Gene Mutation: Change in genes caused by change in structure of the DNA.
- Base(s) in DNA is (are) changed to other(s), or a base(s) is (are) added or removed.
- Rearrangements of the chromosomes occur and are also considered.
- Mutations can occur at a spontaneous rate, or a far greater rate when there is exposure to mutagenic agents.
- Exposure to too high a level of mutagens can threaten the survival of a species; e.g., frog species in the states of Washington and Oregon are facing extinction. This appears to be caused by reduced atmospheric ozone, and the resulting increased level of ultraviolet radiation.
Mutations are heritable changes in genetic material.
A silent mutation is a change in a nucleotide that does not result in a changed the amino acid sequence, i.e., the new codon just happens to code for the same amino acid.
Macroalterations are large changes, such as duplications, deletions, inversions or rearrangements of a large number of bases.
Microalterations involve single base pairs.
- Transitions: Purine changes to an alternate purine; pyrimidine changes to an alternate pyrimidine.
- Transversions: A position with a pyrimidine changes to have a purine; or, purine to pyrimidine. See Figure 13.8.
- Transitions occur more commonly than transversions even though more theoretical combinations of transversions are possible.
- For example, the only possible transition for the base A is to change to the base G. Two transversions are possible for the base A: C and T.
- The reason transitions are more common is due to the nature of the underlying causes of mutations and to the size of the bases.
- A purine can be altered so that it base pairs like the other. It is impossible for a purine to be altered to resemble a pyrimidine, or vice versa.
- Also, remember that purines always hydrogen bond to pyrimidines. This leaves the same distances between each pair in the middle of the DNA polymer. This reduces the likelihood that these types of mistakes are made by DNA polymerase during replication.
Click the above image to see a transition occur for the forth base. Notice that just one of the strands gets an error. After replication of a double-stranded DNA molecule that has a base change in one site, one of the daughter strands will be correct and the other mutant. Prior to replication, opportunity exists to repair the mistake.
- See the Transition and Transversion figure for an easy way to determine one from the other.
- When a single base pair causes the substitution of one amino acid for another, it is called a missense mutation.
- When a single base pair change, deletion or insertion result in the generation of a premature translation termination because of the generation of a new stop codon, this is called a nonsense mutation.
- Frameshift mutations are the result of a single base, or more, insertion or deletion. The result can be the generation of an entirely different protein from the point within the coding region where it occurs. Because they may involve many bases or just one, frameshifts are sometimes macroalterations and sometimes microalterations.
Another classification of mutations has to due with multicellular organisms where cells might belong to the germ-line, if they contribute genetic information to future generations; or, if part of the body that will never contribute genetic information to future generations, the somatic cell line.
- Somatic mutation is one that will never contribute to the germ-line of the effected individual.
- Germinal mutation is one that occurs in a cell that is a progenitor to a germ cell(s). See Figure 3.4.
- For example, if a human embryo sustains a mutation in a cell that later becomes a germ cell: a sperm or egg, it is a germinal mutation.
- Otherwise, it is a somatic cell mutation.
Another classification system for mutations has to do with using the "normal" or most common state as a reference point. The normal state is called "wild-type".
- The change from wild-type to mutant form is called a forward mutation.
- The change back to wild-type is called a reversion or back mutation.
- A reversion can occur either by true reversion, which is when the DNA is restored to the exact previous form;
- or, by a suppressor mutation, when the wild-type phenotype is restored, protein function is restored, but the DNA is not back in its original form. Can you think of a way that a suppressor mutation might occur?
- Reversions are random events. A forward mutation is caused by changing anyone of the bases, or by adding or deleting bases. However, a true reversion is when that exact change is reversed. Therefor it makes sense that the relative frequencies for these classifications of mutations are as follows:
Forward mutations > suppressor > true reversions.
Another classification of mutations is based on the functional effects of the change.
- Morphological Mutants have altered shape.
- Lethal Mutants die as a result of having the mutation, can be dominant or recessive, but most often are recessive. This makes sense because there are so many enzymes an organism must have to live; if both copies are defective, the organism would fail to survive.
- Conditional Mutants are normal under one condition (permissive), but abnormal under another (restrictive). These are extremely useful for studying processes such as development and DNA replication.
- Biochemical Mutants cause defects in biochemical pathways for a substance, which is then deficient. See Figure 13.7.
- Loss-of-Function mutations cause a loss of function that is found normally in wild-type. See Figure 13.6.
- Gain-of-function mutations create a new function not normally found in the wild-type. Hairy-faced people would be an example.
FROM THE ABOVE YOU CAN SEE THAT ANY ONE MUTATION CAN BE DESCRIBED AS BEING MANY DIFFERENT KINDS OF MUTATIONS. FOR EXAMPLE, ONE MUTATION COULD BE A FORWARD MUTATION THAT IS CONDITIONALLY LETHAL, A POINT MUTATION, A GERMINAL MUTATION, A BIOCHEMICAL MUTATION AND A LOSS-OF-FUNCTION MUTATION.
How can we observe mutations when they occur?
- Haploid organisms are extremely useful for studying mutations and mutagens. When mutations occur, they are often observable. What mutation any haploid organism gets, it expresses, because generally there is only one copy of each gene.
- Diploid organisms are tougher. Recessives, the most common type of mutation, are often invisible. They are masked by the presence of a dominant allele.
- One approach is to use F1s. The sudden appearance among the F1 of the recessive phenotype provides a measure of mutation rates.
- In Paramecia, autogamy can be used. Extra credit will be given if you can accurately recount the class lecture coverage of autogomy in Paramecium tetraurelia and its usefulness for mutagenesis studies.
- In male humans and Drosophila, genes on the X can be observed. So, the mutation frequency can be determined by studying males. See Figure 13.12.
- There is a well developed system in Drosophila that allows a certain X to be studied. That X is mutagenized in the males, then passed through an F1 female and then to the next generation of males. See Figure 13.5.
Mutation Methods for Prokaryotes
First, some background:
- Heterotrophs need organic forms of carbon.
- Autotrophs can get carbon from CO2.
- Auxotrophs have certain nutritional requirements caused by a mutation. The wild-type of a certain auxotroph is a prototroph. The prototroph doesn't have the specific nutritional requirement of its auxotrophic counterpart.
- Some auxotrophs have what is called a conditional-lethal mutation (as briefly described in the previous section).
- Complete medium (media is plural; medium is singular) is one that contains all possible requirements needed for growth.
- Minimal medium is one that contains the bare requirements, usually for the prototroph. A selective medium is one that is minimal, plus it has the requirement for a certain auxotroph.
Nutritional Mutants in Prokaryotes
- Met+ strain is the prototroph that does not need methione (met) to live; in contrast the Met- strain, which must have met to survive.
- Gal+ can use galactose as an energy source; Gal- cannot use galactose.
Resistance and Sensitivity
- Pens fail to grow (and eventually die) in the presence of penicillin.
- Penr grow in the presence of penicillin.
- Many other antibiotic resistances occur in certain bacteria. This is a problem for human health.
Penicillin can be used to isolate auxotrophic mutants. For example, to get met- mutants from met+ prototrophs:
- Grow large number of bacteria; transfer to medium lacking methione and containing penicillin.
- Let stand for a predetermined length of time.
- The met+ bacteria that don't need met, grow, and growing kills them. Those that need methione can't grow and live, if you get them out of there before they starve to death.
- Wash the bacteria out of the Penicillin containing medium and transfer all the bacteria to a medium that contains methione.
- Those that survived are those that had mutated to met- before the exposure to penicillin.
- What you did is select for the mutants, making it so that they were the only survivors.
This procedure is extremely useful.
The Genetic Basis for Bacterial Antibiotic Tolerance
Replica Plating is a method for screening bacteria after selection. It involves transferring bacteria from discreet colonies on a plate to another plate while maintaining their relative positions to each other. The plate transferred to usually contains another medium useful for selection or screening.
Before 1952, it was thought that bacteria adapted to living in the presence of substances like antibiotics by developing a biochemical tolerance. In fact, some scientists actually failed to come to grips with bacteria being like eukaryotes: organisms that depend on the use of hereditary information.
The Lederbergs' experiment verified that antibiotic resistance in bacteria was do to the presence of genetic variants in the population of bacteria prior to exposure to the specific antibiotic. They also demonstrated that the resistance was heretically transmitted. See Figure 13.3.
Replica Plating is still a widely use and useful technique. We will speak to it further in a future chapter.
Mutation Rate and Mutagens
- The mutation rate is measured several different ways. Each will yield a kind of answer useful for a different purpose.
- When comparing mutation rates, for example to determine if a substance is a mutagen, use the same units, which means, use the same measurement techniques. It is also essential to make direct comparisons.
- Some ways to express mutation rates include the average frequency of a mutational event per nucleotide incorporated, per cell, per gamete, per individual or per generation.
- Also, the gain or loss of a restriction site per division is becoming common due to the recent development of technologies that make this easy.
- A mutagen or mutagenic agent is a substance or effect that increases the mutation rate above the naturally occurring rate. We humans have inadvertently developed some powerful mutagens in recent times. Examples would be nuclear radiation, mustard gas, by-products of combustion, some insecticides used in the past and certain water pollutants.
- The natural or basal mutation rate: In the last chapter, we discussed mutations that occur during DNA replication because of DNA polymerase mistakes. Also, the DNA polymerase fidelity is much higher in some organisms than in others.
- Therefore, mutations will occur at a certain level even with the absence of mutagenic agents.
What are some of the reasons for spontaneous, naturally occurring mutations? One cause is built into the very structures of the DNA monomers.
- Called tautomeric forms, each of the DNA nitrogenous bases can occur in two different forms. For thymine and guanine, one form is the keto, the other the enol. For adenine and cytosine, one is the amino and the other imino form.
- The common forms are the keto and amino forms. See Figure 13.13.
- The other two forms are rare. However, at times, a base will shift from one form to its alternate form. Then it switches back. The "rareness" of the alternate form can be thought of as the time each base spends in one versus the alternate form. It is like when a light bulb blinks.
- Usually, there is no significant problem with a tautomeric shift.
- The base across it is still correct and in the standard tautomeric form.
- If it happens at a certain moment though, during replication, it can cause the wrong base to be incorporated.
- Adenosine can hydrogen bond with cytosine (2 hydrogen bonds) and thymine can hydrogen bond with guanine (3 hydrogen bonds).
- This results in a transition substitution. It is interesting that these kinds of mistakes occur because they give clues to how DNA polymerase decides which base to put in. Apparently, it is the formation of hydrogen bonds that provides the decision making information to the process of DNA polymerization.
- There are other reasons for mistakes to occur as well. See Figure 13.14.
- We spoke briefly about DNA methylation in an earlier section. In prokaryotes, the commonly methylated base is adenine. But, in eukaryotes, the cytosine residue is sometimes methylated.
- Methylation is correlated to reduced gene expression, and the inactive X-chromosome is highly methylated.
- It is the fifth carbon of the cytosine nitrogenous base that gets a methyl group.
- The problem with cytosine in any event is that it spontaneously (without enzyme involvement) deaminates. This means it loses the amino group from the 4th position carbon. Look at the drawing for cytosine without an amino group, but with the methyl group at the 5th carbon. Look familiar? It should. It is thymine!
- If the 5th position is not methylated, it doesn't look like thymine, but like uracil, albeit deoxyribouracil.
- There is a special enzyme that recognizes uracil in DNA and cuts off the nitrogenous base, leaving a phosphodiester link with no nitrogenous base, called an AP site. Another enzyme then recognizes this AP site and makes a cut on the 3' side of it. DNA polymerase jumps onto the gap adding bases to the 3' end. After adding a few bases, it falls off and ligase seals up the gap.
- The big problem is when the cytosine is methylated. Then it becomes thymine, which makes repair a more chancy operation. Which is right, the guanine, on the un-mutated strand or the thymine that was once a cytosine. We know the answer, but does the repair system? Most of the time it does assume that the guanine is correct, but not always.
- Other kinds of mistakes occur naturally. Some involving recombination. When there are repeats of a certain sequence, it is possible to get slippage during homologous recombination. This can cause deletions and duplications.
- When more than one copy of a long sequence exists in tandem with each other, called a tandem repeat, sometimes a misalignment can cause a deletion of one whole copy or a duplication of a whole copy. This mistake happens so often, its frequency is from 100 to 1000 times more common than a point mutation. There are well known examples of these events being the cause of human disease. Fragile-X syndrome, the most common cause of mental retardation in the U.S.A., is an example. See Figure 5.12 in your textbook.
- Many agriculturally important traits, such as seed-size, are thought to involve selection for increases in the number of tandem genes that govern the trait. This would explain why feral or "volunteer" varieties change quickly to resemble the phenotypes of their natural ancestors.
Mutagens or Mutagenic Agents
- These are substances, conditions and forms of energy that significantly increase the frequency of mutations.
- Examples of a forms of energy are ultraviolet light, x-rays, cosmic energy, gamma radiation, alpha particles, beta particles and neutrons.
- Examples of substances that are mutagens are nitrous acid, hydroxylamine, ethyl methanesulfonate, 5-bromouracil, celery, benzo (a) pyrene, acridine dyes, fungally contaminated peanuts or peanut butter, and many, many more.
- Ultraviolet Radiation causes pyrimidine dimers. See Figure 13.23.
- These can block DNA replication.
- Also, dimers interfere with base pairing between the two DNA polymeres.
- U.V. light also generates free-radicals, which create other kinds of genetic damage.
- Gamma and X-Rays: These can act directly on DNA; however, most damage is caused indirectly when molecules around the DNA such as water, are ionized, creating free-radicals, substances with unpaired electrons. Most often, the result is single or double stranded breaks in the DNA molecules. These breaks are hard to repair because they often leave a phosphate tacked onto the 3' OH, where the break occurs.
- Look at the images for some of the mutagens to get a feel for how they cause their damage. See Figure 13.21.
Some mutagens' mode of action fall into distinct classes shared by other mutagens.
- Alkylating agents are chemicals that donate alkly groups to other molecules. Ethyl methanesulfonate (EMS) is an example. See Figure 13.20.
- Base analogs are similar to the actual correct base and so get incorporated into the DNA as would its natural counterpart. The problem is that, if they are more prone to tautomeric shifts than the natural base, the frequency for mutation goes up, substantially. The compound 5-bromouracil is an example of an analog to thymine. It undergoes a tautomeric shift to base pair with guanine instead of adenine, causing a transition. See Figure 13.17; see Figure 13.16.
- Deaminating agents cause the loss of the amino group. We covered spontaneous deamination of cytosine above. Deaminating agents would increase the frequency of cytosine deamination, greatly. Nitrous acid is a deaminating agent. See Figure 13.18.
- Intercalation agents are compounds that can slide between the nitrogenous bases in a DNA molecule. This tends to cause a greater likelihood for slippage during replication, resulting in an increase in frameshift mutations. See Figure 13.19.
- Hydroxylating agents add an OH group to a position on the DNA base cytosine, causing a G:C to A:T transition. See Figure 13.20b in your textbook.
- Viruses that can integrate into the genome are also mutagenic agents.
Screening for Mutagens: The Ames Assay
So how can we test a new or suspect substance to determine if it is a mutagen?
- There are many defined strains of bacteria available, which each have a known mutation.
- The mutation in one strain might be caused by a certain base substitution.
- The mutation in another might be caused by a frameshift.
- However, in order for a strain to be useful for testing a compound, the kind of mutation responsible for the loss-of-function must be known.
- We discussed before how the forward mutation rate is so much higher than the rate for reversion.
- This is not a problem when working with bacteria, because in a short time we can easily grow billions of cells.
- Among the billions will be a few that have reverted.
- If you put a mutagen into the medium your growing the bacteria and the mutagen causes the kind of mutation that is needed to get reversion to occur, then more revertants will be found in that container.
- You put some bacteria into a flask with medium. In the exact same way you place other bacteria into the same medium but include a suspected mutagen. After growing the bacteria in the two flasks for 10 or more generations but to the same populations sizes, you plate out the bacteria onto plates missing the substance that the mutant strain needs. Only mutants will grow on these plates.
- If you find more on the plates that contained the suspected substance, then you have verified it is a mutagen.
- If the numbers are equal, it means that the substance did not cause the kind of mutation needed for that particular type of mutant to revert.
- You must screen a suspect substance with different types of mutations. Screen with bacteria that revert when frameshifts occur to determine if the mutagen causes frameshifts.
- Screen with a base pair substitution of A:T to G:C to determine if this is the kind of mutation the suspect agent might cause.
- If treating any of these kinds of strains yields an increase in the number of revertants, you will know what kind of mutation the agent causes.
It was discovered that some substances that cause cancer in humans are not mutagenic to bacteria. This was a puzzle. Ames discovered that if liver microsomes (fragments of smooth ER) were added to the suspected compounds, and then the combination was added to bacterial strains, the reversion rate increased.
What does this mean?
- It means that the compounds themselves were not mutagens, but the liver changed the compounds into mutagens, in the process of breaking them down.
- The Ames Assay is an assay like what is described above, but often including liver microsomes, to test whether the breakdown byproducts are in fact mutagenic. See Figure 13.25.
There are many different repairs systems used by organisms.
- In the last chapter we discussed the proofreading functions of DNA polymerase, which included 3' to 5' and 5' to 3' exonuclease activity and the ability to check each base-pair after incorporating a new one.
Earlier, we discussed the damage UV light causes to DNA, pyrimidine dimers. These occur when there are two pyrimidines next to each other on the same strand of DNA. Actual covalent bonds form, due to the excitation of the electrons of the benzene rings just after electron/photon coupling.
- This makes hydrogen bonding odd and it therefor makes DNA replication impossible. It does the same for RNA synthesis.
- The dimers must be removed for the cell to successfully divide, or for the gene to be usable.
- Photolyase Repair of pyrimidine dimers is the simplest system to understand, and is quite interesting. An enzyme, photolyase, sits on top of the dimer, then using a wavelength of light longer than U.V. as a source of energy, it breaks the cross-links to restore the DNA to its original form. See Figure 13.26.
- Excision Repair can be considered the second choice for repair of pyrimidine dimers. It is more complicated and involves removing a whole stretch of a strand of DNA, which leaves a section of single-stranded DNA followed before and after by double-stranded. See Figure 13.28.
- A simpler version of excision repair can be found in use for the removal of deoxyuracil. We explained earlier how this gets into the DNA. This repair involves the use of uracil DNA glycosidase, which cuts out the nitrogenous base, leaving the sugar-phosphate backbone. Another enzyme called AP endonuclease along with phosphodiesterase the strand of DNA with the AP base. DNA polymerase I then does what it does and ligase seals up the results. See Figure 13.27.
- Mismatched DNA is when the base pairing is wrong, an A base pairing with a C for example. Mismatch repair, which is well understood in E. coli, involves the binding of the DNA by a protein that recognizes a mismatch. Then a complex system evaluates which strand of DNA is most likely to be correct. The assumption of this system is the old strand is correct. Old from young are determined by methylation modifications of some of the bases. The old strand is likely to be methylated, the new one, not. If both are, things become more random (but not random). Cuts are made way up or down stream from the mismatch and exonuclease then removes the bases from the "wrong" strand and DNA Polymerase I and ligase do what they do. See Figure 13.29.
- Postreplication repair involves recombination between single-strands of daughter double-stranded DNA near the replication fork. See Figure 13.30.
- Error-Prone repair will be are last example of the sample of the many repair systems. It is also called error prone repair. It eliminates gaps as a last ditch effort to preserve a chance at life. It basically adds whatever bases, but at least usually the right number of bases. It is probably responsible for most of the observed transversions.
Cancer: Mutations can cause cancer. Proto-oncogenes are genes that help regulate cell proliferation. Mutations can make things go a little crazy. Cells dividing out of control leads to tumors.
Mutations can be useful to mankind's evolution as well. But mutation induction for generation of useful mutations works best for organisms with high reproductive rates, and certainly not for humans.
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