YEAST PHEROMONE GENES STRUCTURE, FUNCTION, AND CONTROL

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Alpha-factor is an oligopeptide pheromone of thirteen amino acids which is produced by one of the haploid cell types of yeast (MAT Alpha cells). Cells of the opposite mating type (MAT Alpha) respond to Alpha-factor by arresting in the G1 phase of the cell cycle, the point at which cells are able to mate. Analysis of a cloned Alpha-factor structural gene (MAF Alpha) indicates that this gene encodes a pre-pro-poly-Alpha-factor precursor with four tandem repeats of Alpha-factor, which requires several processing steps for the production of mature Alpha-factor. Hybridization studies utilizing the MAF Alpha DNA indicate that there may be additional Alpha-factor structural genes (MAF Alpha') in the yeast genome. The aims of this proposal are to answer the following questions: 1) Is Alpha-factor required for mating? 2) Does the Alpha-factor precursor itself play a role in mating? 3) How many active Alpha-factor structural genes are present in the yeast genome? 4) If more than one is present, are they all required for mating? 5) What steps are involved in processing and secretion of Alpha-factor and which regions of the precursor are required for these steps? 6) How is the cell-type-specificity of Alpha-factor production determined? The techniques to be used include the cloning and DNA sequencing of the MAF Alpha' genes in order to determine their structure, in vivo tests of their activity, in vitro mutagenesis followed by gene replacement in order to assess phenotypes (i.e. to determine the roles of Alpha-factor and its precursor in mating and the role of the different parts of the precursor in the processing and secretion pathway), and the construction of lacZ fusions to the 5' flanking regions of these genes, followed by deletion analysis, to determine the regions of the gene involved in regulation and expression. Also, an attempt will be made to clone the Alpha-factor structural gene. Alpha-factor is the analogous pheromone produced by MAT Alpha cells. It is an eleven amino acids oligopeptide, which exists in two forms which differ by a single amino acid substitution. My results indicating that the MAF Alpha gene encodes a poly-Alpha-factor precursor suggest that the two forms of Alpha-factor may be encoded in a single gene which codes for a poly-Alpha-factor precursor. The Alpha-factor structural gene will be sequenced to determine whether this is the case and questions similar to those posed for Alpha-factor will be examined.