates the decrease in fluorescence as the temperature increases due to the dissociation of double-stranded DNA. The bottom panel illustrates the first derivative plot. Each peak in this example illustrates a different allele. The double peaks represent the presence of the 2 distinct alleles in the amplification products.
Credit: Seans Potato Business (CC-BY-SA 3.0)
When using Sybr Green, we need to ensure that the PCR is specific so that the fluorescence measurement truly reflect amplification of our gene of interest. At the end of each qPCR run (~40 cycles), a melt curve is performed. A melting curve (or dissociation curve) comes from constant measurements as the temperature is increased. As temperature increases, the DNA strands start to denature and fluorescence will begin to decrease. After complete separation of DNA strands, the fluorescence will again remain constant. The way this curve is viewed is through a derivative plot where the inflection in fluorescence reading is reported as the melting temperature (Tm).
This melt curve illustrates each sample contains the same specific product with a melting temperature of 83.51°C.
Any peaks in this plot refer to a specific PCR product. If multiple peaks appear, the results will not be valid as they do not directly measure a single product.
Expression Measurements
Differential gene expression refers to transcriptional programs activated by the cell under various conditions. “Differential” refers to a comparison of two or more states or timepoints. Using mRNA as an indirect measurement of protein, one can ascertain which proteins are linked to these different states. In eukaryotes, this can be assessed by enriching total RNA for polyA-containing mature mRNA. Through the use of oligo-(dT) containing resin, mRNA can be separated from non-protein encoding RNA. Likewise, performing a reverse-transcription using an oligo-(dT) primer will create a stable complementary DNA (cDNA) molecule that can be used with PCR. Using qPCR in this way is called RT-PCR or reverse-transcription polymerase chain reaction where specific primer pairs are used to amplify a small portion of a known gene.
Hybridization-Based Methods and Microarrays:
Credit: FrozenMan (CC-BY-SA 4.0)
Prior to RT-PCR, the expression of individual genes was assessed through a hybridization-based approach. This method called for running RNA on an agarose gel and transferring the size-fractionated RNAs onto a membrane through a method called “blotting”. This transferred RNA was then hybridized to a radioactively labeled probe for a specific gene (corresponding to the reverse complementary sequence) and visualized by exposure to X-ray film in a process called Northern Blotting. The intensity of the band would be proportional to the amount of mRNA corresponding to the gene of interest. Re-probing with a housekeeping gene like actin would be used as a loading control to illustrate that a similar amount of total RNA was loaded into each well. Differences in sizes of the mRNA on the Northern Blot also revealed differences in splice variants of mature mRNA in the different states.
Credit: Jeremy Seto (CC-BY-NC-SA)
This technique was later adapted using non-radioactive methods. Using these non-radioactive methods, the reverse protocol was developed to measure multiple gene targets. By systematically immobilizing gene-specific probes onto a membrane or a microscope slide, an array of targets can be produced. In the simplest paradigm of having 2 states (control or experimental), cDNA from each sample can be used to generate fluorescent RNA that can hybridize to immobilized probes. Using 2 different fluorescent markers allows for the competitive hybridization onto the array whereby the fluorescent signal in each channel can reveal the differential gene expression of the two states in a 2-color microarray. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.02%3A_Amplifying_DNA_Using_the_Polymerase_Chain_Reaction/17.2.01%3A_qPCR.txt |
DNA fingerprinting is routinely used today to establish paternity, in the diagnosis of inherited disorders, and for use in criminal cases. DNA fingerprinting enables forensic investigators to determine whether two DNA samples originate from the same individual. Not all of the DNA present is used in this analysis. Restriction enzymes act as molecular scissors and are used to cleave DNA molecules at specific points. Over 2,500 different restriction enzymes have been identified. These enzymes are produced by bacteria and are used to destroy foreign DNA such as bacteriophages - viruses that infect and replicate within a bacterium.
For example the restriction enzyme EcoR1, isolated from E. coli, cuts DNA at the sequence GAATTC.
The length and the number of the fragments produced depends upon the frequency and the distance between the recognition sites. This distinct pattern is known as restriction fragment length polymorphisms (RFLP’s) which are unique to each individual therefore forming a DNA fingerprint. After DNA samples are cut by restriction enzymes, the fragments are separated using gel electrophoresis. PCR, polymerase chain reaction, can be used to analysis very small or degraded samples. This enzyme amplifies even trace amounts of DNA
present. The length of the segments analyzed are much smaller and the repeat sites are called microsatellites.
The phosphate group of the DNA molecule is negatively charged which gives the fragments an overall negative charge. In an electrical current the negatively charged fragments will be attracted to the positive pole. Smaller fragments will migrate faster and further in a given time period. Therefore the fragments are separated by size. After separation radioactive markers are added which are complementary to the separated fragments. Photographic film is placed over the gel and the areas that are exposed to the radioactive markers darken. This generates a series of lines that resemble a bar code. The film then becomes the DNA fingerprint. In this experiment we will be using a mixture of dyes that will simulate the migration of DNA fragments.
17.3.02: PCR-Based Mutagenesis
Molecular cloning was the first method available to isolate a gene of interest and make many copies of it to obtain sufficient amounts of the DNA to study. Today, there is a faster and easier way to obtain large amounts of a DNA sequence of interest -the polymerase chain reaction (PCR). PCR allows one to use the power of DNA replication to amplify DNA enormously in a short period of time. As you know, cells replicate their DNA before they divide, and in doing so, double the amount of the cell’s DNA. PCR essentially mimics cellular DNA replication in the test tube, repeatedly copying the target DNA over and over, to produce large quantities of the desired DNA.
Selective Replication
In contrast to cellular DNA replication, which amplifies all of a cell’s DNA during a replication cycle, PCR does targeted amplification to replicate only a segment of DNA bounded by the two primers that determine where DNA polymerase begins replication. Figure 8.34 illustrates the process. Each cycle of PCR involves three steps, denaturing, annealing and extension, each of which occurs at a different temperature.
The Starting Materials
Since PCR is, basically, replication of DNA in a test-tube, all the usual ingredients needed for DNA replication are required:
• A template (the DNA containing the target sequence that is being copied)
• Primers (to initiate the synthesis of the new DNA strands)
• Thermostable DNA polymerase (to carry out the synthesis). The polymerase needs to be heat stable, because heat is used to separate the template DNA strands in each cycle.
• dNTPs (DNA nucleotides to build the new DNA strands).
• The template is the DNA that contains the target you want to amplify (the "target" is the specific region of the DNA you want to amplify).
The primers are short synthetic single-stranded DNA molecules whose sequence matches a region flanking the target sequence. It is possible to chemically synthesize DNA molecules of any given base sequence, to use as primers. To make primers of the correct sequence that will bind to the template DNA, it is necessary to know a little bit of the template sequence on either side of the region of DNA to be amplified. DNA polymerases and dNTPs are commercially available from biotechnology supply companies.
First, all of the reagents are mixed together. Primers are present in millions of fold excess over the template. This is important because each newly made DNA strand starts from a primer. The first step of the process involves separating the strands of the target DNA by heating to near boiling.
Next, the solution is cooled to a temperature that favors complementary DNA sequences finding each other and making base pairs, a process called annealing. Since the primers are present in great excess, the complementary sequences they target are readily found and base-paired to the primers. These primers direct the synthesis of DNA. Only where a primer anneals to a DNA strand will replication occur, since DNA polymerases require a primer to begin synthesis of a new strand.
Figure 8.36 - A PCR thermocycler system. Wikipedia
Extension
In the third step in the process, the DNA polymerase replicates DNA by extension from the 3’ end of the primer, making a new DNA strand. At the end of the first cycle, there are twice as many DNA molecules, just as in cellular replication. But in PCR, the process is repeated, usually for between 25 and 30 cycles. At the end of the process, there is a theoretical yield of 230 (over 1 billion times) more DNA than there was to start. (This enormous amplification power is the reason that PCR is so useful for forensic investigations, where very tiny amounts of DNA may be available at a crime scene.)
The temperature cycles are controlled in a thermocycler, which repeatedly raises and lowers temperatures according to the set program. Since each cycle can be completed in a couple of minutes, the entire amplification can be completed very rapidly. The resulting DNA is analyzed on a gel to ensure that it is of the expected size, and depending on what it is to be used for, may also be sequenced, to be certain that it is the desired fragment.
Mutagenesis
PCR is frequently used to obtain gene sequences to be cloned into vectors for protein expression, for example. Besides simplicity and speed, PCR also has other advantages. Because primers can be synthesized that differ from the template sequence at any given position, it is possible to use PCR for site-directed mutagenesis. That is, PCR can be used to mutate a gene at a desired position in the sequence. This allows the proteins encoded by the normal and mutant genes to be expressed, purified and compared.
Analysis of gene expression
PCR can also be used to measure gene expression. Where in PCR the amount of amplified product is not determined till the end of all the cycles, a variation called quantitative real-time PCR is used, in which the accumulation of product is measured at each cycle. This is possible because real-time PCR machines have a detector module that can measure the levels of a fluorescent marker in the reaction, with the amount of fluorescence proportional to the amount of amplified product. By following the accumulation of product over the cycles it is possible to calculate the amount of starting template. To measure gene expression, the template used is mRNA reverse-transcribed into cDNA (see below). This coupling of reverse transcription with quantitative real-time PCR is called qRT-PCR. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.03%3A_Creating_Correcting_and_Analyzing_Genetic_Variation/17.3.01%3A_DNA_Fingerprinting.txt |
The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders.
CRSPR
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, short repeated sequences found in prokaryotic DNA, separated by spacer sequences derived from past encounters with, for example, a bacteriophage. Like the glass slipper left behind by Cinderella that was later used to identify her, the pieces of the invader's sequences are a way for the bacteria to identify the virus if it attacks again. Inserted into the bacterial genome, these sequences can later be transcribed into a guide RNA that matches, and base-pairs with, sections of the viral genome if it was encountered again. A nuclease associated with the guide RNA then cleaves the sequence base-paired with the guide RNA. (The nucleases are named Cas for CRISPR-associated.)
The essential elements of this system are a guide RNA that homes in on the target sequence and a nuclease that can make a cut in the sequence that is bound by the guide RNA. By engineering guide RNAs complementary to a target gene, it is possible to target the nuclease to cleave within that gene. In the CRISPR/Cas9 system, the Cas9 endonuclease cuts both strands of the gene sequence targeted by the guide RNA (Figure \(1\)). This generates a double-strand break that the cell attempts to repair.
As you may remember, double-strand breaks in DNA can be repaired by simple, nonhomologous end joining (NHEJ) or by homologous recombination. When a break is fixed by NHEJ, there is good chance that there will be deletions or insertions that will inactivate the gene they are in. Thus, targeted cleavage of a site by CRISPR/Cas9 can easily and specifically inactivate a gene, making it easy to characterize the gene's function.
But, what if you wished to simply mutate the gene at a specific site to study the effect of the mutation? This, too, can be achieved. If a homologous sequence bearing the specific mutation is provided, homologous recombination can repair the break, and at the same time insert the exact mutation desired. It is obvious that if you can insert a mutation as just described, it should be possible to correct a mutation in the genome by cleaving at the appropriate spot and providing the correct sequence as a template for repair by homologous recombination. The simplicity of the system holds great promise for curing genetic diseases.
Scientists have also come up with some creative variations on the CRISPR/Cas9 system. For instance, one variant inactivates the nuclease activity of Cas9. The guide RNA in this system pairs with the target sequence, but the Cas9 does not cleave it. Instead, the Cas9 blocks the transcription of the downstream gene (Figure \(2\)) This method allows specific genes to be turned off without actually altering the DNA sequence.
Another variation also uses a disabled Cas9, but this time, the Cas9 is fused to a transcriptional activation domain. In this situation, the guide RNA positions the Cas9-activator domain in a place where it can enhance transcription from a specific promoter (Figure \(3\)). Other variations on this theme attach histone-modifying enzymes or DNA methylases to the inactive Cas9. Again, the guide RNA positions the Cas9 in the desired spot, and the enzyme attached to Cas9 can methylate the DNA or modify the histones in that region.
CRISPR has already been used to edit genomes in a wide variety of species (and in human cell cultures). It may not be long before the technique is approved for clinical use. In the meanwhile, CRISPR is transforming molecular biology. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.03%3A_Creating_Correcting_and_Analyzing_Genetic_Variation/17.3.03%3A_Genome_Editing_%28CRISPR%29.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/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.04%3A_Constructing_and_Using_Transgenic_Organisms/17.4.01%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/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.04%3A_Constructing_and_Using_Transgenic_Organisms/17.4.02%3A_Transgenic_Plants.txt |
Bioremediation is a waste management technique that involves the use of organisms such as plants, bacteria, and fungi to remove or neutralize pollutants from a contaminated site. According to the United States EPA, bioremediation is a “treatment that uses naturally occurring organisms to break down hazardous substances into less toxic or non toxic substances”.
Bioremediation is widely used to treat human sewage and has also been used to remove agricultural chemicals (pesticides and fertilizers) that leach from soil into groundwater. Certain toxic metals, such as selenium and arsenic compounds, can also be removed from water by bioremediation. Mercury is an example of a toxic metal that can be removed from an environment by bioremediation. Mercury is an active ingredient of some pesticides and is also a byproduct of certain industries, such as battery production. Mercury is usually present in very low concentrations in natural environments but it is highly toxic because it accumulates in living tissues. Several species of bacteria can carry out the biotransformation of toxic mercury into nontoxic forms. These bacteria, such as Pseudomonas aeruginosa, can convert Hg2+ to Hg, which is less toxic to humans.
Probably one of the most useful and interesting examples of the use of prokaryotes for bioremediation purposes is the cleanup of oil spills. The importance of prokaryotes to petroleum bioremediation has been demonstrated in several oil spills in recent years, such as the Exxon Valdez spill in Alaska (1989) (Figure \(1\)), the Prestige oil spill in Spain (2002), the spill into the Mediterranean from a Lebanon power plant (2006,) and more recently, the BP oil spill in the Gulf of Mexico (2010). To clean up these spills, bioremediation is promoted by adding inorganic nutrients that help bacteria already present in the environment to grow. Hydrocarbon-degrading bacteria feed on the hydrocarbons in the oil droplet, breaking them into inorganic compounds. Some species, such as Alcanivorax borkumensis, produce surfactants that solubilize the oil, while other bacteria degrade the oil into carbon dioxide. In the case of oil spills in the ocean, ongoing, natural bioremediation tends to occur, inasmuch as there are oil-consuming bacteria in the ocean prior to the spill. Under ideal conditions, it has been reported that up to 80 percent of the nonvolatile components in oil can be degraded within 1 year of the spill. Researchers have genetically engineered other bacteria to consume petroleum products; indeed, the first patent application for a bioremediation application in the U.S. was for a genetically modified oil-eating bacterium.
There are a number of cost/efficiency advantages to bioremediation, which can be employed in areas that are inaccessible without excavation. For example, hydrocarbon spills (specifically, oil spills) or certain chlorinated solvents may contaminate groundwater, which can be easier to treat using bioremediation than more conventional approaches. This is typically much less expensive than excavation followed by disposal elsewhere, incineration, or other off-site treatment strategies. It also reduces or eliminates the need for “pump and treat”, a practice common at sites where hydrocarbons have contaminated clean groundwater. Using prokaryotes for bioremediation of hydrocarbons also has the advantage of breaking down contaminants at the molecular level, as opposed to simply chemically dispersing the contaminant. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.05%3A_Environmental_Applications/17.5.01%3A_Bioremediation.txt |
Resolution of the global water pollution crisis requires multiple approaches to improve the quality of our fresh water and move towards sustainability. The most deadly form of water pollution, pathogenic microorganisms that cause waterborne diseases, kills almost 2 million people in underdeveloped countries every year. The best strategy for addressing this problem is proper sewage (wastewater) treatment. Untreated sewage is not only a major cause of pathogenic diseases, but also a major source of other pollutants, including oxygen-demanding waste, nutrients (N and P, particularly), and toxic heavy metals. Wastewater treatment is done at a sewage treatment plant in urban areas and through a septic tank system in rural areas.
The main purpose of sewage (wastewater) treatment is to remove organic matter (oxygen-demanding waste) and kill bacteria. Special methods also can be used to remove nutrients and other pollutants. The numerous steps at a conventional sewage treatment plant include pretreatment (screening and removal of sand and gravel), primary treatment (settling or floatation to remove organic solids, fat, and grease), secondary treatment (aerobic bacterial decomposition of organic solids), tertiary treatment (bacterial decomposition of nutrients and filtration), disinfection (treatment with chlorine, ozone, ultraviolet light, or bleach to kill most microbes), and either discharge to surface waters (usually a local river) or reuse for some other purpose, such as irrigation, habitat preservation, and artificial groundwater recharge (Figure \(1\)).
The concentrated organic solid produced during primary and secondary treatment is called sludge, which is treated in a variety of ways including landfill disposal, incineration, use as fertilizer, and anaerobic bacterial decomposition, which is done in the absence of oxygen. Anaerobic decomposition of sludge produces methane gas, which can be used as an energy source. To reduce water pollution problems, separate sewer systems (where street runoff goes to rivers and only wastewater goes to a wastewater treatment plant) are much better than combined sewer systems, which can overflow and release untreated sewage into surface waters during heavy rain. Some cities such as Chicago, Illinois have constructed large underground caverns and also use abandoned rock quarries to hold storm sewer overflow. After the rain stops, the stored water goes to the sewage treatment plant for processing.
A septic tank system is an individual sewage treatment system for homes in typically rural settings. The basic components of a septic tank system (Figure \(2\)) include a sewer line from the house, a septic tank (a large container where sludge settles to the bottom and microorganisms decompose the organic solids anaerobically), and the drain field (network of perforated pipes where the clarified water seeps into the soil and is further purified by bacteria). Water pollution problems occur if the septic tank malfunctions, which usually occurs when a system is established in the wrong type of soil or maintained poorly.
For many developing countries, financial aid is necessary to build adequate sewage treatment facilities. The World Health Organization estimates an estimated cost savings of between \$3 and \$34 for every \$1 invested in clean water delivery and sanitation. The cost savings are from health care savings, gains in work and school productivity, and prevented deaths. Simple and inexpensive techniques for treating water at home include chlorination, filters, and solar disinfection. Another alternative is to use constructed wetlands technology (marshes built to treat contaminated water), which is simpler and cheaper than a conventional sewage treatment plant.
Bottled water is not a sustainable solution to the water crisis. Bottled water is not necessarily any safer than the U.S. public water supply, it costs on average about 700 times more than U.S. tap water, and every year it uses approximately 200 billion plastic and glass bottles that have a relatively low rate of recycling. Compared to tap water, it uses much more energy, mainly in bottle manufacturing and long-distance transportation. If you don’t like the taste of your tap water, then please use a water filter instead of bottled water!
CLEAN WATER ACT
During the early 1900s rapid industrialization in the U.S. resulted in widespread water pollution due to free discharge of waste into surface waters. The Cuyahoga River in northeast Ohio caught fire numerous times, including a famous fire in 1969 that caught the nation’s attention. In 1972 Congress passed one of the most important environmental laws in U.S. history, the Federal Water Pollution Control Act, which is more commonly called the Clean Water Act. The purpose of the Clean Water Act and later amendments is to maintain and restore water quality, or in simpler terms to make our water swimmable and fishable. It became illegal to dump pollution into surface water unless there was formal permission and U.S. water quality improved significantly as a result. More progress is needed because currently the EPA considers over 40,000 U.S. water bodies as impaired, most commonly due to pathogens, metals, plant nutrients, and oxygen depletion. Another concern is protecting groundwater quality, which is not yet addressed sufficiently by federal law.
17.5C: Genomics and Biofuels
Microbial genomics can be used to create new biofuels.
Learning Objectives
• Explain the process of creating new biofuels by using microbial genomics
Key Points
• Microorganisms can encode new enzymes and produce new organic compounds that can be used as biofuels.
• Genomic analysis of the fungus Pichia will allow optimization of its use in fermenting ethanol fuels.
• Analysis of the microbes in the hindgut of termites have found 500 genes that may be useful in enzymatic destruction of cellulose.
• Genetic markers have been used in forensic analysis, like in 2001 when the FBI used microbial genomics to determine a specific strain of anthrax that was found in several pieces of mail.
• Genomics is used in agriculture to develop plants with more desirable traits, such as drought and disease resistance.
Key Terms
• renewable resource: a natural resource such that it is replenished by natural processes at a rate comparable to its rate of consumption by humans or other users
• biofuel: any fuel that is obtained from a renewable biological resource
Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population ‘s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped.
For microbial biomass breakdown, many candidates have already been identified. These include Clostridia species for their ability to degrade cellulose, and fungi that express genes associated with the decomposition of the most recalcitrant features of the plant cell wall, lignin, the phenolic “glue” that imbues the plant with structural integrity and pest resistance. The white rot fungus Phanerochaete chrysosporium produces unique extracellular oxidative enzymes that effectively degrade lignin by gaining access through the protective matrix surrounding the cellulose microfibrils of plant cell walls.
Another fungus, the yeast Pichia stipitis, ferments the five-carbon “wood sugar” xylose abundant in hardwoods and agricultural harvest residue. Pichia‘s recently-sequenced genome has revealed insights into the metabolic pathways responsible for this process, guiding efforts to optimize this capability in commercial production strains. Pathway engineering promises to produce a wider variety of organisms able to ferment the full repertoire of sugars derived from cellulose and hemicellulose and tolerate higher ethanol concentrations to optimize fuel yields. For instance, the hindgut contents of nature’s own bioreactor, the termite, has yielded more than 500 genes related to the enzymatic deconstruction of cellulose and hemicellulose. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.05%3A_Environmental_Applications/17.5.02%3A_Water_Treatment.txt |
Protein Expression
Recombinant DNA technology has many uses in basic scientific research to better understand the nature of living things. As a tool, recombinant DNA technology can be used to express proteins towards medical applications. Prior to biotechnology, type I diabetes (insulin-dependent) was treated by injection of insulin isolated from the pancreas of pigs. With the ability to express human proteins inside bacteria, yeast, and other cells, sacrificing pigs for porcine insulin is no longer necessary.
Bacterial expression vector pGEX-3X contains the AmpR gene, the origin of replication, MCS downstream of the hybrid lac/trp promoter (tac) and the coding sequence for glutathione-S-transferase (GST). GST acts as a tag that is fused directly with the protein from the gene of interest and used to purify the protein with a glutathione resin.
Bacteria or other cells can be engineered to express proteins through the process of cloning and transformation. Bacteria are advantageous because of their rapid life cycle and ease of growth. A bacterial expression vector contains the basic plasmid features: the origin of replication as well as antibiotic resistance gene. Often, an affinity tag will be used to aid in the purification of the protein. An example in the vector above shows the GST (glutathione-s-transferase) tag that can be purified with glutathione resin. Expression is only the first problem since bacteria are also synthesizing proteins that are required for the bacteria to grow and divide. Injecting these proteins in addition to insulin would cause an immune reaction that could be deadly. Therefore, it is required that overexpressed proteins be purified and isolated from other undesirable proteins.
Credit: Stewart EJ, Madden R, Paul G, Taddei F (CC-SA 3.0).
Criteria for Choosing an Expression System
Protein expression systems have inherent advantages and disadvantages. The table above summarizes the comparison of the various cellular systems of production (Fernandez & Hoeffler, 1999).
Purification
Different methods of isolation can be applied depending on the properties of the protein. Ion exchange chromatography is useful if the protein of interest has a specific charge that will interact with a resin packed with the opposite charge.
Immunoprecipitation
Immunoprecipitation: Column is packed with Protein-A agarose which binds to antibodies. Cell lysates are then loaded onto the columns where they flow through and are allowed to interact with the antibody. Washes are performed to remove the non-specifically bound proteins. An elution buffer is used to disrupt the interaction of the antibody to the protein target.
Affinity Purification
Affinity purification employs the use of specific antibodies that bind to the protein of interest very tightly to retain it on a column. With these techniques, the protein retained on the resin is washed numerous times to remove other proteins that are non-specifically sticking. A change in pH or ionic conditions then is used to disrupt the interaction with the resin and elute the proteins from the column. Proteins that are engineered to contain tags can be purified by antibodies specific to those tags. Also, the addition of 6 or more consecutive Histidine residues to the end of a protein makes them susceptible to purification with Nickel-NTA resin or Cobalt purification. In these cases, the 6XHis tag associates with these metal ions on the resin are selectively adhered.
Nickel NTA resin coordinating the capture of a 6His tagged protein.
Size Exclusion
Credit: Mydriatic (CC-BY-SA 3.0)
Credit: Takometer(CC-BY 2.5)
Most of you are familiar with water purification filters. Before using these filters, you soak them in water and dark residue leaks out. This dark residue is activated charcoal. The activated charcoal has tiny microscopic pores that trap small items like ions and other particles. The primary goal of these filters is to remove metals and chlorine that are found in tap water. The porous nature of activated charcoal renders it useful for trapping molecules in water purification systems.
The process used to trap these small particles is called size exclusion. Unlike agarose gel electrophoresis where the smaller particles navigate through the matrix faster, size exclusion resins trap the smaller molecules.
The smaller the molecule, the longer they spend within the pores as they traverse through the matrix.
Significance of Purification
Credit: Hans Hillewaert (CC-BY)
All injectable drugs must be clean of endotoxins from bacteria. Purification of the protein of interest from bacterial lysates removes the dangerous pathogenic materials from that would otherwise activate host immune reactivity.
The horseshoe crab (Limulus polyphemus) performs a special function in the ecosystem by providing eggs for migratory birds to feed on. This organism also houses a special cell type in its hemolymph. The Limulus amoebocyte lysate (LAL) test is the most sensitive assay of detecting endotoxins from bacteria. Amoebocytes are collected from these organisms for use on testing batches of injectable drugs to ensure proper purification and safety. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.06%3A_Medical_Applications/17.6.01%3A_Recombinant_Protein_Production.txt |
Bright Field Microscopy
How can we confirm that a person has a specific chromosomal abnormality? The first method was simply to obtain a sample of their cells, stain the chromosomes with Giemsa dye, and examine the results with a light microscope (Figure \(1\)). Each chromosome can be recognized by its length, the location of its centromere, and the characteristic pattern of purple bands produced by the Giemsa. For example, if mitotic cells from a person consistently contained forty seven chromosomes in total with three chromosome 21s this would be indicative of Down syndrome. Bright field microscopy does has its limitations though - it only works with mitotic chromosomes and many chromosome rearrangements are either too subtle or too complex for even a skilled cytogeneticist to discern.
Fluorescence In Situ Hybridization
The solution to these problems was fluorescence in situ hybridization (FISH). The technique is similar to a Southern blot in that a single stranded DNA probe is allowed to hybridize to denatured target DNA (see Section 8.6). However, instead of the probe being radioactive it is fluorescent and instead of the target DNA being restriction fragments on a nylon membrane it is denatured chromosomes on a glass slide. Because there are several fluorescent colours available it is common to use more than one probe at the same time. Typically the chromosomes are also labeled with a fluorescent stain called DAPI which gives them a uniform blue colour. If the chromosomes have come from a mitotic cell it is possible to see all forty six of them spread out in a small area. Alternatively, if the chromosomes are within the nucleus of an interphase cell they appear together within a large blue circle.
Using FISH to Diagnose Down Syndrome
Most pregnancies result in healthy children. However in some cases there is an elevated chance that the fetus has trisomy-21. Older women are at a higher risk because the non-disjunction events that lead to trisomy become more frequent with age. The second consideration is what the fetus looks like during an ultrasound examination. Fetuses with trisomy-21 and some other chromosome abnormalities have a swelling in the back of the neck called a nuchal translucency. If either or both factors is present the woman may choose amniocentesis. In this test some amniotic fluid is withdrawn so that the fetal cells within it can be examined. Figure \(2\) shows a positive result for trisomy-21. Based upon this image the fetus has two X chromosomes and three chromosome 21s and therefore has a karyotype of 47,XX,+21.
Using FISH to Diagnose Cri-du-Chat Syndrome
A physician may suspect that a patient has a specific genetic condition based upon the patient's physical appearance, mental abilities, health problems, and other factors. FISH can be used to confirm the diagnosis. For example, Figure \(3\) shows a positive result for cri-du-chat syndrome. The probes are binding to two long arms of chromosome 5 but only one short arm. One of the chromosome 5s must therefore be missing part of its short arm.
Newer Techniques
FISH is an elegant technique that produces dramatic images of our chromosomes. Unfortunately, FISH is also expensive, time consuming, and requires a high degree of skill. For these reasons, FISH is slowly being replaced with PCR and DNA chip based methods. Versions of these techniques have been developed that can accurately quantify a person's DNA. For example a sample of DNA from a person with Down syndrome will contain 150% more DNA from chromosome 21 than the other chromosomes. Likewise DNA from a person with cri-du-chat syndrome will contain 50% less DNA from the end of chromosome 5. These techniques are very useful if the suspected abnormality is a deletion, a duplication, or a change in chromosome number. They are less useful for diagnosing chromosome inversions and translocations because these rearrangements often involve no net loss or gain of genes.
In the future all of these techniques will likely be replaced with DNA sequencing. Each new generation of genome sequencing machines can sequence more DNA in less time. Eventually it will be cheaper just to sequence a patient's entire genome than to use FISH or PCR to test for specific chromosome defects. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.06%3A_Medical_Applications/17.6.02%3A_Fluorescence_In_Situ_Hybridization.txt |
2D gels are one way of surveying a broad spectrum of molecules simultaneously. Other approaches to doing the same thing involve what are called microarrays. DNA microarrays, for example, can be used to determine all of the genes that are being expressed in a given tissue, simultaneously. Microarrays employ a grid (or array) made of rows and columns on a glass slide, with each box of the grid containing many copies of a specific molecule, say a single-stranded DNA molecule corresponding to the sequence of a single unique gene. As an example, consider scanning the human genome for all of the known mRNA sequences and then synthesizing single stranded DNAs complementary to each mRNA. Each complementary DNA sequence would have its own spot on the matrix. The position of each unique gene sequence on the grid is known and the entire grid would represent all possible genes that are expressed. Then for a simple gene expression analysis, one could take a tissue (say liver) and extract the mRNAs from it. These mRNAs represent all the genes that are being expressed in the liver at the time the extract was made.
The mRNAs can easily be tagged with a colored dye (say blue). The mixture of tagged mRNAs is then added to the array and base-pairing conditions are created to allow complementary sequences to find each other. When the process is complete, each liver mRNA should have bound to its corresponding gene on the array, creating a blue spot in that box on the grid. Since it is known which genes are in which box, a blue spot in a box indicates that the gene in that box was expressed in the liver. The presence and abundance of each mRNA may then readily determined by measuring the amount of blue dye at each box of the grid. A more powerful analysis could be performed with two sets of mRNAs, each with a different colored tag (say blue and yellow). One set of mRNAs could come from the liver of a vegetarian (tagged blue) and the other from a meat eater (tagged yellow), for example. The mRNAs are mixed and then added to the array and complementary sequences are once again allowed to form duplexes. After unhybridized mRNAs are washed away, the plate is analyzed. Blue spots in grid boxes correspond to mRNAs present in the vegetarian liver, but not in that of the meat eater. Green spots (blue plus yellow) would correspond to mRNAs present in equal abundance in the two livers. The intensity of each spot would also give information about the relative amounts of each mRNA in the tissues. Similar analyses could be done, using cDNAs instead of mRNA. Peptide microarrays have peptides bonded to the glass slide instead of DNA and can be used to study the binding of proteins or other molecules to the peptides.
17.6.04: Immunoassays
Enzyme-linked immunosorbent assay (ELISA) is a solid-phase enzyme immunoassay used to detect the presence of a substance in solution.
Learning Objectives
• Describe how the Enzyme-linked immunosorbent assay (ELISA) can be used to detect and quantitate antigens, antibodies and allergens
Key Points
• ELISA is a quantitative technique that measures serum concentration of antigens, antibodies, and allergens.
• Standard ELISA uses antibody-antigen-antibody trapping principle with the second antibody coupled to an enzyme. If the complex is formed, the enzyme converts a clear solution into a colored one that can be measured with a spectrophotometer.
• ELISA is performed in a muti-well microtiter plate. In addition to the test solution, standard solutions are added with known antigen concentration. These solutions will be used to infer the concentration of the antigen being tested.
Key Terms
• spectrophotometrically: By using spectrophotometry.
• epitope: That part of a biomolecule (such as a protein) that is the target of an immune response.
Enzyme-linked immunosorbent assay (ELISA) is a method of quantifying an antigen immobilized on a solid surface. ELISA uses a specific antibody with a covalently coupled enzyme. The amount of antibody that binds the antigen is proportional to the amount of antigen present, which is determined by spectrophotometrically measuring the conversion of a clear substance to a colored product by the coupled enzyme.
Several variations of ELISA, seen in, exist but the most commonly used method is the sandwich ELISA. The sandwich assay uses two different antibodies that are reactive with different epitopes on the antigen with a concentration that needs to be determined. A fixed quantity of one antibody is attached to a series of replicate solid supports, such as plastic microtiter multi-well plate. Test solutions containing antigen at an unknown concentration are added to the wells and allowed to bind. Unbound antigen is removed by washing, and a second antibody which is linked to an enzyme is allowed to bind. This second antibody-enzyme complex constitutes the indicator system of the test. The antigen serves as bridge, so the more antigen in the test solution, the more enzyme-linked antibody will bind. The test solution is used in parallel with a series of standard solutions with known concentrations of antigen that serve as control and reference. The results obtained from the standard solutions are used to construct a binding curve of the second antibody as a function of antigen concentration. The concentration of antigens can be inferred from absorbance readings of standard solutions. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.06%3A_Medical_Applications/17.6.03%3A_Microarrays.txt |
It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes provides methods to treat the disease.
Genetic Diagnosis and Gene Therapy
The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, women diagnosed with breast cancer are usually advised to have a biopsy so that the medical team can determine the genetic basis of cancer development. Treatment plans are based on the findings of genetic tests that determine the type of cancer. If the cancer is caused by inherited gene mutations, other female relatives are also advised to undergo genetic testing and periodic screening for breast cancer. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.
Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA (Figure 1). More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID).
Here’s a website to help you learn more information on SCID and its gene therapy trials.
Production of Vaccines, Antibiotics, and Hormones
Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory. Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.
Antibiotics are a biotechnological product. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.
Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. In addition, human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector.
Learning Objectives
Transgenic organisms possess DNA from a different species, usually generated by molecular cloning techniques. Vaccines, antibiotics, and hormones are examples of products obtained by recombinant DNA technology. Transgenic plants are usually created to improve characteristics of crop plants.
Contributors and Attributions
CC licensed content, Shared previously | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.06%3A_Medical_Applications/17.6.05%3A_Gene_Therapy.txt |
Agricultural biotechnology is a range of tools, including traditional breeding techniques, that alter living organisms, or parts of organisms, to make or modify products; improve plants or animals; or develop microorganisms for specific agricultural uses. Modern biotechnology today includes the tools of genetic engineering. Genetic engineering is the name for certain methods that scientists use to introduce new traits or characteristics to an organism (known also as genetically modified organism or GMO). For example, plants may be genetically modified to produce characteristics to enhance the growth or nutritional profile of food crops.
Benefits of Genetic Engineering
Advocates of modern biotechnology and generic engineering say that the application of biotechnology in agriculture has resulted in benefits to farmers, producers, and consumers.
Enhanced nutrition. Advances in biotechnology may provide consumers with foods that are nutritionally-enriched (Figure below) or longer-lasting, or that contain lower levels of certain naturally occurring toxicants present in some food plants. Developers are using biotechnology to try to reduce saturated fats in cooking oils, reduce allergens in foods, and increase disease-fighting nutrients in foods. Biotechnology may also be used to conserve natural resources, enable animals to more effectively use nutrients present in feed, decrease nutrient runoff into rivers and bays, and help meet the increasing world food and land demands.
Figure \(1\): White rice and Golden rice. Genetically engineered “Golden Rice” contains up to 35 μg β-carotene per gram of rice.
Cheaper and more manageable production. Biotechnology may provide farmers with tools that can make production cheaper and more manageable. For example, some biotechnology crops can be engineered to tolerate specific herbicides, which make weed control simpler and more efficient. Other crops have been engineered to be resistant to specific plant diseases and insect pests, which can make pest control more reliable and effective, and/or can decrease the use of synthetic pesticides. These crop production options can help countries keep pace with demands for food while reducing production costs.
Improved pest control. Biotechnology has helped to make both insect pest control and weed management safer and easier while safeguarding crops against disease. For example, genetically engineered insect-resistant cotton has allowed for a significant reduction in the use of persistent, synthetic pesticides that may contaminate groundwater and the environment. In terms of improved weed control, herbicide-tolerant soybeans, cotton, and corn enable the use of reduced-risk herbicides that break down more quickly in soil and are non-toxic to wildlife and humans.
Concerns about Genetically modified Organisms
The complexity of ecological systems presents considerable challenges for experiments to assess the risks and benefits and inevitable uncertainties of GMOs. Assessing such risks is extremely difficult, because both natural and human-modified systems are highly complex, and fraught with uncertainties that may not be clarified until long after an experimental introduction has been concluded. Critics of GMOs warn that the cultivation of GMOs, with their potential benefits and hazards to the environment, should be carefully considered within broader ecosystems.
Interbreeding with native species. When the genetically modified organisms are allowed to breed with the organisms which are not genetically engineered, then these organisms may affect the genetic of non-genetically engineered organisms. Due to this reason the whole ecological system might get affected. The main concern is that genetically modified organisms might lead the non-GM organisms to extinction and reduce biodiversity.
GM food labeling. In order to verify whether people have been harmed over the years by consuming GMF, specifically in countries like the US where people's dietary are mainly composed of such products, the law for mandatory labeling is highly required. However, the labeling is not just about health issue rather, it is about consumer rights to make an informed choice. Although a consensual system on GMO labeling is crucial, it seems unlikely that an internationally agreed labeling system can be set up in proximate future. Nevertheless, different GMO labeling schemes have been established in different countries, ranging from stringent to extremely lenient or even non existent legislations. While the EU has established strict labeling regulations, in the US, Canada and Argentina, three big producers of GMO food, such laws have been put forward but not enacted by these governments. A proper labeling represents the “GM” word, along with additional information on changed characteristics and the external source of the inserted gene. Negative labeling such as “GM free” is not suggested, because it might give the wrong impression to the consumers. The law for compulsory labeling of genetically modified food products has been established in more than 40 countries. Surveys commissioned by different organizations have shown that people across the world are seeking for transparency and consumer choice and believe that compulsory labeling scheme on GM ingredients is highly required: 88% Canadians, 92% Americans and 93% French.
Consumers right to choose. The International Federation of Organic Agriculture Movement has made stringent efforts to keep GMOs out of organic production, yet some US organic farmers have found their corn (maize) crops, including seeds, to contain detectable levels of genetically engineered DNA. The organic movement is firm in its opposition to any use of GMOs in agriculture, and organic standards explicitly prohibit their use. The farmers, whose seed is contaminated, have been under rigid organic certification, which assures that they did not use any kind of genetically modified materials on their farms. Any trace of GMOs must have come from outside their production areas. While the exact origin is unclear at this time, it is most likely that the pollution has been caused by pollen drift from GMO-fields in surrounding areas. However, the contamination may have also come from the seed supply. Seed producers, who intended to supply GMO-free seed, have also been confronted with genetic contamination and cannot guarantee that their seed is 100% GMO-free.
Ecological long-term effects. The Bt corn produces wind-borne pollen that kills the caterpillars of the Monarch butterfly. If the life cycles of this butterfly are disrupted, the Monarch butterflies might be endangered. Agriculture might be affected as the weeds acquire the modified genes to become more competitive. The risk of the evolution of common plant viruses to become more resistant or form new strains will be greatly increased. If genetic modification is carried out extensively, new viruses with greater potential to harm humankind may evolve, and the probability of this occurring can be quite high.
Human health risk. At least some of the genes used in GMOs may not have been used in the food supply before, so GM foods may pose a potential risk for human health. Much of the GM production currently grown worldwide is destined for animal feed. The FAO has concluded that risks to human and animal health from the use of GM crops and enzymes derived from GM microorganisms as animal feed are negligible. But scientists acknowledge that little is known about the long-term safety of consuming food made from GM products. WHO recognizes the need for continued safety assessments on genetically modified foods before they are marketed to prevent risks to human health and for continued monitoring.
The potential of GM crops to be allergenic is one of the main suspected adverse health effects. Many scientific data indicate that animals fed by GMO crops have been harmed or even died. Rats exposed to transgenic potatoes or soy had abnormal young sperm; cows, goats, buffalo, pigs and other livestock grazing on Bt-maize, GM cottonseed and certain biotech corn showed complications including early deliveries, abortions, infertility and also many died. However, this is a controversial subject as studies conducted by company producing the biotech crops did not show any negative effects of GM crops on mice.
Although Agri-biotech companies do not accept the direct link between the GMOs consumption and human health problems, there are some examples given by the opponents. For example: The foodborne diseases such as soy allergies have increased over the past 10 years in USA and UK and an epidemic of Morgellons disease in the US. There are also reports on hundreds of villagers and cotton handlers who developed skin allergy in India. Recent studies have revealed that Bacillus thuringiensis corn expresses an allergenic protein which alters overall immunological reactions in the body. The aforementioned reports performed by independent GMO researchers have lead to a concern about the risks of GMOs and the inherent risks associated with the genetic technology.
Intellectual property rights are one of the important factors in the current debate on GMOs. The GM crops are patented by Agri-business companies leading to monopolization of the global agricultural food and controlling distribution of the world food supply. Social activists believe that the hidden reason why biotech companies are eager to produce GMO crops is because they can be privatized, unlike ordinary crops which are the natural property of all humanity. It is argued for example that to achieve this monopoly, the large Agri-biotech company, Monsanto, has taken over small seed companies in the past 10 years and has become the biggest Agri-biotech Corporation in the world. The patent right for vegetable forms of life also affect the livelihoods of family farmers as they are required to sign a contract preventing them from saving and re-planting the seeds, thus they have to pay for seeds each year.
Critics, thus advise that the risks for the introduction of a GMOs into each new ecosystem need to be examined on a case-by-case basis, alongside appropriate risk management measures, such as through the precautionary principle in the Cartagena Protocol and the IPPC’s Pest Risk Assessment (PRA). | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/17%3A_Biotechnology/17.07%3A_Agricultural_Applications.txt |
• 18.1: Mapping Genomes
Genome mapping is similar to solving a big, complicated puzzle with pieces of information coming from laboratories all over the world. Genetic maps provide an outline for the location of genes within a genome, and they estimate the distance between genes and genetic markers on the basis of the recombination frequency during meiosis. Physical maps provide detailed information about the physical distance between the genes. The most detailed information is available through sequence mapping.
• 18.2: Sequencing Genomes
Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by some diseases, and they are using whole-genome sequencing to get to the bottom of the problem. Whole-genome sequencing is a process that determines the DNA sequence of an entire genome. Whole-genome sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease.
• 18.3: Genome Projects
• 18.4: Genome Annotation and Databases
• 18.5: Comparative and Functional Genomics
• 18.6: Applications of Genomics
The introduction of DNA sequencing and whole genome sequencing projects, particularly the Human Genome project, has expanded the applicability of DNA sequence information. Genomics is now being used in a wide variety of fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics. The most commonly known application of genomics is to understand and find cures for diseases.
18: Genomics
The study of nucleic acids began with the discovery of DNA, progressed to the study of genes and small fragments, and has now exploded to the field of genomics. Genomics is the study of entire genomes, including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species. The advances in genomics have been made possible by DNA sequencing technology. Just as information technology has led to Google Maps that enable us to get detailed information about locations around the globe, genomic information is used to create similar maps of the DNA of different organisms.
Mapping Genomes
Genome mapping is the process of finding the location of genes on each chromosome. The maps that are created are comparable to the maps that we use to navigate streets. A genetic map is an illustration that lists genes and their location on a chromosome. Genetic maps provide the big picture (similar to a map of interstate highways) and use genetic markers (similar to landmarks). A genetic marker is a gene or sequence on a chromosome that shows genetic linkage with a trait of interest. The genetic marker tends to be inherited with the gene of interest, and one measure of distance between them is the recombination frequency during meiosis. Early geneticists called this linkage analysis.
Physical maps get into the intimate details of smaller regions of the chromosomes (similar to a detailed road map) (Figure \(1\)). A physical map is a representation of the physical distance, in nucleotides, between genes or genetic markers. Both genetic linkage maps and physical maps are required to build a complete picture of the genome. Having a complete map of the genome makes it easier for researchers to study individual genes. Human genome maps help researchers in their efforts to identify human disease-causing genes related to illnesses such as cancer, heart disease, and cystic fibrosis, to name a few. In addition, genome mapping can be used to help identify organisms with beneficial traits, such as microbes with the ability to clean up pollutants or even prevent pollution. Research involving plant genome mapping may lead to methods that produce higher crop yields or to the development of plants that adapt better to climate change.
Genetic maps provide the outline, and physical maps provide the details. It is easy to understand why both types of genome-mapping techniques are important to show the big picture. Information obtained from each technique is used in combination to study the genome. Genomic mapping is used with different model organisms that are used for research. Genome mapping is still an ongoing process, and as more advanced techniques are developed, more advances are expected. Genome mapping is similar to completing a complicated puzzle using every piece of available data. Mapping information generated in laboratories all over the world is entered into central databases, such as the National Center for Biotechnology Information (NCBI). Efforts are made to make the information more easily accessible to researchers and the general public. Just as we use global positioning systems instead of paper maps to navigate through roadways, NCBI allows us to use a genome viewer tool to simplify the data mining process.
CONCEPT IN ACTION
Online Mendelian Inheritance in Man (OMIM) is a searchable online catalog of human genes and genetic disorders. This website shows genome mapping, and also details the history and research of each trait and disorder. Click the link to search for traits (such as handedness) and genetic disorders (such as diabetes).
Whole Genome Sequencing
Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by many diseases and researchers are using whole genome sequencing to get to the bottom of the problem. Whole genome sequencing is a process that determines the DNA sequence of an entire genome. Whole genome sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease. Several laboratories now provide services to sequence, analyze, and interpret entire genomes.
In 2010, whole genome sequencing was used to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, a whole genome sequence revealed a defect in a pathway that controls apoptosis (programmed cell death). A bone marrow transplant was used to overcome this genetic disorder, leading to a cure for the boy. He was the first person to be successfully diagnosed using whole genome sequencing.
The first genomes to be sequenced, such as those belonging to viruses, bacteria, and yeast, were smaller in terms of the number of nucleotides than the genomes of multicellular organisms. The genomes of other model organisms, such as the mouse (Mus musculus), the fruit fly (Drosophila melanogaster), and the nematode (Caenorhabditis elegans) are now known. A great deal of basic research is performed in model organismsbecause the information can be applied to other organisms. A model organism is a species that is studied as a model to understand the biological processes in other species that can be represented by the model organism. For example, fruit flies are able to metabolize alcohol like humans, so the genes affecting sensitivity to alcohol have been studied in fruit flies in an effort to understand the variation in sensitivity to alcohol in humans. Having entire genomes sequenced helps with the research efforts in these model organisms (Figure \(2\)).
The first human genome sequence was published in 2003. The number of whole genomes that have been sequenced steadily increases and now includes hundreds of species and thousands of individual human genomes.
Applying Genomics
The introduction of DNA sequencing and whole genome sequencing projects, particularly the Human Genome Project, has expanded the applicability of DNA sequence information. Genomics is now being used in a wide variety of fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics. The most commonly known application of genomics is to understand and find cures for diseases.
Predicting Disease Risk at the Individual Level
Predicting the risk of disease involves screening and identifying currently healthy individuals by genome analysis at the individual level. Intervention with lifestyle changes and drugs can be recommended before disease onset. However, this approach is most applicable when the problem arises from a single gene mutation. Such defects only account for about 5 percent of diseases found in developed countries. Most of the common diseases, such as heart disease, are multifactorial or polygenic, which refers to a phenotypic characteristic that is determined by two or more genes, and also environmental factors such as diet. In April 2010, scientists at Stanford University published the genome analysis of a healthy individual (Stephen Quake, a scientist at Stanford University, who had his genome sequenced); the analysis predicted his propensity to acquire various diseases. A risk assessment was done to analyze Quake’s percentage of risk for 55 different medical conditions. A rare genetic mutation was found that showed him to be at risk for sudden heart attack. He was also predicted to have a 23 percent risk of developing prostate cancer and a 1.4 percent risk of developing Alzheimer’s disease. The scientists used databases and several publications to analyze the genomic data. Even though genomic sequencing is becoming more affordable and analytical tools are becoming more reliable, ethical issues surrounding genomic analysis at a population level remain to be addressed. For example, could such data be legitimately used to charge more or less for insurance or to affect credit ratings?
Genome-wide Association Studies
Since 2005, it has been possible to conduct a type of study called a genome-wide association study, or GWAS. A GWAS is a method that identifies differences between individuals in single nucleotide polymorphisms (SNPs) that may be involved in causing diseases. The method is particularly suited to diseases that may be affected by one or many genetic changes throughout the genome. It is very difficult to identify the genes involved in such a disease using family history information. The GWAS method relies on a genetic database that has been in development since 2002 called the International HapMap Project. The HapMap Project sequenced the genomes of several hundred individuals from around the world and identified groups of SNPs. The groups include SNPs that are located near to each other on chromosomes so they tend to stay together through recombination. The fact that the group stays together means that identifying one marker SNP is all that is needed to identify all the SNPs in the group. There are several million SNPs identified, but identifying them in other individuals who have not had their complete genome sequenced is much easier because only the marker SNPs need to be identified.
In a common design for a GWAS, two groups of individuals are chosen; one group has the disease, and the other group does not. The individuals in each group are matched in other characteristics to reduce the effect of confounding variables causing differences between the two groups. For example, the genotypes may differ because the two groups are mostly taken from different parts of the world. Once the individuals are chosen, and typically their numbers are a thousand or more for the study to work, samples of their DNA are obtained. The DNA is analyzed using automated systems to identify large differences in the percentage of particular SNPs between the two groups. Often the study examines a million or more SNPs in the DNA. The results of GWAS can be used in two ways: the genetic differences may be used as markers for susceptibility to the disease in undiagnosed individuals, and the particular genes identified can be targets for research into the molecular pathway of the disease and potential therapies. An offshoot of the discovery of gene associations with disease has been the formation of companies that provide so-called “personal genomics” that will identify risk levels for various diseases based on an individual’s SNP complement. The science behind these services is controversial.
Because GWAS looks for associations between genes and disease, these studies provide data for other research into causes, rather than answering specific questions themselves. An association between a gene difference and a disease does not necessarily mean there is a cause-and-effect relationship. However, some studies have provided useful information about the genetic causes of diseases. For example, three different studies in 2005 identified a gene for a protein involved in regulating inflammation in the body that is associated with a disease-causing blindness called age-related macular degeneration. This opened up new possibilities for research into the cause of this disease. A large number of genes have been identified to be associated with Crohn’s disease using GWAS, and some of these have suggested new hypothetical mechanisms for the cause of the disease.
Pharmacogenomics
Pharmacogenomics involves evaluating the effectiveness and safety of drugs on the basis of information from an individual's genomic sequence. Personal genome sequence information can be used to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype. Studying changes in gene expression could provide information about the gene transcription profile in the presence of the drug, which can be used as an early indicator of the potential for toxic effects. For example, genes involved in cellular growth and controlled cell death, when disturbed, could lead to the growth of cancerous cells. Genome-wide studies can also help to find new genes involved in drug toxicity. The gene signatures may not be completely accurate, but can be tested further before pathologic symptoms arise.
Metagenomics
Traditionally, microbiology has been taught with the view that microorganisms are best studied under pure culture conditions, which involves isolating a single type of cell and culturing it in the laboratory. Because microorganisms can go through several generations in a matter of hours, their gene expression profiles adapt to the new laboratory environment very quickly. On the other hand, many species resist being cultured in isolation. Most microorganisms do not live as isolated entities, but in microbial communities known as biofilms. For all of these reasons, pure culture is not always the best way to study microorganisms. Metagenomics is the study of the collective genomes of multiple species that grow and interact in an environmental niche. Metagenomics can be used to identify new species more rapidly and to analyze the effect of pollutants on the environment (Figure \(3\)). Metagenomics techniques can now also be applied to communities of higher eukaryotes, such as fish.
Creation of New Biofuels
Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population’s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped. This vast genetic resource holds the potential to provide new sources of biofuels (Figure \(4\)).
Mitochondrial Genomics
Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and is often used to study evolutionary relationships. Another feature that makes studying the mitochondrial genome interesting is that in most multicellular organisms, the mitochondrial DNA is passed on from the mother during the process of fertilization. For this reason, mitochondrial genomics is often used to trace genealogy.
Genomics in Forensic Analysis
Information and clues obtained from DNA samples found at crime scenes have been used as evidence in court cases, and genetic markers have been used in forensic analysis. Genomic analysis has also become useful in this field. In 2001, the first use of genomics in forensics was published. It was a collaborative effort between academic research institutions and the FBI to solve the mysterious cases of anthrax (Figure \(5\)) that was transported by the US Postal Service. Anthrax bacteria were made into an infectious powder and mailed to news media and two U.S. Senators. The powder infected the administrative staff and postal workers who opened or handled the letters. Five people died, and 17 were sickened from the bacteria. Using microbial genomics, researchers determined that a specific strain of anthrax was used in all the mailings; eventually, the source was traced to a scientist at a national biodefense laboratory in Maryland.
Genomics in Agriculture
Genomics can reduce the trials and failures involved in scientific research to a certain extent, which could improve the quality and quantity of crop yields in agriculture (Figure \(6\)). Linking traits to genes or gene signatures helps to improve crop breeding to generate hybrids with the most desirable qualities. Scientists use genomic data to identify desirable traits, and then transfer those traits to a different organism to create a new genetically modified organism, as described in the previous module. Scientists are discovering how genomics can improve the quality and quantity of agricultural production. For example, scientists could use desirable traits to create a useful product or enhance an existing product, such as making a drought-sensitive crop more tolerant of the dry season.
Proteomics
Proteins are the final products of genes that perform the function encoded by the gene. Proteins are composed of amino acids and play important roles in the cell. All enzymes (except ribozymes) are proteins and act as catalysts that affect the rate of reactions. Proteins are also regulatory molecules, and some are hormones. Transport proteins, such as hemoglobin, help transport oxygen to various organs. Antibodies that defend against foreign particles are also proteins. In the diseased state, protein function can be impaired because of changes at the genetic level or because of direct impact on a specific protein.
A proteome is the entire set of proteins produced by a cell type. Proteomes can be studied using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. The study of the function of proteomes is called proteomics. Proteomics complements genomics and is useful when scientists want to test their hypotheses that were based on genes. Even though all cells in a multicellular organism have the same set of genes, the set of proteins produced in different tissues is different and dependent on gene expression. Thus, the genome is constant, but the proteome varies and is dynamic within an organism. In addition, RNAs can be alternatively spliced (cut and pasted to create novel combinations and novel proteins), and many proteins are modified after translation. Although the genome provides a blueprint, the final architecture depends on several factors that can change the progression of events that generate the proteome.
Genomes and proteomes of patients suffering from specific diseases are being studied to understand the genetic basis of the disease. The most prominent disease being studied with proteomic approaches is cancer (Figure \(7\)). Proteomic approaches are being used to improve the screening and early detection of cancer; this is achieved by identifying proteins whose expression is affected by the disease process. An individual protein is called a biomarker, whereas a set of proteins with altered expression levels is called a protein signature. For a biomarker or protein signature to be useful as a candidate for early screening and detection of a cancer, it must be secreted in body fluids such as sweat, blood, or urine, so that large-scale screenings can be performed in a noninvasive fashion. The current problem with using biomarkers for the early detection of cancer is the high rate of false-negative results. A false-negative result is a negative test result that should have been positive. In other words, many cases of cancer go undetected, which makes biomarkers unreliable. Some examples of protein biomarkers used in cancer detection are CA-125 for ovarian cancer and PSA for prostate cancer. Protein signatures may be more reliable than biomarkers to detect cancer cells. Proteomics is also being used to develop individualized treatment plans, which involves the prediction of whether or not an individual will respond to specific drugs and the side effects that the individual may have. Proteomics is also being used to predict the possibility of disease recurrence.
The National Cancer Institute has developed programs to improve the detection and treatment of cancer. The Clinical Proteomic Technologies for Cancer and the Early Detection Research Network are efforts to identify protein signatures specific to different types of cancers. The Biomedical Proteomics Program is designed to identify protein signatures and design effective therapies for cancer patients.
Summary
Genome mapping is similar to solving a big, complicated puzzle with pieces of information coming from laboratories all over the world. Genetic maps provide an outline for the location of genes within a genome, and they estimate the distance between genes and genetic markers on the basis of the recombination frequency during meiosis. Physical maps provide detailed information about the physical distance between the genes. The most detailed information is available through sequence mapping. Information from all mapping and sequencing sources is combined to study an entire genome.
Whole genome sequencing is the latest available resource to treat genetic diseases. Some doctors are using whole genome sequencing to save lives. Genomics has many industrial applications, including biofuel development, agriculture, pharmaceuticals, and pollution control.
Imagination is the only barrier to the applicability of genomics. Genomics is being applied to most fields of biology; it can be used for personalized medicine, prediction of disease risks at an individual level, the study of drug interactions before the conduction of clinical trials, and the study of microorganisms in the environment as opposed to the laboratory. It is also being applied to the generation of new biofuels, genealogical assessment using mitochondria, advances in forensic science, and improvements in agriculture.
Proteomics is the study of the entire set of proteins expressed by a given type of cell under certain environmental conditions. In a multicellular organism, different cell types will have different proteomes, and these will vary with changes in the environment. Unlike a genome, a proteome is dynamic and under constant flux, which makes it more complicated and more useful than the knowledge of genomes alone.
Glossary
biomarker
an individual protein that is uniquely produced in a diseased state
genetic map
an outline of genes and their location on a chromosome that is based on recombination frequencies between markers
genomics
the study of entire genomes, including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species
metagenomics
the study of the collective genomes of multiple species that grow and interact in an environmental niche
model organism
a species that is studied and used as a model to understand the biological processes in other species represented by the model organism
pharmacogenomics
the study of drug interactions with the genome or proteome; also called toxicogenomics
physical map
a representation of the physical distance between genes or genetic markers
protein signature
a set of over- or under-expressed proteins characteristic of cells in a particular diseased tissue
proteomics
study of the function of proteomes
whole genome sequencing
a process that determines the nucleotide sequence of an entire genome | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.01%3A_Mapping_Genomes.txt |
Skills to Develop
• Describe three types of sequencing
• Define whole-genome sequencing
Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by some diseases, and they are using whole-genome sequencing to get to the bottom of the problem. Whole-genome sequencing is a process that determines the DNA sequence of an entire genome. Whole-genome sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease. Several laboratories now provide services to sequence, analyze, and interpret entire genomes.
For example, whole-exome sequencing is a lower-cost alternative to whole genome sequencing. In exome sequencing, only the coding, exon-producing regions of the DNA are sequenced. In 2010, whole-exome sequencing was used to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, whole-exome sequencing was performed, which revealed a defect in a pathway that controls apoptosis (programmed cell death). A bone-marrow transplant was used to overcome this genetic disorder, leading to a cure for the boy. He was the first person to be successfully treated based on a diagnosis made by whole-exome sequencing. Today, human genome sequencing is more readily available and can be completed in a day or two for about \$1000.
Strategies Used in Sequencing Projects
The basic sequencing technique used in all modern day sequencing projects is the chain termination method (also known as the dideoxy method), which was developed by Fred Sanger in the 1970s. The chain termination method involves DNA replication of a single-stranded template with the use of a primer and a regular deoxynucleotide (dNTP), which is a monomer, or a single unit, of DNA. The primer and dNTP are mixed with a small proportion of fluorescently labeled dideoxynucleotides (ddNTPs). The ddNTPs are monomers that are missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain (Figure \(1\)).
Each ddNTP is labeled with a different color of fluorophore. Every time a ddNTP is incorporated in the growing complementary strand, it terminates the process of DNA replication, which results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is processed by gel electrophoresis after being separated into single strands, the multiple newly replicated DNA strands form a ladder because of the differing sizes. Because the ddNTPs are fluorescently labeled, each band on the gel reflects the size of the DNA strand and the ddNTP that terminated the reaction. The different colors of the fluorophore-labeled ddNTPs help identify the ddNTP incorporated at that position. Reading the gel on the basis of the color of each band on the ladder produces the sequence of the template strand (Figure \(2\)).
Early Strategies: Shotgun Sequencing and Pair-Wise End Sequencing
In shotgun sequencing method, several copies of a DNA fragment are cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments are then sequenced using the chain-sequencing method. Then, with the help of a computer, the fragments are analyzed to see where their sequences overlap. By matching up overlapping sequences at the end of each fragment, the entire DNA sequence can be reformed. A larger sequence that is assembled from overlapping shorter sequences is called a contig. As an analogy, consider that someone has four copies of a landscape photograph that you have never seen before and know nothing about how it should appear. The person then rips up each photograph with their hands, so that different size pieces are present from each copy. The person then mixes all of the pieces together and asks you to reconstruct the photograph. In one of the smaller pieces you see a mountain. In a larger piece, you see that the same mountain is behind a lake. A third fragment shows only the lake, but it reveals that there is a cabin on the shore of the lake. Therefore, from looking at the overlapping information in these three fragments, you know that the picture contains a mountain behind a lake that has a cabin on its shore. This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.
Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to the development of double-barrel shotgun sequencing, more formally known as pairwise-end sequencing. In pairwise-end sequencing, both ends of each fragment are analyzed for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.
Next-generation Sequencing
Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing, which is a group of automated techniques used for rapid DNA sequencing. These automated low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 500 base pairs) in the span of one day. These sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order.
Evolution Connection: Comparing Sequences
A sequence alignment is an arrangement of proteins, DNA, or RNA; it is used to identify regions of similarity between cell types or species, which may indicate conservation of function or structures. Sequence alignments may be used to construct phylogenetic trees. The following website uses a software program called BLAST (basic local alignment search tool).
Under “Basic Blast,” click “Nucleotide Blast.” Input the following sequence into the large "query sequence" box: ATTGCTTCGATTGCA. Below the box, locate the "Species" field and type "human" or "Homo sapiens". Then click “BLAST” to compare the inputted sequence against known sequences of the human genome. The result is that this sequence occurs in over a hundred places in the human genome. Scroll down below the graphic with the horizontal bars and you will see short description of each of the matching hits. Pick one of the hits near the top of the list and click on "Graphics". This will bring you to a page that shows where the sequence is found within the entire human genome. You can move the slider that looks like a green flag back and forth to view the sequences immediately around the selected gene. You can then return to your selected sequence by clicking the "ATG" button.
Use of Whole-Genome Sequences of Model Organisms
The first genome to be completely sequenced was of a bacterial virus, the bacteriophage fx174 (5368 base pairs); this was accomplished by Fred Sanger using shotgun sequencing. Several other organelle and viral genomes were later sequenced. The first organism whose genome was sequenced was the bacterium Haemophilus influenzae; this was accomplished by Craig Venter in the 1980s. Approximately 74 different laboratories collaborated on the sequencing of the genome of the yeast Saccharomyces cerevisiae, which began in 1989 and was completed in 1996, because it was 60 times bigger than any other genome that had been sequenced. By 1997, the genome sequences of two important model organisms were available: the bacterium Escherichia coli K12 and the yeast Saccharomyces cerevisiae. Genomes of other model organisms, such as the mouse Mus musculus, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and humans Homo sapiens are now known. A lot of basic research is performed in model organisms because the information can be applied to genetically similar organisms. A model organism is a species that is studied as a model to understand the biological processes in other species represented by the model organism. Having entire genomes sequenced helps with the research efforts in these model organisms. The process of attaching biological information to gene sequences is called genome annotation. Annotation of gene sequences helps with basic experiments in molecular biology, such as designing PCR primers and RNA targets.
Link to Learning
Click through each step of HHMI genome sequencing site.
Uses of Genome Sequences
DNA microarrays are methods used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and identify sequences. Almost one million genotypic abnormalities can be discovered using microarrays, whereas whole-genome sequencing can provide information about all six billion base pairs in the human genome. Although the study of medical applications of genome sequencing is interesting, this discipline tends to dwell on abnormal gene function. Knowledge of the entire genome will allow future onset diseases and other genetic disorders to be discovered early, which will allow for more informed decisions to be made about lifestyle, medication, and having children. Genomics is still in its infancy, although someday it may become routine to use whole-genome sequencing to screen every newborn to detect genetic abnormalities.
In addition to disease and medicine, genomics can contribute to the development of novel enzymes that convert biomass to biofuel, which results in higher crop and fuel production, and lower cost to the consumer. This knowledge should allow better methods of control over the microbes that are used in the production of biofuels. Genomics could also improve the methods used to monitor the impact of pollutants on ecosystems and help clean up environmental contaminants. Genomics has allowed for the development of agrochemicals and pharmaceuticals that could benefit medical science and agriculture.
It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely. Otherwise, it could be easy to misuse the power of such knowledge, leading to discrimination based on a person's genetics, human genetic engineering, and other ethical concerns. This information could also lead to legal issues regarding health and privacy.
Summary
Whole-genome sequencing is the latest available resource to treat genetic diseases. Some doctors are using whole-genome sequencing to save lives. Genomics has many industrial applications including biofuel development, agriculture, pharmaceuticals, and pollution control. The basic principle of all modern-day sequencing strategies involves the chain termination method of sequencing.
Although the human genome sequences provide key insights to medical professionals, researchers use whole-genome sequences of model organisms to better understand the genome of the species. Automation and the decreased cost of whole-genome sequencing may lead to personalized medicine in the future.
Glossary
chain termination method
method of DNA sequencing using labeled dideoxynucleotides to terminate DNA replication; it is also called the dideoxy method or the Sanger method
contig
larger sequence of DNA assembled from overlapping shorter sequences
deoxynucleotide
individual monomer (single unit) of DNA
dideoxynucleotide
individual monomer of DNA that is missing a hydroxyl group (–OH)
DNA microarray
method used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and identify sequences
genome annotation
process of attaching biological information to gene sequences
model organism
species that is studied and used as a model to understand the biological processes in other species represented by the model organism
next-generation sequencing
group of automated techniques used for rapid DNA sequencing
shotgun sequencing
method used to sequence multiple DNA fragments to generate the sequence of a large piece of DNA
whole-genome sequencing
process that determines the DNA sequence of an entire genome | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.02%3A_Sequencing_Genomes.txt |
Vitruvian Man
The drawing in Figure \(2\), named Vitruvian Man, was created by Leonardo da Vinci in 1490. It was meant to show normal human body proportions. Vitruvian Man is used today to represent a different approach to the human body. It symbolizes a scientific research project that began in 1990, exactly 500 years after da Vinci created the drawing. That project, named the Human Genome Project, is the largest collaborative biological research project ever undertaken.
What Is the Human Genome?
The human genome refers to all the DNA of the human species. Human DNA consists of 3.3 billion base pairs and is divided into more than 20,000 genes onto 23 pairs of chromosomes. The human genome also includes noncoding sequences (e.g. intergenic region) of DNA, as shown in Figure \(2\).
Discovering the Human Genome
Scientists now know the sequence of all the DNA base pairs in the entire human genome. This knowledge was attained by the Human Genome Project (HGP), a \$3 billion, international scientific research project that was formally launched in 1990. The project was completed in 2003, two years ahead of its 15-year projected deadline.
Determining the sequence of the billions of base pairs that make up human DNA was the main goal of the HGP. Another goal was mapping the location and determining the function of all the genes in the human genome. There are only about 20,500 genes in human beings.
A Collaborative Effort
Funding for the HGP came from the U.S. Department of Energy and the National Institutes of Health as well as from foreign institutions. The actual research was undertaken by scientists in 20 universities in the U.S., United Kingdom, Australia, France, Germany, Japan, and China. A private U.S. company named Celera also contributed to the effort. Although Celera had hoped to patent some of the genes it discovered, this was later denied.
Reference Genome of the Human Genome Project
In 2003, the HGP published the results of its sequencing of DNA as a human reference genome. Figure \(4\) illustrates the process of DNA sequencing. The details of this image are out of the scope of this concept and book. The sequence of the human DNA is stored in databases available to anyone on the Internet. The U.S. National Center for Biotechnology Information (NCBI), part of the NIH, as well as comparable organizations in Europe and Japan, maintain the genomic sequences in a database known as Genbank. Protein sequences are also maintained in this database. The sequences in these databases are the combined sequences of anonymous donors, and as such do not yet address the individual differences that make us unique. However, the known sequence does lay the foundation to identify the unique differences among all of us. Most of the currently identified variations among individuals will be single nucleotide polymorphisms or SNPs. An SNP (pronounced "snip") is a DNA sequence variation occurring at a single nucleotide in the genome. For example, two sequenced DNA fragments from different individuals, GGATCTA to GGATTTA, contain a difference in a single nucleotide. If this, base change occurs in a gene, the base change then results in two alleles: the C allele and the T allele. Remember an allele is an alternative form of a gene. Almost all common SNPs have only two alleles. The effect of these SNPs on protein structure and function and any effect on the resulting phenotype are an extensive field of study.
Benefits of the Human Genome Project
The sequencing of the human genome holds benefits for many fields, including molecular medicine and human evolution.
• Knowing the human DNA sequence can help us understand many human diseases. For example, it is helping researchers identify mutations linked to different forms of cancer. It is also yielding insights into the genetic basis of cystic fibrosis, liver diseases, blood-clotting disorders, and Alzheimer's disease, among others.
• The human DNA sequence can also help researchers tailor medications to individual genotypes. This is called personalized medicine, and it has led to an entirely new field called pharmacogenomics. Pharmacogenomics, also called pharmacogenetics, is the study of how our genes affect the way we respond to drugs. You can read more about pharmacogenomics in the Feature below.
• The analysis of similarities between DNA sequences from different organisms is opening new avenues in the study of evolution. For example, analyses are expected to shed light on many questions about the similarities and differences between humans and our closest relatives the nonhuman primates.
Ethical, Legal, and Social Issues of the Human Genome Project
From its launch in 1990, the HGP proactively established and funded a separate committee to oversee potential ethical, legal, and social issues associated with the project. A major concern was the possible use of the knowledge generated by the project to discriminate against people. One issue was the fear that employers and health insurance companies would refuse to hire or insure people based on their genetic makeup, for instance, if they had genes that increased their risk of getting certain diseases. In response, in 1996, the U.S. passed the Health Insurance Portability and Accountability Act (HIPAA). It protects against unauthorized, nonconsensual release of individually identifiable health information to any entity not actively engaged in providing healthcare to a patient. This was followed in 2008 by the Genetic Information Nondiscrimination Act (GINA), which specifically prohibits genetic discrimination by health insurance companies and workplaces.
Review
1. Describe the human genome.
2. What is the Human Genome Project?
3. Identify two main goals of the Human Genome Project.
4. What is the reference genome of the Human Genome Project? What is it based on?
5. Explain how knowing the sequence of DNA bases in the human genome is beneficial for molecular medicine.
6. What was one surprising finding of the Human Genome Project?
7. Why do you think scientists didn’t just sequence the DNA from a single person for the Human Genome Project? Along those lines, why do you think it is important to include samples from different ethnic groups and genders in genome sequencing efforts?
8. True or False. The sequenced human genome does not include noncoding regions — it only includes actual genes.
9. True or False. Knowing the sequence of the human genome can give insight into human evolution.
10. What is pharmacogenomics?
1. If a patient were to have pharmacogenomics done to optimize their medication, what do you think the first step would be?
2. List one advantage and one disadvantage of pharmacogenomics.
11. There are approximately 20,000 human
1. base pairs
2. nucleotides
3. alleles
4. genes
12. Explain how the sequencing of the human genome relates to ethical concerns about genetic discrimination.
Explore More
For years, scientists have had the challenge of sequencing the human genome. Learn more about the human genome project here:
Attributions
1. Vitruvian man public domain via Wikimedia Commons
2. Human genome to genes by LoStrangolatore, CC BY 3.0 via Wikimedia Commons
3. Human genome project by National Human Genome Research Institute (NHGRI), licensed CC BY 2.0 via Wikimedia Commons
4. Text adapted from Human Biology by CK-12 licensed CC BY-NC 3.0 | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.03%3A_Genome_Projects/18.3.01%3A_The_Human_Genome_Project.txt |
Genome annotation is the identification and understanding of the genetic elements of a sequenced genome.
LEARNING OBJECTIVES
Define genome annotation
Key Points
• Once a genome is sequenced, all of the sequencings must be analyzed to understand what they mean.
• Critical to annotation is the identification of the genes in a genome, the structure of the genes, and the proteins they encode.
• Once a genome is annotated, further work is done to understand how all the annotated regions interact with each other.
Key Terms
• BLAST: In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences.
• in silico: In computer simulation or in virtual reality
Genome projects are scientific endeavors that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, an archaean, a protist, or a virus). They annotate protein-coding genes and other important genome-encoded features. The genome sequence of an organism includes the collective DNA sequences of each chromosome in the organism. For a bacterium containing a single chromosome, a genome project will aim to map the sequence of that chromosome.
Once a genome is sequenced, it needs to be annotated to make sense of it. An annotation (irrespective of the context) is a note added by way of explanation or commentary. Since the 1980’s, molecular biology and bioinformatics have created the need for DNA annotation. DNA annotation or genome annotation is the process of identifying the locations of genes and all of the coding regions in a genome and determining what those genes do.
Genome annotation is the process of attaching biological information to sequences. It consists of two main steps: identifying elements on the genome, a process called gene prediction, and attaching biological information to these elements. Automatic annotation tools try to perform all of this by computer analysis, as opposed to manual annotation (a.k.a. curation) which involves human expertise. Ideally, these approaches co-exist and complement each other in the same annotation pipeline (process). The basic level of annotation is using BLAST for finding similarities, and then annotating genomes based on that. However, nowadays more and more additional information is added to the annotation platform. The additional information allows manual annotators to deconvolute discrepancies between genes that are given the same annotation. Some databases use genome context information, similarity scores, experimental data, and integrations of other resources to provide genome annotations through their Subsystems approach. Other databases rely on both curated data sources as well as a range of different software tools in their automated genome annotation pipeline.
Structural annotation consists of the identification of genomic elements: ORFs and their localization, gene structure, coding regions, and the location of regulatory motifs. Functional annotation consists of attaching biological information to genomic elements: biochemical function, biological function, involved regulation and interactions, and expression.
These steps may involve both biological experiments and in silico analysis. Proteogenomics based approaches utilize information from expressed proteins, often derived from mass spectrometry, to improve genomics annotations. A variety of software tools have been developed to permit scientists to view and share genome annotations. Genome annotation is the next major challenge for the Human Genome Project, now that the genome sequences of human and several model organisms are largely complete. Identifying the locations of genes and other genetic control elements is often described as defining the biological “parts list” for the assembly and normal operation of an organism. Scientists are still at an early stage in the process of delineating this parts list and in understanding how all the parts “fit together. ” | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.04%3A_Genome_Annotation_and_Databases/18.4.01%3A_Genome_Annotation.txt |
Source: BiochemFFA_7_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Introduction
For many years, scientists wondered about the nature of the information that directed the activities of cells. What kind of molecules carried the information, and how was the information passed on from one generation to the next? Key experiments, done between the 1920s and the 1950s, established convincingly that this genetic information was carried by DNA. In 1953, with the elucidation of the structure of DNA, it was possible to begin investigating how this information is passed on, and how it is used.
Genomes
We use the word “genome” to describe all of the genetic material of the cell. That is, a genome is the entire sequence of nucleotides in the DNA that is in all of the chromosomes of a cell. When we use the term genome without further qualification, we are generally referring to the chromosomes in the nucleus of a eukaryotic cell. As you know, eukaryotic cells have organelles like mitochondria and chloroplasts that have their own DNA (Figure 7.1 & 7.2). These are referred to as the mitochondrial or chloroplast genomes to distinguish them from the nuclear genome.
Starting in the 1980s, scientists began to determine the complete sequence of the genomes of many organisms, in the hope of better understanding how the DNA sequence specifies cellular functions. Today, the complete genome sequences have been determined for thousands of species from all domains of life, and many more are in the process of being worked out by groups of scientists across the world.
Global genome initiative
The Global Genome Initiative, a collaborative effort to sequence at least one species from each of the 9,500 described invertebrate, vertebrate, and plant families is one of many such ventures. The information from these various efforts is collected in enormous online repositories, so that it is freely available to scientists. As the sequence databases compile ever more information, the fields of computational biology and bioinformatics have arisen, to analyze and organize the data in a way that helps biologists understand what the information in DNA means in the cellular context.
Genes
It has been known for many years that phenotypic traits are controlled by specific regions of the DNA that were termed “genes”. Thus, DNA was envisioned as a long string of nucleotides, in which certain regions, the genes, were separated by non-coding regions that were simply referred to as intergenic sequences (inter=between; genic=of genes). Early experiments in molecular biology suggested a simple relationship between the DNA sequence of a gene and its product, and led scientists to believe that each gene carried the information for a single protein. Changes, or mutations in the base sequence of a gene would be reflected in changes in the gene product, which in turn, would manifest itself in the phenotype or observable trait. This simple picture, while still useful, has been modified by subsequent discoveries that demonstrated that the use of genetic information by cells is somewhat more complicated. Our definition of a gene is also evolving to take new knowledge into consideration.
Figure 7.4 - Human genes sorted by class
Matters of size
A common-sense assumption about genomes would be that if genes specify proteins, then the more proteins an organism made, the more genes it would need to have, and thus, the larger its genome would be. Comparison of various genomes shows, surprisingly, that there is not necessarily a direct relationship between the complexity of an organism and the size of its genome (Figure 7.5). To understand how this could be true, it is necessary to recognize that while genes are made up of DNA, all DNA does not consist of genes (for purposes of our discussion, we define a gene as a section of DNA that encodes an RNA or protein product). In the human genome, less than 2% of the total DNA seems to be the sort of coding sequence that directs the synthesis of proteins. For many years, non-coding DNA in genomes was believed to be useless, and was described as “junk DNA” although it was perplexing that there seemed to be so much “useless” sequence. Recent discoveries have, however, demonstrated that much of this so-called junk DNA may play important roles in evolution, as well as in regulation of gene expression.
Introns
So, what is all the non-coding DNA doing there? We know that even coding regions in our DNA are interrupted by non-coding sequences called introns. This is true of most eukaryotic genomes. An examination of genes in eukaryotes shows that non-coding intron sequences can be much longer than the coding sections of the gene, or exons. Most exons are relatively small, and code for fewer than a hundred amino acids, while introns can vary in size from several hundred base-pairs to many kilobase-pairs (thousands of base-pairs) in length. For many genes in humans, there is much more of intron sequence than coding (a.k.a. exon) sequence. Intron sequences account for roughly a quarter of the genome in humans.
Other non-coding sequences
What other kinds of non-coding sequences are there? One function for some DNA sequences that do not encode RNA or proteins is in specifying when and to what extent a gene is used, or expressed. Such regions of DNA are called regulatory regions and each gene has one or more regulatory sequences that control its expression. However, regulatory sequences do not account for all the rest of the DNA in our genomes, either.
Transposable sequences
Surprisingly, almost half of the human genome appears to consist of several kinds of repetitive sequences. Many of the repetitive sequences are known to be transposable elements (transposons), sections of DNA that can move around within the genome. Sometimes referred to as “jumping genes” these transposable elements can move from one chromosomal location to another, either through a simple “cut and paste” mechanism that cuts the sequence out of one region of the DNA and inserts it into another location, or through a process called retrotransposition involving an RNA intermediate.
LINES & SINES
There are millions of copies of each of two major classes of such transposable elements, the LINEs (Long Interspersed Elements) and SINEs (Short Interspersed Elements) in our genomes.
LINEs and SINEs are both a kind of transposable element called retrotransposons, sequences that are copied into RNA, then reverse transcribed back into DNA before being inserted into new locations. This movement is typically not sequence specific, meaning that the transposons can be inserted randomly in the genome, in many cases within coding regions. As might be expected, this can disrupt the function of the gene. Transposons may also insert within regulatory regions, and change the expression of the genes they control. As a major cause of mutation in genomes, transposons play an important role in evolution.
Finally, recent findings have shown that much of the genome is transcribed into RNAs, even though only about 2% encodes proteins. What are the RNAs that do not encode proteins? Ribosomal RNAs (Figure 7.7) and transfer RNAs, together with the small nuclear RNAs that function in splicing, account for some of these non-translated transcripts, but not all. The remaining RNAs are regulatory RNAs, small molecules that play an important role in regulating gene expression. As we understand more about genomes, it is becoming evident that the so-called “junk” DNA is anything but.
18.4.03: The ENCODE Project
The human genome was sequenced in 2003, an important step in understanding the blueprint of life. However, before this information can be fully utilized, the location, identity, and function of all protein- encoding and non-protein-encoding genes must be determined. Moreover, the human genome has many other functional elements, ranging from promotors, regulatory sequences, and other factors that determine chromatin structure. These must also be determined to fully understand the human genome.
The ENCODE (Encyclopedia of DNA Elements) project aims to solve these problems by delineating all functional elements of the human genome. To accomplish this goal, a consortium was formed to guide the project. The consortium aimed to advance and develop technologies for annotating the human genome with higher accuracy, completeness, and cost-effectiveness, along with more standardization.They also aimed to develop a series of computational techniques to parse and analyze the data obtained.
To accomplish this goal, a pilot project was launched. The ENCODE pilot project aimed to study 1% of the human genome in depth, roughly from 2003 to 2007. From 2007 to 2012, the ENCODE project ramped up to annotate the entire genome. Finally, from 2012 onwards, the ENCODE project aims further increases in all dimensions: deeper sequencing, more assays, more transcription factors, etc.
This chapter will describe some of the experimental and computational techniques used in the ENCODE project. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.04%3A_Genome_Annotation_and_Databases/18.4.02%3A_Non-Coding_Elements.txt |
Genomics is a field that studies the entire collection of an organism’s DNA or genome. It involves sequencing, analyzing, and comparing the information contained within genomes. Since sequencing has become much less expensive and more efficient, vast amounts of genomic information is now available about a wide variety of organisms, but particularly microbes, with their smaller genome size. In fact, the biggest bottleneck currently is not the lack of information but the lack of computing power to process the information!
Sequencing
Sequencing, or determining the base order of an organism’s DNA or RNA, is often one of the first steps to finding out detailed information about an organism. A bacterial genome can range from 130 kilobase pairs (kbp) to over 14 Megabase pairs (Mbp), while a viral genome ranges from 0.859 to 2473 kbp. For comparison, the human genome contains about 3 billion base pairs.
Shotgun sequencing
Shotgun sequencing initially involves construction of a genomic library, where the genome is broken into randomly sized fragments that are inserted into vectors to produce a library of clones. The fragments are sequenced and then analyzed by a computer, which searches for overlapping regions to form a longer stretch of sequence. Eventually all the sequences are aligned to give the complete genome sequence. Errors are reduced because many of the clones contain identical or near identical sequences, resulting in good “coverage” of the genome.
Shotgun Sequencing. By Commins, J., Toft, C., Fares, M. A. [CC BY-SA 2.5], via Wikimedia Commons
Second generation DNA sequencing
Second-generation DNA sequencing uses massively parallel methods, where multiple samples are sequenced side-by-side. DNA fragments of a few hundred bases each are amplified by PCR and then attached to small bead, so that each bead carries several copies of the same section of DNA. The beads are put into a plate containing more than a million wells, each with one bead, and the DNA fragments are sequenced.
Third- and fourth-generation DNA sequencing
Third-generation DNA sequencing involves the sequencing of single molecules of DNA. Fourth-generation DNA sequencing, also known as “post light sequencing,” utilizes methods other than optical detection for sequencing.
Bioinformatics
After sequencing, it is time to make sense of the information. The field of bioinformatics combines many fields together (i.e. biology, computer science, statistics) to use the power of computers to analyze information contained in the genomic sequence. Locating specific genes within a genome is referred to as genome annotation.
Open Reading Frames (ORFS)
An open reading frame or ORF denotes a possible protein-coding gene. For double-stranded DNA, there are six reading frames to be analyzed, since the DNA is read in sets of three bases at a time and there are two strands of DNA. An ORF typically has at least 100 codons before a stop codon, with 3’ terminator sequences. A functional ORF is one that is actually used by the organism to encode a protein. Computers are used to search the DNA sequence looking for ORFs, with those presumed to encode protein further analyzed by a bioinformaticist.
It is often helpful for the sequence to be compared against a database of sequences coding for known proteins. GenBank is a database of over 200 billion base pairs of sequences that scientists can access, to try and find matches to the sequence of interest. The database search tool BLAST (basic local alignment search tool) has programs for comparing both nucleotide sequences and amino acid sequences, providing a ranking of results in order of decreasing similarity.
BLAST Results.
Comparative Genomics
Once the sequences of organisms have been obtained, meaningful information can be gathered using comparative genomics. For this genomes are assessed for information regarding size, organization, and gene content.
Comparison of the genome of microbial strains has given scientists a better picture regarding the genes that organisms pick up. A group of multiple strains share a core genome, genes coding for essential cellular functions that they all have in common. The pan genome represents all the genes found in all the members of species, so provides a good idea of the diversity of a group. Most of these “extra” genes are probably picked up by horizontal gene transfer.
Comparative genomics also shows that many genes are derived as a result of gene duplication. Genes within a single organism that likely came about because of gene duplication are referred to as paralogs. In many cases one of the genes might be altered to take on a new function. It is also possible for gene duplication to be found in different organisms, as a result of acquiring the original gene from a common ancestor. These genes are called orthologs.
Functional Genomics
The sequence of a genome and the location of genes provide part of the picture, but in order to fully understand an organism we need an idea of what the cell is doing with its genes. In other words, what happens when the genes are expressed? This is where functional genomics comes in – placing the genomic information in context.
The first step in gene expression is transcription or the manufacture of RNA. Transcriptome refers to the entire complement of RNA that a cell can make from its genome, while proteome refers to all the proteins encoded by an organisms’ genome, in the final step of gene expression.
Microarrays
Microarrays or gene chips are solid supports upon which multiple spots of DNA are placed, in a grid-like fashion. Each spot of DNA represents a single gene or ORF. Known fragments of nucleic acid are labeled and used as probes, with a signal produced if binding occurs. Microarrays can be used to determine what genes might be turned on or off under particular conditions, such as comparing the growth of a bacterial pathogen inside the host versus outside of the host.
Proteomics
The study of the proteins of an organism (or the proteome) is referred to as proteomics. Much of the interest focuses on functional proteomics, which examines the functions of the cellular proteins and the ways in which they interact with one another.
One common technique used in the study of proteins is two-dimensional gel electrophoresis, which first separates proteins based on their isoelectric points. This is accomplished by using a pH gradient, which separates the proteins based on their amino acid content. The separated proteins are then run through a polyacrylamide gel, providing the second dimension as proteins are separated by size.
Structural proteomics focuses on the three-dimensional structure of proteins, which is often determined by protein modeling, using computer algorithms to predict the most likely folding of the protein based on amino acid information and known protein patterns.
Metabolomics
Metabolomics strives to identify the complete set of metabolic intermediates produced by an organism. This can be extremely complicated, since many metabolites are used by cells in multiple pathways.
Metagenomics
Metagenomics or environmental genomics refers to the extraction of pooled DNA directly from a specific environment, without the initial isolation and identification of organisms within that environment. Since many microbial species are difficult to culture in the laboratory, studying the metagenome of an environment allows scientists to consider all organisms that might be present. Taxa can even be identified in the absence of organism isolation using nucleic acid sequences alone, where the taxon is known as phylotype.
Key Words
genomics, sequencing, shotgun sequencing, genomic library, second generation DNA sequencing, massively parallel methods, third- and fourth-generation DNA sequencing, bioinformatics, genome annotation, open reading frame/ORF, functional ORF, GenBank, BLAST/basic local alignment search tool, comparative genomics, core genome, pan genome, paralog, ortholog, functional genomics, transcriptome, proteome, microarray/gene chips, probe, proteomics, functional proteomics, two-dimensional gel electrophoresis, structural proteomics, metabolomics, metagenomics/environmental genomics, metagenome, phylotype.
Study Questions
1. What does the field of genomics encompass?
2. What is shotgun sequencing and how does this allow for the complete sequencing of an organism’s genome?
3. What are the basic differences among 2nd, 3rd, and 4th generation sequencing?
4. What is an open reading frame and how can scientists use it to determine information about a genome and its products?
5. How does functional genomics differ from comparative genomics? What are the tools used in functional genomics and what information can be obtained from each? | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.05%3A_Comparative_and_Functional_Genomics/18.5.01%3A_Comparative_Genomics.txt |
Consider a matrix containing all of the known gene sequences in a genome. To make such a matrix for analysis, one would need to make copies of every gene, either by chemical synthesis or by using the polymerase chain reaction. The strands of the resulting DNAs would then be separated to obtain single-stranded sequences that could be attached to the chip. Each box of the grid would contain sequence from one gene. With this grid, one could analyze the transcriptome - all of the mRNAs being made in selected cells at a given time. For a simple analysis, one could take a tissue (say liver) and extract all the mRNAs from it. This mRNA population represents all the genes that were being expressed in the liver cells at the time the RNA was extracted. These RNAs should be able to hybridize (base-pair) with their corresponding genes on the microarray. Genes that were not being expressed would have no mRNAs to bind to their corresponding genes on the grid.
Figure 8.25 - Copying and labeling of transcriptome. Image by Taralyn Tan
In practice, the mRNAs are not used directly, but are copied into single-stranded DNA copies called cDNAs. The cDNAs are tagged with a fluorescent dye and added to the microarray under conditions that allow base pairing so that the cDNAs can find and base pair with complementary sequences on the matrix (Figure 8.26). The matrix is then washed to remove unhybridized cDNAs. The presence/absence/abundance of each mRNA is then readily determined by measuring the amount of dye at each box of the grid.
Figure 8.26 - Add labeled cDNAs to microarray plate. Image by Taralyn Tan
In Figure 8.27, a fluorescent cDNA has bound to the spot on the far right in the third row of the grid. This means that the sequence of the cDNA was complementary to the sequence of the gene sequence immobilized at that spot. Because the identity of the genes at each position on the grid is known, we then know that the sample contained mRNA that corresponded to that particular gene. In other words, that gene was being expressed in the cells from which the mRNAs were obtained.
A more powerful analysis could be performed with two sets of mRNAs simultaneously. . One set of cDNAs could come from a cancerous tissue and the other from a non-cancerous tissue, for example. The cDNAs derived from each sample is marked with a different color (say green for normal and red for cancerous) (Figure 8.25). The cDNAs are mixed and then added to the matrix and complementary sequences are once again allowed to form duplexes (Figure 8.27).
Figure 8.28 - Microarray analysis comparing gene expression in normal and cancer cells. Wikipedia
Unhybridized cDNAs are washed away and then the plate is analyzed. Red grid boxes correspond to an mRNA present in the cancerous tissue, but not in the non-cancerous tissue. Green grid boxes correspond to an mRNA present in the non-cancerous tissue, but not in the cancerous tissue. Yellow would correspond to mRNAs present in equal abundance in the two tissues (Figure 8.28). The intensity of each spot also gives information about the relative amounts of each mRNA in each tissue.
Figure 8.29 - Automated high throughput sequencer. Wikipedia
The same principle used for nucleic acid microarrays can be adapted for analyzing other molecules. For example, polypeptides could be bonded to the glass slide instead of DNA to create a protein chip. Protein chips are useful for studying the interactions of proteins with other molecules as well as for diagnostics.
RNA-Seq Technique
Like microarrays, a newer method called RNA-Seq, is a tool for simultaneously detecting and quantitating all of the transcripts in a given sample. This method relies on recently developed sequencing technologies called next-generation sequencing, or deep sequencing. These techniques allow for rapid, parallel sequencing of millions of DNA fragments and can, thus, be used not only for genomic DNA, but also to sequence all of the reverse-transcribed RNAs from a given sample.
To determine all the protein-coding genes that were being expressed in a particular set of cells under specific physiological conditions, all of the mRNA would first be extracted and reverse-transcribed into cDNA. This step is similar to the preparation of samples for microarrays. However, at this point, the cDNAs are fragmented into smaller pieces, and have small sequencing adapters attached at either end. The fragments are then subjected to high-throughput sequencing, to obtain short sequences from all of the fragments. These data are aligned against the genome sequence and used to measure the level of expression of different genes. RNA-Seq offers some advantages over microarrays. With microarrays, an RNA can only be detected if the gene sequence corresponding to it is present on the grid. In RNA-Seq every RNA present in the sample is sequenced, so detection of RNAs is not limited by the probes on a chip. RNA-Seq is more sensitive than microarrays and offers a much larger range over which gene expression can be measured accurately. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.05%3A_Comparative_and_Functional_Genomics/18.5.02%3A_Transcriptomics.txt |
Learning Objectives
• Describe the techniques used in proteomics to analyze proteins
Basic Techniques in Protein Analysis
The ultimate goal of proteomics is to identify or compare the proteins expressed in a given genome under specific conditions, study the interactions between the proteins, and use the information to predict cell behavior or develop drug targets. Just as the genome is analyzed using the basic technique of DNA sequencing, proteomics requires techniques for protein analysis. The basic technique for protein analysis, analogous to DNA sequencing, is mass spectrometry.
Mass Spectrometry
Mass spectrometry is used to identify and determine the characteristics of a molecule. It is a technique in which gas phase molecules are ionized and their mass-to-charge ratio is measured by observing acceleration differences of ions when an electric field is applied. Lighter ions will accelerate faster and be detected first. If the mass is measured with precision, then the composition of the molecule can be identified. In the case of proteins, the sequence can be identified. The challenge of techniques used for proteomic analyses is the difficulty in detecting small quantities of proteins, but advances in spectrometry have allowed researchers to analyze very small samples of protein. Variations in protein expression in diseased states, however, can be difficult to discern. Proteins are naturally-unstable molecules, which makes proteomic analysis much more difficult than genomic analysis.
X-ray crystallography and Nuclear Magnetic Resonance
X-ray crystallography enables scientists to determine the three-dimensional structure of a protein crystal at atomic resolution. Crystallographers aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X-rays onto an electronic detector that is the same type used to capture images in a digital camera. After each blast of X-rays, lasting from a few seconds to several hours, the researchers precisely rotate the crystal by entering its desired orientation into the computer that controls the X-ray apparatus. This enables the scientists to capture in three dimensions how the crystal scatters, or diffracts, X-rays. The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation to calculate the position of every atom in the crystallized molecule. The result is a three-dimensional digital image of the molecule.
Another protein imaging technique, nuclear magnetic resonance (NMR), uses the magnetic properties of atoms to determine the three-dimensional structure of proteins. NMR spectroscopy is unique in being able to reveal the atomic structure of macromolecules in solution, provided that highly-concentrated solution can be obtained. This technique depends on the fact that certain atomic nuclei are intrinsically magnetic. The chemical shift of nuclei depends on their local environment. The spins of neighboring nuclei interact with each other in ways that provide definitive structural information that can be used to determine complete three-dimensional structures of proteins.
Protein Microarrays and Two- Hybrid Screening
Protein microarrays have also been used to study interactions between proteins. These are large-scale adaptations of the basic two-hybrid screen. The premise behind the two-hybrid screen is that most eukaryotic transcription factors have modular activating and binding domains that can still activate transcription even when split into two separate fragments, as long as the fragments are brought within close proximity to each other. Generally, the transcription factor is split into a DNA-binding domain (BD) and an activation domain (AD). One protein of interest is genetically fused to the BD and another protein is fused to the AD. If the two proteins of interest bind each other, then the BD and AD will also come together and activate a reporter gene that signals interaction of the two hybrid proteins.
Western Blot
The western blot, or protein immunoblot, is a technique that combines protein electrophoresis and antibodies to detect proteins in a sample. A western blot is fairly quick and simple compared to the above techniques and, thus, can serve as an assay to validate results from other experiments. The protein sample is first separated by gel electrophoresis, then transferred to a nitrocellulose or other type of membrane, and finally stained with a primary antibody that specifically binds the protein of interest. A fluorescent or radioactive-labeled secondary antibody binds to the primary antibody and provides a means of detection via either photography or x-ray film, respectively.
Key Points
• Mass Spectrometry is a technique that is useful for determining the size of a protein or protein complex.
• X-ray crystallography and NMR are techniques useful for determining the 3-D structure of a protein or protein complex.
• Protein microarrays are useful for determining protein-protein interactions.
Key Terms
• microarray: any of several devices containing a two-dimensional array of small quantities of biological material used for various types of assays
• reporter gene: a gene that researchers attach to a regulatory sequence of another gene of interest and whose product is easily identifiable in assays | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.05%3A_Comparative_and_Functional_Genomics/18.5B%3A_Basic_Techniques_in_Protein_Analysis.txt |
Skills to Develop
• Explain pharmacogenomics
• Define polygenic
The introduction of DNA sequencing and whole genome sequencing projects, particularly the Human Genome project, has expanded the applicability of DNA sequence information. Genomics is now being used in a wide variety of fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics. The most commonly known application of genomics is to understand and find cures for diseases.
Predicting Disease Risk at the Individual Level
Predicting the risk of disease involves screening currently healthy individuals by genome analysis at the individual level. Intervention with lifestyle changes and drugs can be recommended before disease onset. However, this approach is most applicable when the problem resides within a single gene defect. Such defects only account for approximately 5 percent of diseases in developed countries. Most of the common diseases, such as heart disease, are multi-factored or polygenic, which is a phenotypic characteristic that involves two or more genes, and also involve environmental factors such as diet. In April 2010, scientists at Stanford University published the genome analysis of a healthy individual (Stephen Quake, a scientist at Stanford University, who had his genome sequenced); the analysis predicted his propensity to acquire various diseases. A risk assessment was performed to analyze Quake’s percentage of risk for 55 different medical conditions. A rare genetic mutation was found, which showed him to be at risk for sudden heart attack. He was also predicted to have a 23 percent risk of developing prostate cancer and a 1.4 percent risk of developing Alzheimer’s. The scientists used databases and several publications to analyze the genomic data. Even though genomic sequencing is becoming more affordable and analytical tools are becoming more reliable, ethical issues surrounding genomic analysis at a population level remain to be addressed.
Art Connection
In 2011, the United States Preventative Services Task Force recommended against using the PSA test to screen healthy men for prostate cancer. Their recommendation is based on evidence that screening does not reduce the risk of death from prostate cancer. Prostate cancer often develops very slowly and does not cause problems, while the cancer treatment can have severe side effects. The PCA3 test is considered to be more accurate, but screening may still result in men who would not have been harmed by the cancer itself suffering side effects from treatment. What do you think? Should all healthy men be screened for prostate cancer using the PCA3 or PSA test? Should people in general be screened to find out if they have a genetic risk for cancer or other diseases?
Pharmacogenomics and Toxicogenomics
Pharmacogenomics, also called toxicogenomics, involves evaluating the effectiveness and safety of drugs on the basis of information from an individual's genomic sequence. Genomic responses to drugs can be studied using experimental animals (such as laboratory rats or mice) or live cells in the laboratory before embarking on studies with humans. Studying changes in gene expression could provide information about the transcription profile in the presence of the drug, which can be used as an early indicator of the potential for toxic effects. For example, genes involved in cellular growth and controlled cell death, when disturbed, could lead to the growth of cancerous cells. Genome-wide studies can also help to find new genes involved in drug toxicity. Personal genome sequence information can be used to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype. The gene signatures may not be completely accurate, but can be tested further before pathologic symptoms arise.
Microbial Genomics: Metagenomics
Traditionally, microbiology has been taught with the view that microorganisms are best studied under pure culture conditions, which involves isolating a single type of cell and culturing it in the laboratory. Because microorganisms can go through several generations in a matter of hours, their gene expression profiles adapt to the new laboratory environment very quickly. In addition, the vast majority of bacterial species resist being cultured in isolation. Most microorganisms do not live as isolated entities, but in microbial communities known as biofilms. For all of these reasons, pure culture is not always the best way to study microorganisms. Metagenomics is the study of the collective genomes of multiple species that grow and interact in an environmental niche. Metagenomics can be used to identify new species more rapidly and to analyze the effect of pollutants on the environment (Figure \(2\)).
Microbial Genomics: Creation of New Biofuels
Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population’s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped. Microorganisms are used to create products, such as enzymes that are used in research, antibiotics, and other anti-microbial mechanisms. Microbial genomics is helping to develop diagnostic tools, improved vaccines, new disease treatments, and advanced environmental cleanup techniques.
Mitochondrial Genomics
Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and is often used to study evolutionary relationships. Another feature that makes studying the mitochondrial genome interesting is that the mitochondrial DNA in most multicellular organisms is passed on from the mother during the process of fertilization. For this reason, mitochondrial genomics is often used to trace genealogy.
Information and clues obtained from DNA samples found at crime scenes have been used as evidence in court cases, and genetic markers have been used in forensic analysis. Genomic analysis has also become useful in this field. In 2001, the first use of genomics in forensics was published. It was a collaborative attempt between academic research institutions and the FBI to solve the mysterious cases of anthrax communicated via the US Postal Service. Using microbial genomics, researchers determined that a specific strain of anthrax was used in all the mailings.
Genomics in Agriculture
Genomics can reduce the trials and failures involved in scientific research to a certain extent, which could improve the quality and quantity of crop yields in agriculture. Linking traits to genes or gene signatures helps to improve crop breeding to generate hybrids with the most desirable qualities. Scientists use genomic data to identify desirable traits, and then transfer those traits to a different organism. Scientists are discovering how genomics can improve the quality and quantity of agricultural production. For example, scientists could use desirable traits to create a useful product or enhance an existing product, such as making a drought-sensitive crop more tolerant of the dry season.
Summary
Imagination is the only barrier to the applicability of genomics. Genomics is being applied to most fields of biology; it is being used for personalized medicine, prediction of disease risks at an individual level, the study of drug interactions before the conduct of clinical trials, and the study of microorganisms in the environment as opposed to the laboratory. It is also being applied to developments such as the generation of new biofuels, genealogical assessment using mitochondria, advances in forensic science, and improvements in agriculture.
Art Connections
Figure \(1\): In 2011, the United States Preventative Services Task Force recommended against using the PSA test to screen healthy men for prostate cancer. Their recommendation is based on evidence that screening does not reduce the risk of death from prostate cancer. Prostate cancer often develops very slowly and does not cause problems, while the cancer treatment can have severe side effects. The PCA3 test is considered to be more accurate, but screening may still result in men who would not have been harmed by the cancer itself suffering side effects from treatment. What do you think? Should all healthy men be screened for prostate cancer using the PCA3 or PSA test? Should people in general be screened to find out if they have a genetic risk for cancer or other diseases?
Answer
There are no right or wrong answers to these questions. While it is true that prostate cancer treatment itself can be harmful, many men would rather be aware that they have cancer so they can monitor the disease and begin treatment if it progresses. And while genetic screening may be useful, it is expensive and may cause needless worry. People with certain risk factors may never develop the disease, and preventative treatments may do more harm than good.
Glossary
metagenomics
study of the collective genomes of multiple species that grow and interact in an environmental niche
pharmacogenomics
study of drug interactions with the genome or proteome; also called toxicogenomics
polygenic
phenotypic characteristic caused by two or more genes
pure culture
growth of a single type of cell in the laboratory
18.06: Applications of Genomics
Learning Objectives
• Explain the ways in which cancer proteomics may lead to better treatments
Genomes and proteomes of patients suffering from specific diseases are being studied to understand the genetic basis of diseases. The most prominent set of diseases being studied with proteomic approaches is cancer. Proteomic approaches are being used to improve screening and early detection of cancer, which is achieved by identifying proteins whose expression is affected by the disease process.
An individual protein that indicates disease is called a biomarker, whereas a set of proteins with altered expression levels is called a protein signature. For a biomarker or protein signature to be useful as a candidate for early screening and detection of a cancer, it must be secreted in body fluids (e.g. sweat, blood, or urine) such that large-scale screenings can be performed in a non-invasive fashion. The current problem with using biomarkers for the early detection of cancer is the high rate of false-negative results. A false-negative is an incorrect test result that should have been positive. In other words, many cases of cancer go undetected, which makes biomarkers unreliable. Some examples of protein biomarkers used in cancer detection are CA-125 for ovarian cancer and PSA for prostate cancer. Protein signatures may be more reliable than biomarkers to detect cancer cells.
Proteomics is also being used to develop individualized treatment plans, which involves the prediction of whether or not an individual will respond to specific drugs and the side effects that the individual may experience. In addition, proteomics can be used to predict the possibility of disease recurrence. The National Cancer Institute has developed programs to improve the detection and treatment of cancer. The Clinical Proteomic Technologies for Cancer and the Early Detection Research Network are efforts to identify protein signatures specific to different types of cancers. The Biomedical Proteomics Program is designed to identify protein signatures and design effective therapies for cancer patients.
Key Points
• Identifying those proteins whose expression is affected by disease processes can be used to improve screening and early detection of cancer.
• Different biomarkers and protein signatures are being used to analyze each type of cancer.
• A future goal of cancer proteomics is to have a personalized treatment plan for each individual.
Key Terms
• biomarker: a substance used as an indicator of a biological state, most commonly disease
Contributions and Attributions
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44558/latest...ol11448/latest. License: CC BY: Attribution
• Proteomics/Introduction to Proteomics. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Proteom..._to_Proteomics. License: CC BY-SA: Attribution-ShareAlike
• proteomics. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/proteomics. License: CC BY-SA: Attribution-ShareAlike
• proteome. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/proteome. License: CC BY-SA: Attribution-ShareAlike
• genomics. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/genomics. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Genomics and Proteomics. November 12, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m45485/latest/. License: CC BY: Attribution
• Structural Biochemistry/Proteins/Western Blotting. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Structu...stern_Blotting. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44558/latest...ol11448/latest. License: CC BY: Attribution
• Structural Biochemistry/Proteins/NMR Spectroscopy. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Structu...R_Spectroscopy. License: CC BY-SA: Attribution-ShareAlike
• Proteomics/Protein Identification - Mass Spectrometry. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Proteom...s_Spectrometry. License: CC BY-SA: Attribution-ShareAlike
• Structural Biochemistry/Proteins/X-ray Crystallography. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Structu...rystallography. License: CC BY-SA: Attribution-ShareAlike
• Two-hybrid screening. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Two-hybrid_screening. License: CC BY-SA: Attribution-ShareAlike
• reporter gene. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/reporter%20gene. License: CC BY-SA: Attribution-ShareAlike
• microarray. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/microarray. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Genomics and Proteomics. November 12, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m45485/latest/. License: CC BY: Attribution
• OpenStax College, Genomics and Proteomics. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44558/latest...e_17_05_01.jpg. License: CC BY: Attribution
• Proteomics/Protein Identification - Mass Spectrometry. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Proteom...s_Spectrometry. License: CC BY-SA: Attribution-ShareAlike
• Structural Biochemistry/Proteins/X-ray Crystallography. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Structu...rystallography. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Biology. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44558/latest...ol11448/latest. License: CC BY: Attribution
• biomarker. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/biomarker. License: CC BY-SA: Attribution-ShareAlike
• OpenStax College, Genomics and Proteomics. November 12, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m45485/latest/. License: CC BY: Attribution
• OpenStax College, Genomics and Proteomics. October 16, 2013. Provided by: OpenStax CNX. Located at: http://cnx.org/content/m44558/latest...e_17_05_01.jpg. License: CC BY: Attribution
• Proteomics/Protein Identification - Mass Spectrometry. Provided by: Wikibooks. Located at: http://en.wikibooks.org/wiki/Proteom...s_Spectrometry. License: CC BY-SA: Attribution-ShareAlike
• Structural Biochemistry/Proteins/X-ray Crystallography. Provided by: Wikibooks. Located at: en.wikibooks.org/wiki/Structu...rystallography. License: CC BY-SA: Attribution-ShareAlike
• Cancer biomarkers. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Cancer_biomarkers. License: Public Domain: No Known Copyright | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/18%3A_Genomics/18.06%3A_Applications_of_Genomics/18.6C%3A_Cancer_Proteomics.txt |
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/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.01%3A_The_Process_of_Development/19.1.01%3A_Embryonic_Development.txt |
Learning Objectives
• Describe the stages of the cell cycle
• Discuss how the cell cycle is regulated
• Describe the implications of losing control over the cell cycle
• Describe the stages of mitosis and cytokinesis, in order
So far in this chapter, you have read numerous times of the importance and prevalence of cell division. While there are a few cells in the body that do not undergo cell division (such as gametes, red blood cells, most neurons, and some muscle cells), most somatic cells divide regularly. A somatic cell is a general term for a body cell, and all human cells, except for the cells that produce eggs and sperm (which are referred to as germ cells), are somatic cells. Somatic cells contain two copies of each of their chromosomes (one copy received from each parent).
A homologous pair of chromosomes is the two copies of a single chromosome found in each somatic cell. The human is a diploid organism, having 23 homologous pairs of chromosomes in each of the somatic cells. The condition of having pairs of chromosomes is known as diploidy. Cells in the body replace themselves over the lifetime of a person. For example, the cells lining the gastrointestinal tract must be frequently replaced when constantly “worn off” by the movement of food through the gut. But what triggers a cell to divide, and how does it prepare for and complete cell division? The cell cycle is the sequence of events in the life of the cell from the moment it is created at the end of a previous cycle of cell division until it then divides itself, generating two new cells.
The Cell Cycle
One “turn” or cycle of the cell cycle consists of two general phases: interphase, followed by mitosis and cytokinesis. Interphase is the period of the cell cycle during which the cell is not dividing. The majority of cells are in interphase most of the time. Mitosis is the division of genetic material, during which the cell nucleus breaks down and two new, fully functional, nuclei are formed. Cytokinesis divides the cytoplasm into two distinctive cells.
Interphase
A cell grows and carries out all normal metabolic functions and processes in a period called G1 (Figure 1). G1 phase (gap 1 phase) is the first gap, or growth phase in the cell cycle. For cells that will divide again, G1 is followed by replication of the DNA, during the S phase. The S phase (synthesis phase) is period during which a cell replicates its DNA.
After the synthesis phase, the cell proceeds through the G2 phase. The G2 phase is a second gap phase, during which the cell continues to grow and makes the necessary preparations for mitosis. Between G1, S, and G2 phases, cells will vary the most in their duration of the G1 phase. It is here that a cell might spend a couple of hours, or many days. The S phase typically lasts between 8-10 hours and the G2 phase approximately 5 hours. In contrast to these phases, the G0 phase is a resting phase of the cell cycle. Cells that have temporarily stopped dividing and are resting (a common condition) and cells that have permanently ceased dividing (like nerve cells) are said to be in G0.
The Structure of Chromosomes
Billions of cells in the human body divide every day. During the synthesis phase (S, for DNA synthesis) of interphase, the amount of DNA within the cell precisely doubles. Therefore, after DNA replication but before cell division, each cell actually contains two copies of each chromosome. Each copy of the chromosome is referred to as a sister chromatid and is physically bound to the other copy. The centromere is the structure that attaches one sister chromatid to another. Because a human cell has 46 chromosomes, during this phase, there are 92 chromatids (46 × 2) in the cell. Make sure not to confuse the concept of a pair of chromatids (one chromosome and its exact copy attached during mitosis) and a homologous pair of chromosomes (two paired chromosomes which were inherited separately, one from each parent) (Figure 2).
Mitosis
The mitotic phase of the cell typically takes between 1 and 2 hours. During this phase, a cell undergoes two major processes. First, it completes mitosis, during which the contents of the nucleus are equitably pulled apart and distributed between its two halves. Cytokinesis then occurs, dividing the cytoplasm and cell body into two new cells. Mitosis is divided into four major stages that take place after interphase (Table 1) and in the following order: prophase, metaphase, anaphase, and telophase. The process is then followed by cytokinesis.
Table 1. Cell Division: Mitosis Followed by Cytokinesis
Phase Illustration Key Events Micrograph
Prophase Chromosomes condense and become visible
Spindle fibers emerge from the centrosomes
Nuclear envelope breaks down
Centrosomes move toward opposite poles
Prometaphase Chromosomes continue to condense
Kinetochores appear at the centromeres
Mitotic spindle microtubules attach to kinetochores
Metaphase Chromosomes are lined up at the metaphase plate
Each sister chromatid is attached to a spindle fiber originating from opposite poles
Anaphase Centromeres split in two
Sister chromatids (now called chromosomes) are pulled toward opposite poles
Certain spindle fibers begin to elongate the cell
Telophase Chromosomes arrive at opposite poles and begin to decondense
Nuclear envelope surrounds each set of chromosomes
The mitotic spindle breaks down
Spindle fibers continue to push poles apart
Cytokinesis Animal cells: a cleavage furrow separates the daughter cells
Plant cells: a cell plate, the precursor to a new cell wall, separates the daughter cells
Prophase is the first phase of mitosis, during which the loosely packed chromatin coils and condenses into visible chromosomes. During prophase, each chromosome becomes visible with its identical partner attached, forming the familiar X-shape of sister chromatids. The nucleolus disappears early during this phase, and the nuclear envelope also disintegrates. A major occurrence during prophase concerns a very important structure that contains the origin site for microtubule growth. Recall the cellular structures called centrioles that serve as origin points from which microtubules extend. These tiny structures also play a very important role during mitosis. A centrosome is a pair of centrioles together. The cell contains two centrosomes side-by-side, which begin to move apart during prophase. As the centrosomes migrate to two different sides of the cell, microtubules begin to extend from each like long fingers from two hands extending toward each other. The mitotic spindle is the structure composed of the centrosomes and their emerging microtubules. Near the end of prophase there is an invasion of the nuclear area by microtubules from the mitotic spindle. The nuclear membrane has disintegrated, and the microtubules attach themselves to the centromeres that adjoin pairs of sister chromatids. The kinetochore is a protein structure on the centromere that is the point of attachment between the mitotic spindle and the sister chromatids. This stage is referred to as late prophase or “prometaphase” to indicate the transition between prophase and metaphase.
Metaphase is the second stage of mitosis. During this stage, the sister chromatids, with their attached microtubules, line up along a linear plane in the middle of the cell. A metaphase plate forms between the centrosomes that are now located at either end of the cell. The metaphase plate is the name for the plane through the center of the spindle on which the sister chromatids are positioned. The microtubules are now poised to pull apart the sister chromatids and bring one from each pair to each side of the cell.
Anaphase is the third stage of mitosis. Anaphase takes place over a few minutes, when the pairs of sister chromatids are separated from one another, forming individual chromosomes once again. These chromosomes are pulled to opposite ends of the cell by their kinetochores, as the microtubules shorten. Each end of the cell receives one partner from each pair of sister chromatids, ensuring that the two new daughter cells will contain identical genetic material.
Telophase is the final stage of mitosis. Telophase is characterized by the formation of two new daughter nuclei at either end of the dividing cell. These newly formed nuclei surround the genetic material, which uncoils such that the chromosomes return to loosely packed chromatin. Nucleoli also reappear within the new nuclei, and the mitotic spindle breaks apart, each new cell receiving its own complement of DNA, organelles, membranes, and centrioles. At this point, the cell is already beginning to split in half as cytokinesis begins.
Cytokinesis
The cleavage furrow is a contractile band made up of microfilaments that forms around the midline of the cell during cytokinesis. (Recall that microfilaments consist of actin.) This contractile band squeezes the two cells apart until they finally separate. Two new cells are now formed. One of these cells (the “stem cell”) enters its own cell cycle; able to grow and divide again at some future time. The other cell transforms into the functional cell of the tissue, typically replacing an “old” cell there. Imagine a cell that completed mitosis but never underwent cytokinesis. In some cases, a cell may divide its genetic material and grow in size, but fail to undergo cytokinesis. This results in larger cells with more than one nucleus. Usually this is an unwanted aberration and can be a sign of cancerous cells.
Cell Cycle Control
A very elaborate and precise system of regulation controls direct the way cells proceed from one phase to the next in the cell cycle and begin mitosis. The control system involves molecules within the cell as well as external triggers. These internal and external control triggers provide “stop” and “advance” signals for the cell. Precise regulation of the cell cycle is critical for maintaining the health of an organism, and loss of cell cycle control can lead to cancer.
Mechanisms of Cell Cycle Control
As the cell proceeds through its cycle, each phase involves certain processes that must be completed before the cell should advance to the next phase. A checkpoint is a point in the cell cycle at which the cycle can be signaled to move forward or stopped. At each of these checkpoints, different varieties of molecules provide the stop or go signals, depending on certain conditions within the cell. A cyclin is one of the primary classes of cell cycle control molecules (Figure 3). A cyclin-dependent kinase (CDK) is one of a group of molecules that work together with cyclins to determine progression past cell checkpoints. By interacting with many additional molecules, these triggers push the cell cycle forward unless prevented from doing so by “stop” signals, if for some reason the cell is not ready. At the G1 checkpoint, the cell must be ready for DNA synthesis to occur. At the G2 checkpoint the cell must be fully prepared for mitosis. Even during mitosis, a crucial stop and go checkpoint in metaphase ensures that the cell is fully prepared to complete cell division. The metaphase checkpoint ensures that all sister chromatids are properly attached to their respective microtubules and lined up at the metaphase plate before the signal is given to separate them during anaphase.
The Cell Cycle Out of Control: Implications
Most people understand that cancer or tumors are caused by abnormal cells that multiply continuously. If the abnormal cells continue to divide unstopped, they can damage the tissues around them, spread to other parts of the body, and eventually result in death. In healthy cells, the tight regulation mechanisms of the cell cycle prevent this from happening, while failures of cell cycle control can cause unwanted and excessive cell division. Failures of control may be caused by inherited genetic abnormalities that compromise the function of certain “stop” and “go” signals. Environmental insult that damages DNA can also cause dysfunction in those signals. Often, a combination of both genetic predisposition and environmental factors lead to cancer. The process of a cell escaping its normal control system and becoming cancerous may actually happen throughout the body quite frequently. Fortunately, certain cells of the immune system are capable of recognizing cells that have become cancerous and destroying them. However, in certain cases the cancerous cells remain undetected and continue to proliferate. If the resulting tumor does not pose a threat to surrounding tissues, it is said to be benign and can usually be easily removed. If capable of damage, the tumor is considered malignant and the patient is diagnosed with cancer.
Try It
Cancer is an extremely complex condition, capable of arising from a wide variety of genetic and environmental causes. Typically, mutations or aberrations in a cell’s DNA that compromise normal cell cycle control systems lead to cancerous tumors. Cell cycle control is an example of a homeostatic mechanism that maintains proper cell function and health. While progressing through the phases of the cell cycle, a large variety of intracellular molecules provide stop and go signals to regulate movement forward to the next phase. These signals are maintained in an intricate balance so that the cell only proceeds to the next phase when it is ready.
The homeostatic control of the cell cycle can be thought of like a car’s cruise control. Cruise control will continually apply just the right amount of acceleration to maintain a desired speed, unless the driver hits the brakes, in which case the car will slow down. Similarly, the cell includes molecular messengers, such as cyclins, that push the cell forward in its cycle. In addition to cyclins, a class of proteins that are encoded by genes called proto-oncogenes provide important signals that regulate the cell cycle and move it forward. Examples of proto-oncogene products include cell-surface receptors for growth factors, or cell-signaling molecules, two classes of molecules that can promote DNA replication and cell division.
In contrast, a second class of genes known as tumor suppressor genes sends stop signals during a cell cycle. For example, certain protein products of tumor suppressor genes signal potential problems with the DNA and thus stop the cell from dividing, while other proteins signal the cell to die if it is damaged beyond repair. Some tumor suppressor proteins also signal a sufficient surrounding cellular density, which indicates that the cell need not presently divide. The latter function is uniquely important in preventing tumor growth: normal cells exhibit a phenomenon called “contact inhibition;” thus, extensive cellular contact with neighboring cells causes a signal that stops further cell division.
These two contrasting classes of genes, proto-oncogenes and tumor suppressor genes, are like the accelerator and brake pedal of the cell’s own “cruise control system,” respectively. Under normal conditions, these stop and go signals are maintained in a homeostatic balance. Generally speaking, there are two ways that the cell’s cruise control can lose control: a malfunctioning (overactive) accelerator, or a malfunctioning (underactive) brake. When compromised through a mutation, or otherwise altered, proto-oncogenes can be converted to oncogenes, which produce oncoproteins that push a cell forward in its cycle and stimulate cell division even when it is undesirable to do so.
For example, a cell that should be programmed to self-destruct (a process called apoptosis) due to extensive DNA damage might instead be triggered to proliferate by an oncoprotein. On the other hand, a dysfunctional tumor suppressor gene may fail to provide the cell with a necessary stop signal, also resulting in unwanted cell division and proliferation. A delicate homeostatic balance between the many proto-oncogenes and tumor suppressor genes delicately controls the cell cycle and ensures that only healthy cells replicate. Therefore, a disruption of this homeostatic balance can cause aberrant cell division and cancerous growths.
Contributors and Attributions
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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.
19.2.03: 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/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.02%3A_Cell_Division/19.2.02%3A_Frog_Embryology.txt |
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.
Types of Stem Cells
Several adjectives are used to describe the developmental potential of stem cells; that is, the number of different kinds of differentiated cell that they can become.
1. Totipotent cells. In mammals, totipotent cells have the potential to become any type in the adult body and any cell of the extraembryonic membranes (e.g., placenta). The only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage (as shown by the ability of mammals to produce identical twins, triplets, etc.). In mammals, the expression totipotent stem cells is a misnomer — totipotent cells cannot make more of themselves.
2. Pluripotent stem cells. These are true stem cells, with the potential to make any differentiated cell in the body (but probably not those of the placenta which is derived from the trophoblast).
Three types of pluripotent stem cells occur naturally:
• Embryonic Stem (ES) Cells. These can be isolated from the inner cell mass (ICM) of the blastocyst — the stage of embryonic development when implantation occurs. For humans, excess embryos produced during in vitro fertilization (IVF) procedures are used. Harvesting ES cells from human blastocysts is controversial because it destroys the embryo, which could have been implanted to produce another baby (but often was simply going to be discarded).
• Embryonic Germ (EG) Cells. These can be isolated from the precursor to the gonads in aborted fetuses.
• Embryonic Carcinoma (EC) Cells. These can be isolated from teratocarcinomas, a tumor that occasionally occurs in a gonad of a fetus. Unlike the other two, they are usually aneuploid.
All three of these types of pluripotent stem cells can only be isolated from embryonic or fetal tissue. They can be grown in culture, but only with special methods to prevent them from differentiating.
In mice and rats, embryonic stem cells can also:
• contribute to the formation of a healthy chimeric adult when injected into a blastocyst which is then implanted in a surrogate mother;
• enter the germline of these animals; that is, contribute to their pool of gametes;
• develop into teratomas when injected into immunodeficient (SCID) mice. These tumors produce a wide variety of cell types representing all three germ layers (ectoderm, mesoderm, and endoderm).
Using genetic manipulation in the laboratory, pluripotent stem cells can now be generated from differentiated cells. These induced pluripotent stem cells (iPSCs) are described below.
1. Multipotent stem cells. These are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. Multipotent stem cells are found in adult animals; perhaps most organs in the body (e.g., brain, liver, lungs) contain them where they can replace dead or damaged cells. These adult stem cells may also be the cells that — when one accumulates sufficient mutations — produce a clone of cancer cells.
Stem Cells for Human Therapy
The Dream
Many medical problems arise from damage to differentiated cells. Examples:
• Type 1 diabetes mellitus where the beta cells of the pancreas have been destroyed by an autoimmune attack
• Parkinson's disease; where dopamine-secreting cells of the brain have been destroyed
• Spinal cord injuries leading to paralysis of the skeletal muscles
• Ischemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvation
• Multiple sclerosis with its loss of myelin sheaths around axons
• Blindness caused by damage to the cornea
The great developmental potential of stem cells has created intense research into enlisting them to aid in replacing the lost cells of such disorders. While progress has been slow, some procedures already show promise. Using multipotent "adult" stem cells.
• culturing human epithelial stem cells and using their differentiated progeny to replace a damaged cornea. This works best when the stem cells are from the patient (e.g. from the other eye). Corneal cells from another person (an allograft) are always at risk of rejection by the recipient's immune system.
• the successful repair of a damaged left bronchus using a section of a donated trachea that was first cleansed of all donor cells and then seeded with the recipient's epithelial cells and cartilage-forming cells grown from stem cells in her bone marrow. So far the patient is doing well and needs no drugs to suppress her immune system.
Using differentiated cells derived from embryonic stem (ES) cells. Phase I clinical trials are underway to assess the safety of
• injecting retinal cells derived from ES cells
• into the eyes of young people with an inherited form of juvenile blindness;
• into the eyes of adults with age-related macular degeneration.
• injecting glial cells derived from ES cells into patients paralyzed by spinal cord injuries.
The Immunological Problems
One major problem that must be solved before human stem cell therapy becomes a reality is the threat of rejection of the transplanted cells by the host's immune system (if the stem cells are allografts; that is, come from a genetically-different individual).
A Possible Solution
One way to avoid the problem of rejection is to use stem cells that are genetically identical to the host. This is already possible in the rare situations when the patient has healthy stem cells in an undamaged part of the body (like the stem cells being used to replace damaged corneas). But even where no "autologous" stems cells are available, there may be a solution: using somatic-cell nuclear transfer .
In this technique,
1. An egg has its own nucleus removed and replaced by
2. a nucleus taken from a somatic (e.g., skin) cell of the donor.
3. The now-diploid egg is allowed to develop in culture to the blastocyst stage when
4. embryonic stem cells can be harvested and grown up in culture.
5. When they have acquired the desired properties, they can be implanted in the donor with no fear of rejection.
Using this procedure it possible to not only grow blastocysts but even have these go on to develop into adult animals — cloning — with a nuclear genome identical to that of the donor of the nucleus. The first successful cloning by SCNT was with amphibians. Later, mammals such as sheep (Dolly), cows, mice and others were successfully cloned. And in the 11 November 2007 issue of Science, researchers in Oregon reported success with steps 1–4 in rhesus monkeys (primates like us).
Their procedure:
• Remove the spindle and thus all nuclear material from secondary oocytes at metaphase of meiosis II.
• Fuse each enucleated egg with a skin cell taken from a male monkey.
• Culture until the blastocyst stage is reached.
• Extract embryonic stem cells from the inner cell mass.
• Establish that they have the nuclear genome of the male (but mostly the mitochondrial genome of the female).
• Culture with factors to encourage differentiation: they grew cardiac muscle cells (which contracted), and even neuron-like cells.
• Inject into SCID mice and examine the tumors that formed. These contained cells of all three germ layers: ectoderm, mesoderm, and endoderm.
• However, even after more than 100 attempts, they have not been able to implant their monkey blastocysts in the uterus of a surrogate mother to produce a cloned monkey.
This should reassure people who view with alarm the report in May 2013 by the same workers that they have finally succeeded in producing embryonic stem cells (ESCs) using SCNT from differentiated human tissue. The workers assure us that they will not attempt to implant these blastocysts in a surrogate mother to produce a cloned human. And their failure with monkeys suggests that they would fail even if they did try.
While cloning humans still seems impossible, patient-specific ESCs
• could be used in cell-replacement therapy or, failing that,
• provide the material for laboratory study of the basis of — and perhaps treatment of — genetic diseases.
Whether they will be more efficient and more useful than induced pluripotent stem cells remains to be seen.
Questions that Remain to be Answered
• Imprinted Genes. Sperm and eggs each contain certain genes that carry an "imprint" identifying them later in the fertilized egg as being derived from the father or mother respectively. Creating an egg with a nucleus taken from an adult cell may not allow a proper pattern of imprinting to be established. When the diploid adult nucleus is inserted into the enucleated egg (at least those of sheep and mice), the new nucleus becomes "reprogrammed". What reprogramming actually means still must be learned, but perhaps it involves the proper methylation and demethylation of imprinted genes. For example, the inactive X chromosome in adult female cells must be reactivated in the egg, and this actually seems to happen.
• Aneuploidy. In primates (in contrast to sheep, cattle, and mice), the process of removing the resident nucleus causes molecules associated with the centrosome to be lost as well. Although injecting a donor nucleus allows mitosis to begin, spindle formation may be disrupted, and the resulting cells fail to get the correct complement of chromosomes (aneuploidy).
• Somatic Mutations. This procedure also raises the spectre of amplifying the effect(s) of somatic mutations. In other words, mutations that might be well-tolerated in a single somatic cell of the adult (used to provide the nucleus) might well turn out to be quite harmful when they become replicated in a clone of cells injected later into the patient.
• Political Controversy. The goal of this procedure (which is often called therapeutic cloning even though no new individual is produced) is to culture a blastocyst that can serve as a source of ES cells. But that same blastocyst could theoretically be implanted in a human uterus and develop into a baby that was genetically identical to the donor of the nucleus. In this way, a human would be cloned. And in fact, Dolly and other animals are now routinely cloned this way. The spectre of this is so abhorrent to many that they would like to see the procedure banned despite its promise for helping humans. In fact, many are so strongly opposed to using human blastocysts — even when produced by nuclear transfer — that they would like to limit stem cell research to adult stem cells (even though these are only multipotent).
Possible Solutions to the Ethical Controversy
Induced pluripotent stem cells (iPSCs)
A promising alternative to the use of embryonic stem cells in human therapy are recently-developed methods of genetically reprogramming the nuclei of differentiated adult cells so that they regain the pluripotency of embryonic stem (ES) cells.
In June 2007, three laboratories reported that introducing extra copies of only 4 genes into adult mouse skin cells (fibroblasts) enables them to regain the properties of ES cells. When these cells, named induced pluripotent stem cells (iPSCs for short), were placed in mouse blastocysts, they participated in building all the tissues of the chimeric mice that resulted. (When placed in tetraploid (4n) blastocysts — unable by themselves to develop normally — embryos were formed that thus were clones of the skin cell donor.) The four genes: c-Myc, Sox2, Oct3/4, Klf4.
By 2009, several labs had succeeded in producing fertile adult mice from iPSCs derived from mouse embryonic fibroblasts. This shows that iPSCs are just a capable of driving complete development (pluripotency) as embryonic stem cells.
Reprogramming works in humans, too! Using the same four genes, the Yamanaka lab in Japan reported on 20 November 2007, that they now had reprogrammed human skin cells to become induced pluripotent stem cells (iPSCs). And the Thomson lab in Wisconsin accomplished the same thing using SOX2, OCT4, NANOG, and LIN28.
Further evidence of the remarkable role played by these few genes is the finding that during normal embryonic development of the zebrafish, the same or similar genes (SoxB1, Oct4, Nanog) are responsible for turning on the genes of the zygote. Earlier in development of the blastula, all the genes being expressed (including these) are the mother's — mRNAs and proteins that the mother deposited in the unfertilized egg. It makes sense that the same proteins that can reprogram a differentiated cell into a pluripotent state (iPSCs) are those that produce the pluripotent cells of the early embryo.
These achievements open the possibility of
• creating cells for laboratory study of the basis of genetic diseases.
Examples: researchers have succeeded in deriving iPSCs from
• patients with amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), and then causing them to differentiate into motor neurons (the cells affected in the disease) for study of their properties;
• the skin cells of a patient with an inherited heart disease (long QT syndrome) and causing these to differentiate into beating heart cells for study in the laboratory.
• The Jaenisch lab reported in the 6 March 2009 issue of Cell that they have succeeded in making iPSCs (they call them hiPSCs) from fibroblasts taken from patients with Parkinson's disease. The cells were then differentiated into dopamine-releasing cells — the cells lacking in this disease. What is particularly exciting is that they accomplished this after using the Cre-lox system to remove all the genes (e.g., SOX2, OCT4, KLF4) needed for reprogramming the fibroblasts to an embryonic-stem-cell-like condition.
• Since that report, other laboratories — using other methods — have also created iPSCs from which all foreign DNA (vector and transgenes) has been removed. Not only should such cells be safer to use in therapy, but these results show that the stimulus to reprogram a differentiated cell into a pluripotent state need only be transitory.
• creating patient-specific cell transplants — avoiding the threat of immunological rejection — that could be used for human therapy.
Therapy with iPSCs has already been demonstrated in mice. Three examples:
1. The Jaenisch lab in Cambridge, MA reported (in Science, 21 December 2007) that they had successfully treated knock-in mice that make sickle-cell hemoglobin with the human βS genes (and show many of the signs of sickle-cell disease in humans) by
• harvesting some fully-differentiated fibroblasts from a sickle-cell mouse;
• reprogramming these to become iPSCs by infecting them with Oct4, Sox2, Klf4, and c-Myc;
• then removing (using the Cre-lox system) the c-Myc to avoid the danger of this oncogene later causing cancer in the recipient mice;
• replacing the βS genes in the iPSCs with normal human βA genes;
• coaxing, with a cocktail of cytokines, these iPSCs to differentiate in vitro into hematopoietic (blood cell) precursors;
• injecting these into sickle-cell mice that had been irradiated to destroy their own bone marrow (as is done with human bone marrow transplants). (Although the recipient mice were different animals from the fibroblast donor, they were of the same inbred strain and thus genetically the same — like identical human twins. So the procedure fully qualifies as "patient-specific", i.e., with no danger of the injected cells being rejected by the recipient's immune system.)
The result: all the signs of sickle-cell disease (e.g., anemia) in the treated animals showed marked improvement.
2. In the 25 July 2013 issue of Nature, a team of Japanese scientists report that they were able to manufacture three-dimensional buds of human liver cells. Their process:
• create human iPSCs from human fibroblasts using the techniques described above;
• treat these with the substances needed for them to differentiate in liver cell precursors;
• culture these with a mixture of human endothelial cells and mesenchymal stem cells (to mimic the conditions that occur in normal embryonic development of the liver);
• implant the resulting solid masses (buds) of liver-like cells into immunodeficient mice.
The result: the implanted buds developed a blood supply and the mice began to secrete human albumin, human alpha-1-antitrypsin, and to to detoxify injected chemicals just as human livers do.
3. Workers in the Melton lab at Harvard University reported in the 9 October 2014 issue of Cell that they had succeeded in differentiating large numbers of human beta cells from human iPSCs (as well as from human ES cells). When transplanted into diabetic mice, these cells brought their elevated blood sugar levels back down.
Let us hope that what works in mice can someday be developed into a safe therapy that will work in humans. (In the case of Type 1 diabetes mellitus, however, even patient-derived beta cells will still be at risk of the same autoimmune rejection that caused the disease in the first place.)
Despite these successes, iPSCs may not be able to completely replace the need for embryonic stem cells and may even be dangerous to use in human therapy. Several groups have found that human iPSCs contain mutations as well as epigenetic patterns (e.g., methylation of their DNA) that are not found in embryonic stem cells. Some of the mutations are also commonly found in cancer cells.
Other approaches being explored
• ES cells can be derived from a single cell removed from an 8-cell morula. The success of preimplantation genetic diagnosis (PGD) in humans shows that removing a single cell from the morula does not destroy it — the remaining cells can develop into a blastocyst, implant, and develop into a healthy baby. Furthermore, the single cell removed for PGD can first be allowed to divide with one daughter used for PGD and the other a potential source of an ES cell line.
• In altered nuclear transfer (ANT) — a modified version of SCNT (somatic-cell nuclear transfer) — a gene necessary for later implantation (Cdx2 — encoding a homeobox transcription factor) is turned off (by RNA interference) in the donor nucleus before the nucleus is inserted into the egg. The blastocyst that develops
• has a defective trophoblast that cannot implant in a uterus
• but the cells of the inner cell mass are still capable of developing into cultures of ES cells. (The gene encoding the interfering RNA can then be removed using the Cre/loxP technique.)
• Jose Cibelli and his team at Advanced Cell Technology reported in the 1 February 2002 issue of Science that they had succeeded inIf this form of cloning by parthenogenesis works in humans [It does! — success with unfertilized human eggs was reported in June 2007.], it would have
• stimulating monkey oocytes to begin dividing without completing meiosis II (therefore still 2n)
• growing these until the blastocyst stage, from which they were able to harvest
• ES cells.
• the advantage that no babies could be produced if the blastocyst should be implanted (two identical genomes cannot produce a viable mammal — probably because of incorrect imprinting);
• the disadvantage that it will only help females (because only they can provide an oocyte!) (But men may have a procedure that works for them next.)
• On 24 March 2006, Nature published an online report that a group of German scientists had been able to derive pluripotent stem cells from the stem cells that make spermatogonia in the mouse. Both in vitro and when injected into mouse blastocysts, these cells differentiated in a variety of ways including representatives of all three germ layers. If this could work in humans, it would
• provide a source of stem cells whose descendants would be "patient-specific"; that is, could be transplanted back into the donor (men only!) without fear of immune rejection.
• avoid the controversy surrounding the need to destroy human blastocysts to provide embryonic stem cells.
• The 7 January 2007 issue of Nature Biotechnology reports the successful production of amniotic fluid-derived stem cells ("AFS"). These are present in the amniotic fluid removed during amniocentesis. With the proper culture conditions, they have been shown to be able to differentiate into a variety of cell types includingSo these cells are pluripotent. Although perhaps not as versatile as embryonic stem cells, they are more versatile than adult stem cells.
• ectoderm (neural tissue)
• mesoderm (e.g., bone, muscle)
• endoderm (e.g., liver)
Applied to humans, none of the above procedures would involve the destruction of a potential human life. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.03%3A_Cell_Differentiation/19.3.01%3A_Stem_Cells.txt |
The other pages describe:
• the properties and potential therapeutic applications of embryonic (and other types of) stem cells
• how mouse embryonic stem cells can be used to make transgenic mice
• how the fusion of a differentiated cell from an adult sheep with an enucleated sheep egg can produce a clone of the cell donor ("Dolly")
The techniques used in the early steps of each process have been achieved with human cells.
Thirteen years ago 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 and yolk sac.
The cells of the inner cell mass are considered pluripotent; that is, each is capable of producing descendants representing all of the hundreds of differentiated cell types in the newborn baby, including
• ectodermal cells like neurons and skin (epithelial cells)
• mesodermal cells like striated muscle, smooth muscle, cartilage, and bone
• endodermal cells like the liver and the lining of the intestine
The Process
• Remove the trophoblast cells from a human blastocyst (these were extras not needed for assisted reproductive technology).
• Separate the cells of the inner cell mass and culture them on a plate of "feeder" cells (mouse fibroblasts were used).
• Isolate single cells and grow them as clones.
• Test the clones.
The Results
• Each successful clone maintained a normal human karyotype (unlike most cultured human cells — HeLa cells, for example).
• These cells had high levels of the enzyme telomerase, which maintains normal chromosome length and is characteristic of cells with unlimited potential to divide ("immortal").
• When injected into SCID mice, these cells formed teratomas; tumors containing a mix of differentiated human cell types, including cells characteristic of
• ectoderm
• mesoderm
• endoderm
Note
SCID = severe combined immunodeficiency.
SCID mice lack a functioning immune system (have neither T cells nor B cells) and so cannot reject foreign tissue. Some rare inherited diseases of humans are also called SCID. They produce a similar phenotype but involve different molecular defects.
Human embryonic stem cells have the potential to
• teach us about the process of human embryonic development, its genetic control, etc.
• provide a source of replacement cells to repair damaged human tissue. As the proper signals are discovered, it will be possible to cause these cells to differentiate along a particular pathway, e.g., to form insulin-secreting beta cells of the islets of Langerhans. Such cells might be able to replace lost or non-functioning cells in a human patient (e.g., with Type 1 diabetes mellitus).
However, there are problems that remain to be solved before this hope can be realized.
• Production of human ES cells requires the destruction of the blastocyst, and this is morally-repugnant to many people.
• Cell replacement therapy had better be "patient-specific"; that is, the donated cells should be genetically identical to the recipient. Otherwise, the replaced cells are at risk of being rejected by the host's immune system. [Link to a discussion of "therapeutic cloning" — a method to avoid this.
• ES cells are pluripotent and might differentiate in unwanted ways when introduced into the patient. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.03%3A_Cell_Differentiation/19.3.02%3A_Embryonic_Stem_Cells.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/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.04%3A_Nuclear_Reprogramming/19.4.01%3A_The_Organizer.txt |
Reproductive cloning, possible through artificially-induced asexual reproduction, is a method used to make a clone of an entire organism.
Learning Objectives
• Differentiate reproductive cloning from cellular and molecular cloning
Key Points
• A form of asexual reproduction, parthenogenesis, occurs when an embryo grows and develops without the fertilization of the egg.
• In reproductive cloning, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of an individual of the same species, it will become a zygote that is genetically identical to the donor.
• Reproductive cloning has become successful, but still has limitations as cloned individuals often exhibit facial, limb, and cardiac abnormalities.
• Therapeutic cloning, the cloning of human embryos as a source of embryonic stem cells, has been attempted in order to produce cells that can be used to treat detrimental diseases or defects.
Key Terms
• clone: a living organism produced asexually from a single ancestor, to which it is genetically identical
• stem cell: a primal undifferentiated cell from which a variety of other cells can develop through the process of cellular differentiation
• parthenogenesis: a form of asexual reproduction where growth and development of embryos occur without fertilization
Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals (parents), making it impossible to generate an identical copy or clone of either parent. Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory.
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction. An example of parthenogenesis occurs in species in which the female lays an egg. If the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. If the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor. Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications. There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals (e.g. horses, bulls, and goats) have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities. There have been attempts at producing cloned human embryos as sources of embryonic stem cells. Sometimes referred to as cloning for therapeutic purposes, the technique produces stem cells that attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.04%3A_Nuclear_Reprogramming/19.4.02%3A_Reproductive_Cloning.txt |
Learning Objectives
• Summarize the mechanisms and cell types that establish the body axes
Vertebrate axis formation
During development, as the germ layers form, the ball of cells still retains its spherical shape. However, animal bodies have lateral-medial (left-right), dorsal-ventral (back-belly), and anterior-posterior (head-feet) axes. How are these established? In one of the most seminal experiments ever to be carried out in developmental biology, Spemann and Mangold took dorsal cells from one embryo and transplanted them into the belly region of another embryo. They found that the transplanted embryo now had two notochords: one at the dorsal site from the original cells and another at the transplanted site. This suggested that the dorsal cells were genetically programmed to form the notochord and define the axis. Since then, researchers have identified many genes that are responsible for axis formation. Mutations in these genes leads to the loss of symmetry required for organism development. Animal bodies have externally visible symmetry. However, the internal organs are not symmetric. For example, the heart is on the left side and the liver on the right. The formation of the central left-right axis is an important process during development. This internal asymmetry is established very early during development and involves many genes. Research is still ongoing to fully understand the developmental implications of these genes.
Neural tube
In the developing chordate (including vertebrates), the neural tube is the embryo’s precursor to the central nervous system, which comprises the brain and spinal cord. The neural groove gradually deepens as the neural folds become elevated, and ultimately the folds meet and coalesce in the middle line and convert the groove into a closed tube, the neural tube or neural canal, the ectodermal wall of which forms the rudiment of the nervous system.
Primary and secondary neurulation
The neural tube develops in two ways: primary neurulation and secondary neurulation. Primary neurulation divides the ectoderm into three cell types: the internally located neural tube, the externally located epidermis, and the neural crest cells, which develop in the region between the neural tube and epidermis but then migrate to new locations. Primary neurulation begins after the neural plate forms. The edges of the neural plate start to thicken and lift upward, forming the neural folds. The center of the neural plate remains grounded, allowing a U-shaped neural groove to form. This neural groove sets the boundary between the right and left sides of the embryo. The neural folds pinch in towards the midline of the embryo and fuse together to form the neural tube. In secondary neurulation, the cells of the neural plate form a cord-like structure that migrates inside the embryo and hollows to form the tube.
Each organism uses primary and secondary neurulation to varying degrees. Neurulation in fish proceeds only via the secondary form. In avian species the posterior regions of the tube develop using secondary neurulation and the anterior regions develop by primary neurulation. In mammals, secondary neurulation begins around the 35th somite. Mammalian neural tubes close in the head in the opposite order that they close in the trunk. In the head, neural crest cells migrate, the neural tube closes, and the overlying ectoderm closes. In the trunk, overlying ectoderm closes, the neural tube closes and neural crest cells migrate.
Neural tube subdivisions
Four neural tube subdivisions eventually develop into distinct regions of the central nervous system by the division of neuroepithelial cells: the prosencephalon, the mesencephalon, the rhombencephalon and the spinal cord. The prosencephalon further goes on to develop into the telencephalon (the forebrain or cerebrum) and the diencephalon (the optic vesicles and hypothalamus). The mesencephalon develops into the midbrain. The rhombencephalon develops into the metencephalon (the pons and cerebellum) and the myelencephalon (the medulla oblongata).
For a short time, the neural tube is open both cranially and caudally. These openings, called neuropores, close during the fourth week in the human. Improper closure of the neuropores can result in neural tube defects such as anencephaly or spina bifida. The dorsal part of the neural tube contains the alar plate, which is primarily associated with sensation. The ventral part of the neural tube contains the basal plate, which is primarily associated with motor (i.e., muscle) control.
Signaling molecules and other factors
The neural tube patterns along the dorsal-ventral axis establish defined compartments of neural progenitor cells that lead to distinct classes of neurons. This patterning occurs early in development and results from the activity of several secreted signaling molecules. Sonic hedgehog (Shh) is a key player in patterning the ventral axis, while Bone morphogenic proteins (Bmp) and Wnt family members play an important role in patterning the dorsal axis. Other factors shown to provide positional information to the neural progenitor cells include Fibroblast growth factors (FGF) and Retinoic Acid. Retinoic acid is required ventrally along with Shh to induce Pax6 and Olig2 during differentiation of motor neurons. Three main ventral cell types are established during early neural tube development: the floor plate cells, which form at the ventral midline during the neural fold stage; as well as the more dorsally located motor neurons and interneurons. These cell types are specified by the secretion of Shh from the notochord (located ventrally to the neural tube), and later from the floor plate cells. Shh acts as a morphogen, meaning that it acts in a concentration-dependent manner to specify cell types as it moves further from its source. The different combinations of expression of transcription factors along the dorsal-ventral axis of the neural tube are responsible for creating the identity of the neuronal progenitor cells.
Key Points
• The three axes of the animal body are established in development via the expression of specific sets of genes that regulate which cells will develop into specific structures.
• During development, the dorsal cells are genetically programmed to develop into the notochord and define the axis.
• The neural tube can develop in two ways: primary or secondary neurulation, which are used by organisms in varying degrees to establish the neural tube that will develop into the central nervous system (brain and spinal cord).
• Specific patterns along the neural tube that are established via secretion and production of specific signaling molecules (such as Wnt, Shh, BMP and retinoic acid) play a key role in patterning the dorsal and ventral axes.
Key Terms
• neural tube: hollow longitudinal dorsal tube formed in the folding and subsequent fusion of the opposite ectodermal folds in the embryo that gives rise to the brain and spinal cord
• neurulation: the process by which the beginnings of the vertebrate nervous system is formed in embryos
• anencephaly: a lethal birth defect in which most of the brain and parts of the skull are missing; absence of the encephalon
• notochord: a flexible rodlike structure that forms the main support of the body in the lowest chordates; a primitive spine | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.05%3A_Pattern_Formation/19.5.01%3A_Establishing_Body_Axes.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.
Figure 14.5.1 nanos graph
• 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
Figure 14.5.2 Gene even skipped
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.
Figure 14.5.3 Eve promoter Figure 14.5.4 Eve 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/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.05%3A_Pattern_Formation/19.5.02%3A_Segmentation.txt |
Insect (Drosophila) and frog (Xenopus) development passes through three rather different (although often overlapping) phases: (1) 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. (2) Establishing the main body parts such as the notochord and central nervous system in vertebrates and the segments in Drosophila These are run by genes of the zygote itself.
Now let us look for clues as to how the final working out of the embryo is done. We shall examine four examples:
1. the formation of wings (in Drosophila)
2. the formation of legs (also in Drosophila)
3. the formation of the bones (radius and ulna) of the front limb in mammals (mice)
4. the formation of eyes (probably in all animals)
Wings
The insect body plan consists of head, thorax, and abdomen. The thorax is built from three segments, T1, T2, and T3. Each carries a pair of legs; hence insects are six-legged creatures. In most of the insect orders, T2 and T3 each carry a pair of wings (the honeybee is an example). However, flies belong to the insect order diptera; they have only a single pair of wings (on T2). The third thoracic segment, T3, carries instead a pair of balancing organs called halteres.
In Drosophila, a gene called Ultrabithorax (Ubx) acts within the cells of T3 to suppress the formation of wings. By creating a double mutation in the Ultrabithorax gene (in its introns, as it turned out), Professor E. B. Lewis of Caltech was able to produce flies in which the halteres had been replaced by a second pair of wings. Ultrabithorax (Ubx) is an example of a "selector gene". Selector genes are genes that regulate (turning on or off) the expression of other genes. Thus selector genes act as "master switches" in development.
Wings and all their associated structures are complicated pieces of machinery. Nonetheless, mutations in a single gene, were able to cause the reprogramming of the building of T3 (and deprived the flies of their ability to fly). Selector genes encode transcription factors. Ultrabithorax encodes a transcription factor that is normally expressed at high levels in T3 (as well as in the first abdominal segment) of Drosophila.
These photographs were taken by, and kindly supplied by, Professor Lewis. He has spent his entire career studying selector genes in Drosophila. His life's work was honored when he shared the 1995 Nobel Prize for physiology or medicine.
Legs
Another selector gene, called Antennapedia (Antp), is normally turned "on" (expressed) in the thorax and turned "off" (repressed) in the cells of the head. However, mutations in Antp can cause it to turn on in the head and form a pair of legs where the antennae would normally be.
When you consider the many genes that must be involved in building a complex structure like an insect leg (or wing), it is remarkable that a single gene can switch them all on. It is also clear that once a selector gene turns "on" in certain cells of the embryo, it remains "on" in all the cells derived from those cells. Those cells become irrevocably committed to carrying out the genetic program leading to the formation of a leg or wing.
Homeobox Genes
Most selector genes, including Antp and Ubx, are homeobox genes
Antp, Ubx, and a number of other selector genes have been cloned and sequenced. They all contain within their coding regions a sequence of some 180 nucleotides called a homeobox. The approximately 60 amino acids encoded by the homeobox are called a homeodomain. It mediates DNA binding by these proteins. Many proteins containing homeodomains have been shown to be transcription factors; probably they all are.
The table shows the sequence of 60 amino acids in the homeodomain of the protein encoded by the Drosophila homeobox gene Antennapedia (Antp) compared with the homeodomain encoded by the mouse gene HoxB7; by bicoid (bcd), another homeobox gene in Drosophila; by goosecoid, a homeobox gene in Xenopus; and by mab-5, a homeobox gene in the roundworm Caenorhabditis elegans. A dash indicates that the amino acid at that position is identical to the one in the Antennapedia homeobox domain. Note that the mouse homeobox in HoxB7 differs from the Antp homeobox by only two amino acids (even though some 700 millions years have passed since these animals shared a common ancestor). HoxB6, used in the experiment described in the next section, differs from Antp in only 4 amino acids.
The Hox Cluster
Antp and Ubx are two of 8 homeobox genes that are linked in a cluster on one Drosophila chromosome. All of them encode transcription factors, each with a DNA-binding homeodomain and act in sequential zones of the embryo in the same order that they occur on the chromosome! The entire cluster is designated HOM-C with lab, Pb, Dfd, Scr, and Antp belonging to the ANT-C complex and Ubx, Abd-A, and Abd-B designated the BX-C complex, All animals that have been examined have at least one Hox cluster. Their genes show strong homology to the genes in Drosophila. Mice and humans have 4 Hox clusters (a total of 39 genes in humans) located on four different chromosomes.
• In mice: HoxA, HoxB (shown here), HoxC, HoxD
• In humans: HOXA, HOXB, HOXC, HOXD
As in Drosophila, they act along the developing embryo in the same sequence that they occupy on the chromosome. All the genes in the mammalian Hox clusters show some sequence homology to each other (especially in their homeobox) but very strong sequence homology to the equivalent genes in Drosophila. HoxB7 differs from Antp at only two amino acids, HoxB6 at four. In fact, when the mouse HoxB6 gene is inserted in Drosophila, it can substitute for Antennapedia and produce legs in place of antennae just as mutant Antp genes do. This fascinating result indicates clearly that these selector genes have retained, through millions of years of evolution, their function of assigning particular positions in the embryo, but the structures actually built depend on a different set of genes specific for a particular species.
The Mammalian Skeleton
The foreleg of the mouse and the arm of humans contain a single upper bone, the humerus, and two lower bones, the radius and ulna. The building of the entire arm, including carpals and the phalanges of the fingers, is controlled by Hox cluster genes.
When mice were bred with homozygous mutations for both HoxA11 and HoxD11, they were born with neither radius nor ulna in the forelimbs. Here, then, is another example of the power of selector genes to initiate a whole program, perhaps involving hundreds of other genes, to form a structure as complex as a forelimb. Mice that are homozygous for mutant HoxA10, C10, and D10 genes fail to form a lumbar and sacrum region in their vertebral column ("backbone"). Instead these vertebrae develop ribs like the thoracic vertebrae above them. However, if any one of these 6 Hox alleles is normal, the mice are much less severely affected. This shows the high degree of redundancy of these Hox genes.
Eyes
The compound eye of Drosophila is a marvel of precisely-organized structural elements. No one knows how many genes it takes to make the eye, but it must be a large number. Nevertheless, a single selector gene, eyeless (ey) (named, as is so often the case, for its mutant phenotype) can serve as a master switch turning on the entire cascade of genes needed to build the eye. Through genetic manipulation, it is possible to get the eyeless gene to be expressed in tissues where it is ordinarily not expressed. When eyeless is turned on in cells destined to form
• the insect's antennae, eyes form on the antennae
• wings, extra eyes form on the wings
• legs, eyes form on the legs.
Mice have a gene, small eyes (Sey; also known as Pax6) that is similar in sequence to the Drosophila eyeless gene. As its name suggests, it, too, is involved in eye formation (even though the structure of the mouse eye is entirely different from the compound eye of Drosophila).
However, the sequences of the mouse small eyes gene and the Drosophila eyeless genes are so similar that the mouse gene can substitute for eyeless when introduced into Drosophila. So, like the genes of the Hox clusters, Drosophila eyeless and mouse small eyes have retained, through millions of years of independent evolution, their function of assigning particular positions in the embryo where certain structures should be built, but the structures actually built depend on a different set of genes specific for a particular species.
Humans also have a gene that is homologous to small eyes and eyeless: it is called aniridia. Those rare humans who inherit a single mutant version of aniridia lack irises in their eyes. | textbooks/bio/Introductory_and_General_Biology/Map%3A_Raven_Biology_12th_Edition/19%3A_Cellular_Mechanisms_of_Development/19.06%3A_Evolution_of_Pattern_Formation/19.6.01%3A_Homeobox_Genes.txt |
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