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Unformatted text preview: BIS101/Engebrecht Lecture20 5/14/10 DNA damage, repair and recombination In this class we have examined three processes in eukaryotes that provide genetic change: 1) Meiotic chromosome segregation (Mendel’s law of independent assortment) 2) Genetic recombination 3) Mutation!!!!! We discussed the relationship between DNA damage and mutation. DNA damage is any modification of the DNA that alters the chemical structure of the DNA double helix. This excludes modifications like methylation of bases where specific cellular enzymes are utilized (e.g. CpG methylation). DNA damage can occur at the bases, the sugars and the sugar-phosphate backbone. DNA damage can be cytotoxic, meaning it interferes with essential cellular processes like replication and transcription and therefore leads to cell death or it can be mutagenic, meaning is alters base pairing potential of the damaged base resulting in a change of nucleotide at a given position. A mutation is the heritable change in DNA sequence that results in a change in the sequence of the normal bases. Remember, a mutation may or may not result in a phenotype. The relationship between DNA damage and mutation is complicated and depends on the ability of the cell to either tolerate (meaning not fix the damage) or fix the damage either back to the normal base or to a change in the base (mutation). The ability to fix damage is dependent on a large number of genes that are used to sense and respond to DNA damage, we will discuss a number of these proteins. Are mutations adaptive (ala Lamark) or do they preexist and are selected (ala Darwin)? Luria and Delbruck did an experiment with E. coli and the lytic phage T1 to try to address this issue. They grew E. coli with T1 and looked at the number of resistant colonies that arose (i.e., E. coli mutants that were resistant to infection by T1). They reasoned that if mutations were adaptive there would not be a large variation in mutation frequency when a selective pressure is applied but if the mutations were preexisting then one would see a large variation (or fluctuation) in mutations frequency between different populations. They in fact did see a large fluctuation among the different populations and therefore concluded that mutations were preexisting and random. Figure 14-15 in 8th edition and Figure 15-5 in 9th edition shows a cartoon of this idea. This remains a controversial issue – Dr. Roth on campus is performing research on this topic. This has important implication for evolution. This type of experiment can also tell you the mutation rate as described in your book; however, you do not need to know how to calculate this. Mutations There are many types of mutations. Some of them we have discussed previously but we revisited them again today. We went over in some detail Handout26Mutations. Please know and be able to recognize these mutations (see Clicker questions below). We also discussed mutations with respect to the functional consequence of the mutation on the gene product. A null mutation (or loss-of-function) means that there is either no gene product made or the gene product made is completely nonfunctional. This is contrasted with a leaky or hypomorphic mutation, where either less protein is made or the protein that is made is only partially functional (think about the white gene of Drosophila and the many hypomorphic alleles we examined previously). Gain-of-function mutations are mutations that confer a new property onto the protein and therefore are usually dominant. This could be due to an acquisition of a novel function, increase in function or altered expression. The example we returned to is tRNA nonsense mutations. I also told you about the Antennapedia mutation of Drosophila. This is an interesting gain-of-function dominant allele of the Antennapedia gene that results in a fly that has legs on the head where there should be antenna. This is the result of expression of the Antennapedia gene in the head (normally expression is confined to the body). Interestingly, loss-of-function mutations in the gene result in antenna being formed in the place of legs. Reversion vs. suppression: please see Handout22. Remember, a reversion is the exact correction of the mutant back to the wild-type sequence, while suppression is a second mutation that is able to suppress the original mutation to make it appear wild type. Suppressors can be intragenic (meaning the secondary mutation is in the same gene that the first mutation was in, i.e., frameshift mutations in phage T4) or extragenic meaning in another gene (tRNA suppressor). Suppressor analysis is an important genetic tool to help elucidate function of a gene. Please note that you can distinguish reversion vs. intragenic suppression vs. extragenic suppression by analyzing progeny through crosses. CQ1: A mutant allele of the gene for phyenylalanine hydroxylase contains no detectable activity for that enzyme is a null allele (B). CQ2: A mutant allele of the same gene for phyenylalanine hydroxylase contains 20% activity for that enzyme is a hypomorphic allele (A). CQ3: A mutation that occurred in a plant petal is a somatic mutation (D). CQ4: A prototrophic yeast strain is obtained after mutational treatment of an auxotroph. The prototroph is crossed to a wild-type strain. Some progeny are auxotrophic; therefore, the change must be due to an extragenic suppressor (E). Somatic mutations arise in a somatic cell and therefore are not passed on to the offspring. A somatic mutation can be observed as a mutant sector, that is all cells derived from the cell that had a mutation will exhibit the mutant phenotype and those cells that didn’t derive from the mutant cell will look wild type. The somatic mutations we can see are most likely dominant because most organisms we have discussed are diploid and a mutation will only affect one copy. Since somatic mutations are not inherited, why do we care? Cancer usually arises because of an accumulation of somatic mutations. Germline mutations, on the other hand, are inherited to offspring. (There are heritable mutations that cause cancer, see HNCCP below and the BRACA genes that when mutated result in a predisposition to breast cancer). Mechanisms of Spontaneous mutagenesis = naturally occurring 1) Transposition – insertional mutations caused by transposons (jumping genes, more on this next week). It turns out that half of all spontaneous mutations in Drosophila are due to transposon insertions. 2) Replication errors: we will return to this on Thursday. What you need to know for the midterm: Viral transduction DNA structure Renaturation/Denaturation curves – how to analyze and interpret Chromosome structure Polymerase substrates Directionality of replication and transcription Ordering genes in a metabolic pathway Complementation and intragenic recombination Lac operon regulatory mutations in combination with CAP in presence and absence of glucose/lactose Effect of mutations with respect to transcription, translation and RNA processing ...
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- Spring '08