Hasson, M.S., D. Bllinder, J. Thorner, & D.D. Jenness (1994) Mutational activation of the STE5 gene product bypasses the requirement for G protein (3 and 7 subunits in the yeast pheromone response pathway. Mol. Cell. Biol. 14: 1054-1065.
This article describes the isolation of constitutive STE5 mutations. Such mutations generate constitutive signaling via the mating-type response pathway and cause cell cycle arrest in haploids. Therefore, investigators isolating such mutations must do so in diploid cells. In previous articles, the MATalMATa 'diploid' genotype was reversibly maintained by the introduction of a plasmid-borne copy of the opposite mating-type locus. In this article this is accomplished by a different mechanism.
Chromosome III carries the expressed copy of the MA T locus and two additional but nonexpressed copies of MA T. These so-called silent copies of MA T are located near the telomeres of chromosome III and are referred to as HMLa (left telomere, copy of MATa) and HMRa (right telomere, copy of MATa). HMLa and HMRa are not expressed in wild-type cells because of the repressing effects of the SIR1, 2, 3, and 4 genes. The products of these genes silence the expression of HMLa and HMRa via chromosomal position effects (reviewed in Laurenson & Rine, 1992).
A mutation in any one of the SIR genes relieves the repression at the silent HMLa and HMRa loci. Both loci are expressed and the cell is functionally an a/a diploid. This article uses a temperature-sensitive sir3 mutation to isolate constitutive STE5 mutations. At the permissive temperature Sir3p is functional, the strain expresses only the information at the MAT locus, and the cell is genetically and phenotypically haploid. At the nonpermissive temperature Sir3p is inactive, MAT, HMLa, and HMRa are all expressed, and the cell is phenotypically diploid yet genetically haploid.
1. Describe the strategy outlined here for the isolation of haploid-lethal STE5 mutations. Why are such mutations expected to be dominant to STE51 What is the growth phenotype of sir3ts cells carrying a plasmid-borne STE5Hpt mutation at the permissive and nonpermissive temperatures?
2. STE5 is a large gene. Moreover, the mutagenesis method introduced multiple mutations. Describe how the phenotypically significant alteration in STE5Hpl-l was localized.
3. Describe the growth phenotype of STE5Hpl-2. Is this a generalized effect or specific to a particular phase of the cell cycle? Is this consistent with the expected phenotype of a constitutive STE5 mutation?
4. Describe the method used to assay mating efficiency.
5. List the results in Table 2 that indicate that STE5Hpl-2 suppresses ste2::LEU2. Discuss whether multiple copies of STE5Hpl-2 are needed. What form of suppression is this (by-pass, allele specific, suppression by epistasis) and why?
6. Three alleles of ste4 are tested in Table 2. Compare the mutational alteration in ste4-3, ste4A::LEU2, and ste4::LEU2. Compare the ability of STE5Hpl-2 to suppress each of these alleles. What form of suppression is this (by-pass, allele-specific, suppression by epistasis)? What does this result suggest with regard to the functional relationship between Ste4p and Ste5p?
7. List the results in Table 2 that indicate that STE5Hpl-2 does not suppress ste7::LEU2, stel!A::hisG, or the double fus3-6::LEU2 ksslA::HIS3. What two interpretations of this result are presented?
8. Based on the results in Tables 3 and 4 discuss the following statement. 'Together, the data indicate that the product of the STE5Hp!-l gene can activate the pheromone pathway in the absence of the pheromone receptor and the G protein but that for full activity it requires G/?7' or overexpression.
Laurenson, P. & J. Rine (1992) Silencers, silencing, and heritable transcriptional states. Microbiol. Rev. 56: 543-560.
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