Article 9

Ramer, S.W. & R.W. Davis (1993) A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 90: 452-456.

FUS1 encodes a product required for the fusion of haploid cells during mating. FUS1 expression is induced by exposure to mating type pheromone and induction requires signal transduction via the pheromone response pathway defined by the Ste2, Ste4, Stel8, Ste5, Ste7, Stell, Fus3/Kssl, and Stel2 proteins. Stel2p, a DNA-binding transcription activator, turns on FUS1 transcription by binding to sites in the promoter. In Article 9 the authors use yet again another approach for the isolation of genes involved in mating-type signal transduction and successfully identify a new STE gene.

1. In this article the authors searched for genes that, when overexpressed, cause the constitutive expression of one of the downstream targets of the mating type pheromone pathway FUS1.

(a) Diagram the library vector showing the structure of the insertion site of the yeast DNA fragments. At best, only one in six inserts will produce a product normally expressed in yeast. Explain why one in six is the maximum number.

(b) Diagram the FUS1 reporter construct.

(c) Outline the steps used to identify 'positive' clones. Start with the selection of transformants (assume the selection marker on the vector is LEU2). Be sure to specify the carbon source at each step.

2. 'Preliminary sequence data suggested that this clone might not contain a full-length gene.' Based on the information in the text, diagram the fusion gene found in the novel clone. Indicate the translation start site of the fusion gene. Based on the sequence of STE20 in Figure 3, what residues are present in the protein product of ste20NH.

3. Is overexpressed STE20N dominant or recessive to STE201 What does this suggest with regard to the role of the N-terminal region of Ste20p? Which residues contain the putative kinase domain of Ste20p?

4. Describe the construction of ste20-l and list the complete phenotype.

5. Is Ste20p a positive or negative regulator of the mating-type response pathway? Explain.

6. Describe the expression pattern of STE20.

7. Epistasis analysis was undertaken to place STE20 in the mating type response pathway in relation to the other STE genes.

(a) When mating efficiency is measured, which is epistatic, STE20N or ste4, ste5, stell, or stel21 Where does this place STE20 in the pathway:

(b) When growth arrest is measured, which is epistatic, STE20AN or ste4, ste5, stell, or stel21 Where does this place STE20 in the pathway: STE4-► STE5-- STE 11-► STE7-* ST El 2

(c) Which is epistatic: ste20-Al or overexpression of ST FA'} What phenotype is monitored in this experiment?

(d) Which is epistatic: ste20-Al or overexpression of STE11AN1 What phenotype is monitored in this experiment?

(e) Which is epistatic: ste20-Al or overexpression of STE121 What phenotype is monitored in this experiment?

8. Based on the conflicting results of their epistasis analysis the authors finally settle on the suggestion that STE20 functions 'prior to STE12' in the mating-type signaling pathway. Discuss the possibility that these results suggest a dual function for Ste20p in the mating response. One function is a positive one in the mating-type signaling pathway and the second function is in growth arrest.

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