Article 12

Akada, R., L. Kallal, D.I. Johnson, & J. Kurjan (1996) Genetic relationships between the G protein /?7 complex, Ste5p, Ste20p and Cdc42p: investigation of effector roles in the yeast pheromone response pathway. Genetics 143: 103-117.

1. Summarize the selection scheme designed to identify mutations that enhance the phenotype of ste4-ts mutant. Be sure to include the following.

(a) The genotype of the parent strain(s).

(b) Which alleles of ste4 were used? Why did the authors use temperature sensitive alleles and not a ste4A mutation?

(c) The growth conditions of the first step in the selection process (carbon source, temperature, etc.).

(d) Potential mutants identified in the selection were screened for their ability to mate at the permissive temperature on galactose plates (step 2). What classes of unwanted mutants would be eliminated by this screen? Explain.

(e) Mutants that passed the screen in 'c' were tested further for their ability to mate at the nonpermissive temperature on galactose plates (step 3). What is the purpose of this screen? Explain.

2. Define the term 'synthetic sterile'.

3. Diagram a cross that would allow the isolation of segregants carrying only the ste-x mutation from a ste4-ts ste-x double mutant isolated by this selection process.

4. Describe the cloning strategy used to isolate ste-x complementing plasmids.

5. Which of the secondary screens described above (step 2 or step 3) should have weeded out the SIR mutations? Why did it fail to do so?

6. The stel8 and ste21 mutations were isolated starting with the parental strain carrying ste4-3510. The ste5 and ste20 mutations were isolated starting with the parental strain carrying ste4-299. Nonetheless, the authors state that 'the synthetic sterile effects were not allele specific'.

(a) Diagram a cross that would test whether the stel8-14 mutation (isolated in the ste4-3510 strain) is allele specific.

(b) There is no specific information given as to the position of the alterations in ste4-3510 or ste4-299 nor are we informed as to which other ste4 alleles were tested for the authors to conclude that none of the synthetic sterile mutations were allele specific. Discuss why this information would have been valuable.

(c) The original intent of the search for mutations that are synthetic sterile with ste4-ts mutations was to identify proteins that interact directly with Ste4p. Do the results reported here allow the authors to conclude that Ste4p physically interacts with Ste5p? Explain.

7. Discuss a model of enhancement, other than 'allele-specific enhancement', that might explain the results obtained here. That is, mutations in STE18, STE5, and STE20 enhance the phenotype of a ste4-ts mutation. Keep in mind that epistasis analysis of these genes indicates that they could act at the same step.

8. In a recent study Blondel et al. (1999) identify STE21 as MSN5, a member of the nuclear exportin family. They report that Msn5p (Ste21p) is responsible for the pheromone-stimulated export of Farlp from the nucleus. Discuss how a mutation in MSN5 (STE21) could enhance a ste4-ts mutation. Which of the models of enhancement described in Chapter 9 does this represent?

9. Evaluate the results presented regarding the genetic interaction of STE20 and CDC42, particularly in Table 6 and Figures 6 and 7. Of the models presented in Figure 8, which do you think is most consistent with the results presented in this and the other articles of this case study? Explain.

REFERENCE

Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, & M. Peter (1999) Nuclear export of Farlp in response to pheromones requires the export receptor Msn5p/Ste21p. Genes Dev. 13: 2284-2300.

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