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2005_pset4_ans - 7.06 Problem Set#4 Spring 2005 1 Youre...

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1 7.06 Problem Set #4, Spring 2005 1. You’re doing a mutant hunt in S. cerevisiae (budding yeast), looking for temperature- sensitive mutants that are defective in the cell cycle. You discover a mutant strain that cannot complete the cell cycle at the non-permissive temperature. You determine that the cells are blocked at the G1 to S transition. a. After treating the mutant strain with α -factor at the permissive temperature (25 ° C), you remove the α -factor and switch the cells to the non-permissive temperature (37 ° C). You then isolate different known cyclins via biochemical purification. Which cyclins should be present at high levels in these mutants at the non-permissive temperature? Alpha factor synchronizes cells in G1. When you then shift your cells to high temperature, the cells will arrest just prior to the initiation of S phase. Thus the S phase cyclins should be present at high levels. The S phase cyclins are Clb5 and Clb 6. The levels of the G1 cyclins (Cln1, 2, 3) are also still high. b. You determine that the temperature sensitive mutation is a recessive loss-of-function mutation. Outline an experiment to determine which gene is mutated in your strain. Identify the gene using “cloning by complementation.” For this technique, you transform the mutant yeast with a library (or collection) of vectors, each containing a different gene from wild-type yeast cells. You then plate the transformed mutant yeast, and select for colonies that can now grow at the non-permissive temperature. These colonies can grow because you have provided the wild-type function of the gene that is mutated in these cells from the vector. You then use PCR to amplify the gene that the vector harbors and that confers temperature resistance to these mutant yeasts. You then sequence the insert and use BLAST to analyze the sequence in order to determine which gene is harbored in the vector that complements the defect of your cells. (NOTE: please make sure that you understand why this technique will only work if your mutant phenotype is recessive.) Through your experiment in part b , you determine that the mutation is in SCF, a ubiquitin ligase. To further characterize this mutant you perform the following experiment. You treat two cultures (one of wild-type cells, the other of scf-ts mutant cells) with alpha factor at 25 ° C. You treat two cultures (one of wild-type cells, the other of scf-ts mutant cells) with hydroxyurea at 25 ° C. Finally, you incubate one culture of scf-ts mutant cells at 25 ° C and then shift them up to 37 ° C for the length of one cell cycle. You make cell extracts from these five cell populations and run the extracts on SDS-PAGE gels. You then perform Western blot analyses using different antibodies against several cell cycle proteins. When blotting for Sic1, you see the following result:
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2 WT scf-ts WT scf-ts scf-ts alpha 25C alpha 25C HU 25C HU 25C 25C 37C Sic1 Sic1 c. Explain the results seen in the above gel, including why you see a shift in the
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