Question

1. You are characterizing a temperature-sensitive mutant called nlk1 in Saccharomyces cerevisiae. To examine whether the mutant has a defect in cell cycle progression, you perform two experiments. First, you try flow cytometry with samples of the mutant strain incubated at the permissive or non-permissive temperatures A wildtype strain incubated at each temperature is a used as control. The flow cytometry results are shown below in Panel A. Second, you examine the Nlk1 protein levels throughout the cell cycle in a synchronized cell population when the cells are incubated at a semi-permissive temperature (i.e., conditions where the mutant nlk1 protein is partially inactivated). Again, you use wildtype NIk1 as a control (wildtype NIk1 is functional at this temperature) and examine whether there are differences with the mutant nik1 protein. The protein results are shown in Panel B. Panel A A) Does the nlk1 mutant have a cell cycle defect? If so, at which cell cycle transition is it defective? Provide a short explanation for your answer. (3 points) Wildtype 1N 2N IN 2N nlk1 Permissive Temp Non-permissive Temp. Panel B Wiltype Nik1 mutant nlk1 B) How does the regulation of the mutant nik1 protein levels differ throughout the cell cycle compared to the wildtype Nik1 protein? What does this suggest about how the wildtype NIk1 protein is regulated? What do you predict the mutation in the defective nlk1 protein does? (3 points) C) Based on your answer in B, suggest an experiment that would test your hypothesis about the regulation of the Nik1 protein. (3 points)

Answer 1

1. You are characterizing a temperature-sensitive mutant called nlk1 in Saccharomyces cerevisiae. To examine whether the mutant has a defect in cell cycle progression, you perform two experiments. First, you try flow cytometry with samples of the mutant strain incubated at the permissive or non-permissive temperatures A wildtype strain incubated at each temperature is a used as control. The flow cytometry results are shown below in Panel A. Second, you examine the Nik1 protein levels throughout the cell cycle in a synchronized cell population when the cells are incubated at a semi-permissive temperature (i.e., conditions where the mutant nik1 protein is partially inactivated). Again, you use wildtype Nik1 as a control (wildtype Nik1 is functional at this temperature) and examine whether there are differences with the mutant nlk1 protein. The protein results G1 phase S phase Mphase Wildtype Less M phase are shown in Panel B. Panel A A) Does the nik1 mutant have a cel cycle defect? If so, at which cell cycle transition is it defective? Provide a short explanation for your answer. (3 points) nikt Permissive Temp Non-permissive Temp. nikt Permissive Temp Non-permissive Temp es, In the mutant at non permissive temperature there is a G1/S Panel B arrest. S。the cells ar more in G1 phase in nikl mutant and there is a Wilitype Nilkt mutant nikt large decrease of Cells in Mphase So transition from S to M is defective in this mutant B) In Wild type there is no NLK1 protein during G1 to S transition. But in NLK1 protein is present during G1 to S transition . This means wild type nlk1 is negatively regulated during G1/s transition while mutant NLK1 protein is not. B) How does the regulation of the mutant nik1 protein levels differ throughout the cell cycle compared to the wildtype NIk1 protein? What does this suggest about how the wildtype NIk1 protein is regulated? What do you predict the mutation in the defective nik1 protein does? (3 points) C) Rescue the nlk1 mutant using plasmid containing wildtype nlk1 gene and see whether the resuced mutant gives same result as wild type for these experiments tha alr rn your at an experimen tnat would test your hypothesis about the regulation of the Nik1 protein. (3 points)