2005_pset4 - 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? 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. 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: WT scf-ts WT scf-ts scf-ts alpha 25C alpha 25C HU 25C HU 25C 25C 37C Sic1 Sic1
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2 c. Explain the results seen in the above gel, including why you see a shift in the migration of Sic1, and why a loss of function temperature sensitive mutation in SCF would give these results. Also include in your answer an explanation of how SCF normally interacts with Sic1 and why a loss of SCF function would cause a cell cycle block at the G1 to S transition. d. Briefly describe an experiment to prove that your explanation for the shift in the migration of Sic1 in the above blot is correct. 2. Your colleague identifies an interesting temperature sensitive (ts) mutant “ cdcX-ts ” in S. cerevisiae . He suspects that cdcX might be required for the entry into mitosis. a.
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2005_pset4 - 7.06 Problem Set #4, Spring 2005 1. Youre...

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