Article 10

Leberer, E., D. Dignard, D. Harcus, L. Hougan, M. Whiteway, & D.Y. Thomas (1993) Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 and Farl. Mol. Gen. Genet. 241: 241 -254.

The results described in Article 9 do not clearly indicate the position of STE20 in the mating type pheromone response pathway. The authors of this article hope to gain insight into this question by isolating multicopy suppressors of an ste20 null mutation.

1. Describe the selection/screen designed by the authors for the isolation of multicopy suppressors of ste20.

(a) Describe the genotype of the host strain in detail, specifically the ste20 mutant allele used for the search.

(b) Describe the library. YEp24 carries URA3.

(c) What phenotype will be used to identify clones carrying a multicopy suppressor?

(d) Outline the steps in the selection/screen starting with the selection of transformants.

2. List all the phenotypes of ste20-l that are suppressed by plasmid p24-l.

3. Describe how the authors demonstrated that the multicopy suppressor gene in plasmid p24-l was STE5.

4. Ramer and Davis (1993) (Article 9) reported that overexpression of STE11N did not suppress a ste20 null mutation. Leberer et al. report here that a hyperactive STE11 allele suppresses the mating defect of ste20 mutations. Both articles find that overexpression of STE12 suppresses ste20-1. What type of suppression is this (by-pass, allele specific, or suppression by epistasis)? Where do these results place STE20 in relation to STE11 in the mating-type pheromone response pathway?

5. Compare the structure of the mutant alleles ste20-l and ste20-2. Use diagrams for your answer.

6. What experimental results suggest that ste20-l produces a partially functional product? What is the presumed product of ste20-l (based on your knowledge from Article 9)? Does expression depend on the STE20 promoter? Explain.

7. Summarize the results that indicate the following.

(a) Activation of the mating-type pheromone response pathway by STE5 overproduction is dependent on the partially functional ste20-l allele.

(b) Activation of the mating-type pheromone response pathway by STE5 overproduction is dependent on STE4 and STE18.

8. What type of suppression is the suppression of ste20-l by multicopy STE5 (bypass, allele specific, or suppression by epistasis)?

9. Choose one of the following two models of the relationship of the STE4, STE5, and STE20 genes as best explaining this portion of the mating-type pheromone response pathway. Support your choice using the results presented in this article.

Model 1

Model 2

STE4

STE4

STE20

STE20

STE5

STE5

10. Describe the structural and functional similarities of Ste5p and Farlp. How was the functional similarity demonstrated?

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