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LS50 Genetics Practice Problems April 7, 2016 Problem 1. Saturation of mutants in genetic screens In an effort to identify the loci involved in embryonic development, Nusslein-Volhard and Wieschaus (1984) screened for mutants of D. melanogaster with phenotypes in embryonic development. As more mutants were isolated, the probability of a newly- isolated mutation affecting a novel locus decreased. In this problem, you will assess the claim that continuing the screen would not result in the identification of enough additional loci to warrant further investment in time and effort. 1. Assume that there are n loci involved in embryonic development and that all loci are mutated with equal probability. What is the probability that a newly-isolated embryonic development mutant will not affect a given one of these n loci? 2. What is the probability that, after m mutants have been identified, a given locus has still not been identified by any mutant? 3. The data obtained by Nusslein-Volhard and Wieschaus are given in the table below. The x- and yvalue pairs represent the number of mutants isolated and the number of loci identified, respectively, at various points in their screen. Assuming that n = 63 (i.e., all mutants were identified by the end of the screen), plot your expectations of the number of loci identified alongside the observed values for each point. Comment on the quality of the fit. x 7 19 27 40 54 70 y 7 19 21 25 31 33 x 81 97 115 131 145 154 y 37 44 47 48 53 53 x 158 168 184 196 203 215 y 53 57 59 59 60 60 x 231 243 255 271 y 61 62 63 63 4. So far you have assumed that all loci have an equal probability of being identified by a mutation. Now assume that there are two classes of loci: one class that is highly likely to be identified, and another that is unlikely to be identified. Qualitatively sketch the plot of loci identified vs. mutants isolated assuming that the numbers of loci in each class are equal. How does this compare to the Nusslein-Volhard and Wieschaus data? 1 Problem 3. Neurospora2. Neurospora Like Saccharomyces cerevisiae, the spores produced by Neurospora crassa are contained in a sac called an ascus. Unlike in S. cerevisiae, after meiosis I and II, the ascospores go through one round Like Saccharomyces cerevisiae, the spores produced by Neurospora crassa are contained in a sac called an of mitosis to produce asci with eight spores each. The Neurospora ascus is a rigid cylinder in ascus. Unlike in S. cerevisiae, after meiosis I and II, the ascospores go through one round of mitosis to which, each division, nuclear spindles areascus positioned in acylinder line andindowhich, not overlap. The division, produceduring asci with eight sporesthe each. The Neurospora is a rigid during each result is a linear octad: the order of spores in the ascus reflects their pattern of descent from the nuclear spindles are positioned in a line and do not overlap. The result is a linear octad:athe order of diploid underwent meiosis. spores incell thethat ascus reflects their pattern of descent from a diploid cell that underwent meiosis. Neurospora ascospores are normally black, but but youyou have identified a asingle-locus recessive mutation that Neurospora ascospores are normally black, have identified single-- locus recessive makesmutation the ascospores white. When you cross black and white haploids and sporulate the diploids, you that makes the ascospores white. When you cross black and white haploids and observe several patterns of ascus spore coloration: sporulate the diploids, you observe several patterns of ascus spore coloration: a. Which of the above pattern(s) is/are produced in the absence of recombination and other genomic modifications? your answer by diagramming theofchromosome inheritance 1. Which of the above Explain pattern(s) is/are produced in the absence recombination and otherthat genomic modleads ifications? to this/these pattern(s) during spore formation. Explain your answer by diagramming the chromosome inheritance that leads to this/these pattern(s) during spore formation. b. Which of the above pattern(s) is/are produced as a result of crossover events? Explain 2. Which of the above pattern(s) is/are produced as a result of crossover events? Explain your answer your answer a diagram of chromosome inheritance during spore formation. using a using diagram of chromosome inheritance during spore formation. 3. Which pattern(s) you expect togene see ifconversion gene conversion replication c. Which pattern(s) do youdo expect to see if occursoccurs beforebefore DNA DNA replication in in meiosis I? After DNA replication? Explain your answers with diagrams as above. meiosis I? After DNA replication? Explain your answers with diagrams as above. 4. You encounter one exceptional ascus in which four spores are black and the remaining four spores are dead. Provide one possible explanation this phenotype andand describe an experiment d. You encounter one exceptional ascus in whichfor four spores are black the remaining four to test your sporeshypothesis. are dead. Provide one possible explanation for this phenotype and describe an experiment to test your hypothesis. Problem 3: Reverse Mutant Screening You are studying a non-model yeast species that has no tools for direct genome manipulation and no published genome sequence. Its life cycle is identical to that of S. cerevisiae. You are interested in understanding the role of the YFG1 gene in resistance to iron toxicity in this species. You use an experimental approach where you mutagenize haploid cells by irradiating them with UV light (which primarily induces point mutations), plate them on complete media and then carry out colony PCR using primers flanking the YFG1 locus. Sequencing of the PCR product and alignment with the wild-type sequence allows you to identify mutations in the YFG1 locus. Mutant colonies are then picked and assayed for resistance to iron toxicity (measured as the fraction of cells surviving after exposure) and levels of YFG mRNA. (There are several techniques used to measure the amount of mRNA for a specific transcript in cells). You screen a very large number of colonies (you have robots) and obtain 10 colonies with mutations in the YFG gene. The table below shows a summary of your results. Note that the mutation column shows changes in base pairs, not amino acids; and RNA expression is relative to wild type cells. 2 Colony # wt 1 2 3 4 5 6. 7. 8. 9. 10. Mutation none A15T C115T G52A C200T G15T G120A C700T T300C G52A C545T Survival 60% 0% 60% 60% 0% 30% 0% 70% 60% 0% 80% Expression 1.0 0 1.0 1.0 1.0 0.5 1.0 2.0 1.0 1.0 1.0 As completely as you can, classify the mutations into different classes based on their phenotype and what you can deduce about what causes the phenotype. For example, start by dividing them into loss of function and gain of function subsets. Clearly indicate ambiguity or inconsistency. Problem 4: Transmission genetics You are an orchid geneticist interested in two recessive traits each controlled by a single allele in inbred lineages of orchids in your greenhouse. The traits are purple flowers in one lineage and pale leaves in the other. Starting only with wild-type plants, a single plant with purple flowers and a single plant with pale leaves, describe step by step an experiment you would carry out to determine whether the two alleles are linked. Assume that the plants are isogenic at all other regions and that you can ignore life-?cycle details of orchids and treat them as a generic, sexually reproducing diploid organism. Problem 5: Sporulation pathway You hunt for recessive temperature sensitive mutants that allow cells to sporulate in rich medium (srg = sporulation regulatory genes) and isolate 15 mutants that define 3 complementation groups srg1- srg3. A colleague has been isolating mutants that fail to sporulate under any conditions (spo = sporulation). You jointly decide to examine the interaction between the mutants by making double mutants and seeing whether they can sporulate on rich and on standard medium. You get the following results: Double srg1 srg1 srg1 srg2 srg2 srg2 srg3 srg3 srg3 Mutant spo1 spo2 spo3 spo1 spo2 spo3 spo1 spo2 spo3 Sporulation on rich medium + + + - Sporulation on sporulation medium + + + - Can you produce a genetic pathway that shows how these six genes are involved in regulating sporulation? 3