You are characterizing a temperature-sensitive mutant called nlk1 in Saccharomyc

Question

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 Nlk1 as a control (wildtype Nlk1 is functional at this temperature) and examine whether there are differences with the mutant nlk1 protein. The protein results are shown in Panel B.

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.

B) How does the regulation of the mutant nlk1 protein levels differ throughout the cell cycle compared to the wildtype Nlk1 protein? What does this suggest about how the wildtype Nlk1 protein is regulated? What do you predict the mutation in the defective nlk1 protein does?

C) Based on your answer in B, suggest an experiment that would test your hypothesis about the regulation of the Nlk1 protein.

Explanation / Answer

A) The histogram clearly shows a rise in the peak of negetive controls for nIk1 temperature sensitive mutant cells, when incubated at non-permissive temperatures. This indicates that at the non-permissive temperature, the number of cells which did not show a specific parameter under analysis, have tremendously increased.

The same cells, at a permissive temperature, have shown identical results to that of wild type strains. Therefore, the cells are temperature sensitive, and at non-permissive temperature, they have shown variation.

The defect lies in the G1 - S transition or G1-S boundary.

B) The mutant protein levels are similar in both the wild type and mutant strains throughout the G1, S, M, and S-M transitions states, but only at the G1-S transition stage, there is an expression of the said protein in mutant cells, at this transition.

In the gel given in panel B, there is an absence of any protein band in the G1-S transition stage, in wild type cells.

However, in the mutant cells, an extra band of proteins can be seen in the G1-S transition stage, which indicates the nIk1 protein is periodic and the protein level is peaked at this transition stage.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.