Article 6

Blinder, D., S. Bouvier, & D.D. Jenness (1989) Constitutive mutants in the yeast pheromone response: ordered function of the gene products. Cell 56: 479-486.

1. The results reported in Dietzl & Kurjan (1987) and Whiteway et al. (1989) (Article 5) indicate that the STE genes encode components of a switch regulatory pathway as defined in Chapter 6. Discuss the evidence that supports this conclusion.

2. Explain why the anticipated phenotype of constitutive mutations in the mating-type signaling pathway is haploid lethal.

3. Based on your knowledge of the roles of Ste2p, Gpalp (Scglp), and Ste4p, would you expect dominant or recessive constitutive mutations in STE2, GPA1, and STE41 Explain.

4. Describe the red/white colony-sectoring assay. How is it used here to identify clones carrying potential haploid lethal mutations? Include in your answer an explanation of why candidate haploid lethal mutants would have a non-sectoring phenotype.

5. Describe the procedures undertaken in the analysis of the 87 nonsectoring mutant clones that reduced this number to the final five mutants studied in this article. What phenotypes distinguish haploid lethal mutants affecting the mating-type signaling pathway from other haploid lethal mutants affecting other pathways?

6. For genetic analysis of the haploid lethal mutants, it is essential that cells that have lost the pDJ117 plasmid are capable of mating even though they are incapable of division. Discuss.

7. Describe the experimental procedure used to test dominance of the haploid lethal mutations.

8. Describe the two methods used to demonstrate that the four recessive haploid lethal mutations are alleles of SGC1 (GPA1).

9. Diagram the cross HPL-6 x HIS3. Give the genotype and phenotype (viability and ability to grow in the absence of histidine) of the parents, diploid, and PD, NPD, and TT tetrad spores. (Assume that the mating is between cells of strain 5680-4 (his3) and cells of mutant DB6 that have lost the pD117 plasmid.)

10. Table 3 presents the segregation pattern of the mutant phenotype.

(a) What is the 'mutant phenotype'?

(b) In the column labeled '3:1', what is the phenotype of the '3' spores?

11. In Table 3, the first two rows demonstrate that scgl-4 is epistatic to ste2-3's.

(a) What data allow the authors to make this conclusion?

(b) Of 10 tetrads from the W.T. x ste2-3's cross, two give a 3:1 segregation pattern. What does this indicate and how does this help you in your evaluation of the results of the scgl-4 x ste2-3's cross?

(c) Would you have expected 'cold-sensitive segregants' given the segregation pattern observed? Explain.

12. The results of the cross of STE4hp' x ste5-3's in Table 3 indicate that ste5-3's is epistatic to STE4Hpl.

(a) Specify these results.

(b) What is the phenotype (inviability, morphology, mating efficiency) of the STE4Hpl ste5-3's double mutant segregants at 34°C and at 22°C?

(c) Give the genotype and phenotype of the spores of a 3:1 tetrad.

(d) What is the genotype of the 11 cold-sensitive segregants and is this the expected number given the number of tetrads analyzed? Explain.

13. The authors suggest one possible mechanism for the constitutive activity of the Ste4Hpl mutant protein. Discuss. Given the same model of the pheromone response pathway proposed in Figure 2, suggest an alternate mechanism for this Ste4p constitutive mutant. (Assume that some free #7 is always present.)

REFRENCE

Dietzel, C. & J. Kurjan (1987) The yeast SCG1 gene: a G a-like protein implicated in the a-and »-factor response pathway. Cell 50: 1001-1010.

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