PROBLEM SET #2Micro/Genome 411 1. The following is a schematic diagram (?exploded? view) representing ongoing DNA synthesis at the replication fork. a)Label items 1, 2, 3, and 4 on the figure. b)Indicate the leading and lagging strands. c)Indicate the 3' and 5' ends at the left end of the DNA molecule being replicated. d)What is the function of the molecules depicted as item 2? 2. Sequencing has revealed a cytosine methylase (dcm) site within a gene that you are studying by the same amber suppressor "mutational fingerprinting" method used to analyze spontaneous mutations in lacI. In this case, however, not only does the apparent dcm site (5'- CCAGG-3') not appear as a G:C ? A:T hotspot, it doesn't appear as a source of G:C ? A:T transitions at all. How can you explain this result? 3. Assuming that all possible base pair changes can occur with equal frequency, what would be the expected ratio of transversions to transitions in a large collection of spontaneous mutations? 4. One class of point mutation within an open reading frame typically can't be suppressed by intragenic mutation at a second site. Which class of point mutation is this? 5. Which type of mutation would be more easily measured or quantified in E. coli: loss of the ability to synthesize proline (pro + ? pro ? ) or the opposite (pro ? ? pro + )? Why? 6. The compound 2-aminopurine (2-AP) is a structural analog of adenine. 2-AP normally pairs with thymine in DNA, but can occasionally pair with cytosine as well. What mutational spectrum would you expect as a result of treating cells with 2-AP? 7. You have isogenic strains of E. coli that are wild-type and mutant for photoreactivation. You infect each strain with phage T4 before subjecting them to uv exposure. Do you expect to see any differences in the burst size of phage on the two strains? Why or why not? 8. E. coli cells mutant in the gene for a protein involved in activating the SOS response (RecA ? ) are not efficiently mutagenized by a variety of mutagenic treatments such as irradiation with ultraviolet light that are highly mutagenic to wild-type (i.e., RecA + ) cells. Explain. 9. Predict how the following E. coli mutants compare to wild-type cells in their frequency of spontaneous mutation, frequency of mutation after treatment with ultraviolet (UV) light, and survival after treatment with UV. Remember that no repair system is 100% effective, and the cell often has multiple repair systems to repair the same type of DNA damage. For each mutant below, write ?same,? ?increased? or ?decreased? in the appropriate space. Answers for some of the mutants are shown. (Disregard DNA polymerization errors occurring during repair processes such as excision repair.) Repair system mutation Spontaneous mutation rate UV-inducted mutation rate Survival after UV treatment Photoreactivation ? same increaseddecreased Uracil N-glycosylase ? increasedsame same DNA pol III proofreading ? Mismatch repair ? Overactive dam methylase Excision repair ? same Error-prone repair ? RecA ? decreased (slightly) 10. You are investigating the behavior of E. coli mutants that are constitutive for expression of the SOS response (SOS c ). a) Name two mutations that will result in an SOS c phenotype. Based on their presumed target sizes, which of these mutations do you expect to be the more common of the two? b) Analysis if the mutational "fingerprint" of these SOS c mutants reveals that the most common mutations they create are G:C ? T:A and A:T ? T:A transversions. Remember that these mutations result as a consequence of SOS induction in the complete absence of any damage beyond normal, spontaneous mutation. Why are these particular transversions the most common mutations resulting from unwarranted induction of the SOS response? (HINT: Think about the most common forms of spontaneous chemical damage to DNA and how those might be acted on by error-prone repair.) 11. You isolate an E. coli strain with a Tn10 insertion near to a particular gene K, as shown below. This is the only Tn10 in the cell. The frequency of K + ? K ? mutations is increased in such cells compared to cells with a Tn10 insertion far from the K gene. Diagram the final structures resulting from two different events that would contribute to the increase in mutation frequency. (HINT: Remember that Tn10 transposes by a conservative mechanism.) 12. You have a plasmid containing a conservative transposon that carries ampicillin resistance (Ap r ). The plasmid itself has a temperature-sensitive phenotype such that it can't replicate at 42°C. You introduce this plasmid into a strain of wild-type (Ap s ) E. coli and grow them overnight in LB broth. You spread ~10 7 of these cells onto LB agar containing ampicillin and incubate the plate at 42°C. After 24 hours, approximately 50 colonies are observed. Give three mechanisms that could explain the appearance of these colonies. Tet a b K c d e Tn10 gene K ANSWERS 1. d. The proteins bind to single-stranded DNA and stabilize it and prevent it from re-annealing into a double-stranded duplex, thus maintaining the template for replication. 2. The dcm site is not in the correct reading frame within the gene. Spontaneous deamination of the C met (5'-CC met AGG-3') still results in the sequence 5'-NCTAGGN-3', but the reading frame is such that no amber ("TAG") codon is formed; the mutant base (T) is instead in the second or third position within the reading frame, yielding codons of "CTA" or "NCT", which are not detected by the amber suppression method. 3. For any given base, there are two possible transversions, but only one possible transition. The expected ratio is thus 2:1 (transversions:transitions). (In reality, not all base changes occur with equal frequency, and transversions are relatively rare. 4. Nonsense mutations are suppressed by secondary mutations in different genes (typically tRNA genes). This is intergenic suppression. Nonsense mutations can't be suppressed intragenically except by altering the nonsense codon itself. 5. Gain of function (pro ? ? pro + ) is more easily measured, as mutants can be selected for growth on minimal medium, thus excluding all parental types. Loss of function (pro + ? pro ? ) requires you to screen replica plates containing hundreds of individual colonies in order to identify those relative few that grow with proline added but not on minimal medium alone. 6. 2-AP can be incorporated during DNA replication in place of either an A (2-AP:T pairs) or a G (2-AP:C pairs). Subsequent rounds of replication using the 2-AP-containing strand as a template can thus lead to A:T ? GC transitions (from the 2-AP:T pair) or G:C ? A:T transitions (from the 2-AP:C pair). 5' 3' 7. The photoreactivation mutant should show decreased replication of the phage relative to the wild-type. Mutations induced in the phage genome by uv treatment will not be repaired as efficiently, leading to problems with phage replication. (Possible activation of the SOS response in this host will lead to even more phage mutations, some of which would limit burst size as well.) 8. RecA ? mutants are incapable of inducing the SOS response, and thus will not activate the error-prone repair pathway of DNA synthesis following radiation. The recA ? cells can't replicate over UV-induced lesions in their DNA, and most cells will die as a result. UV treatment simply kills the cells, so (surviving) mutant cells are not recovered and the apparent mutation frequency is lower. 9. Repair system mutation Spontaneous mutation rate UV-inducted mutation rate Survival after UV treatment Photoreactivation ? same increaseddecreased Uracil N-glycosylase ? increasedsame same DNA pol III proofreading ? increasedsame same Mismatch repair ? increasedsame same Overactive dam methylase increasedsame same Excision repair ? same increaseddecreased Error-prone repair ? same decreaseddecreased RecA ? decreased (slightly)decreaseddecreased 10a) LexA knockouts (LexA ? ) and RecA-constitutive activators (RecA*). Of these two, the LexA ? mutation would be the most common as knockout mutations are relatively easy to obtain, while "gain-of function" mutations like RecA* have very small target size and are relatively rare. b) The most common lesions acted on by a constitutive error-prone repair system would be spontaneous depurination sites where G and A bases have been cleaved, leaving a gap in the template. Assuming a preferential insertion of A's opposite gaps or other lesions in the template, error-prone repair would cause pyrimidine (C and T) bases to be replaced with A, resulting in the observed mutational fingerprint. 11. Intramolecular transposition resulting in the deletion of part or all of gene K. (The Tn10 insertion can be anywhere within or to the right of gene K; the diagram shows insertion between d and e.) Intramolecular transposition into gene k resulting in an inversion. 12. i) Transposition of the transposon (and its Ap r marker) into the chromosome. ii) Integration of the entire plasmid into the chromosome by homologous recombination. iii) Reversion (or suppression) of the ts mutation of the plasmid such that it replicates at 42°C. b a 'Kc d eK' Tet + (lost) e Tet + (lost) a b K c d + Kendall Gray Microsoft Word - problem set 2.doc
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