Article 1

MacKay, V. and T.R. Manney (1974a) Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants. Genetics 76: 255-271.

Haploid Saccharomyces cells may be either mating type a or mating type a and mating type is determined by the MA T locus which maps to chromosome III. There are two alleles of MAT: MA7a is present in cells of mating type a and MATa is present in cells of mating type a. The MAT alleles encode different DNA-binding transcription regulators that control the expression of a large battery of unlinked genes involved in cell type determination (a mating type, a mating type, a/a diploid). Haploid cells mate with cells of the opposite mating type but do not undergo meiosis or form spores. Diploid a/a cells undergo meiosis and sporulate under the appropriate conditions but do not mate with any cell type. Mating and sporulation are both very complex processes and require dozens of gene functions. This Case Study focuses on mating and, more specifically, how haploid cells sense and respond to the presence of the mating pheromone produced by cells of the opposite mating type.

Haploid cells respond to the presence of cells of the opposite mating type as follows. First, both partners in the mating pair arrest as unbudded cells in G1 of the cell cycle at Start. Start is the point at which the cell becomes committed to division and is the execution point of Cdc28 cyclin-dependent kinase action. Second, both partners form a mating projection directed toward the mating partner. Cells with this projection are pear-shaped and are quite distinct from budding cells. The shape is referred to as a schmoo. Third, gene expression of a number of mating-specific genes is induced. One of the first of these genes to be identified was a-agglutinin, a surface protein involved in adhesion to the mating partner and produced by a mating type cells. It localizes to the schmoo tip. Most strains of mating type a produce a-agglutinin constitutively but in some strains a-agglutinin expression is induced by the presence of mating type a cells.

At the time that Article 1 was published mating type a cells were known to produce a secreted peptide hormone, or pheromone, called a-factor. Similarly, in Article 1 the authors demonstrate that mating type a cells produce a diffusible factor called a-factor that was later shown to also be a peptide hormone. Thus the mating response described above is likely to be the result of events initiated by the effects of these pheromones on cells of the opposite mating type. Mutations affecting the ability of haploid cells to mate could identify genes encoding components of the pheromone sensing pathway as well as functions required for pheromone production or the process of mating itself. In Article 1, the authors develop a strategy for isolating mutants that are unable to mate, which they call 'sterile' or ste mutants.

1. Using the table below, summarize and compare the phenotypes of a mating type, a mating type, and a la diploid cells.

Phenotype a Mating-type cells a Mating-type cells a la Diploid cells

Cell cycle arrest in response to a-factor

Cell cycle arrest in response to a-factor a-Factor secretion a-Factor secretion

Schmoo formation in response to a-factor

Schmoo formation in response to a-factor a-Agglutinin synthesis

«-Agglutinin synthesis

Sporulation under starvation conditions

2. The CAN1 gene encodes arginine permease. Canavanine is an analogue of the amino acid arginine and is utilized by Saccharomyces in place of arginine for protein synthesis. When this occurs it is toxic to the cells. Based on this description, explain the fact that recessive mutations in CAN1 are resistant to the toxic effects of canavanine.

3. Explain the strategy behind the procedure used here to isolate nonmating clones.

4. Are these ste mutants spontaneous or induced? Explain.

5. Is this a selection or a screen? Explain.

6. Explain why the canl mutation must be in the strain that is mutagenized and not in the strain to which it is mated during the mutant isolation procedure.

7. Explain why it is necessary to screen the canavanine resistant potential ste mutant clones obtained for the following criteria.

(a) The nutritional requirements of the potential mutant are the same as the mutagenized parental strain.

CASE STUDY IV 209

(b) The potential mutant is unable to mate with tester strains of both mating types.

(c) The potential mutant is unable to sporulate.

8. Explain why the mutants obtained from one isolation procedure are unlikely to be independent, i.e. different mutations that arose separately.

9. List the other phenotypes for which the ste mutants were tested.

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