GENETIC REGULATION OF YEAST CELL TYPE

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This proposal examines the molecular basis of differential gene expression in the unicellular eucaryotic organism, the yeast Saccharomyces cerevisiae. Yeast cells exhibit one of three specialized cell types -- a, alpha, and a alpha -- each of which produces a unique set of proteins that create the distinctive cell types. Expression of the structural genes that encode these cell- type-specific proteins is regulated by alleles of a single genetic locus, the mating-type locus (MATa and MATalpha). Our goal is to understand the mechanism by which the regulatory protein alphal, encoded by MATalpha, activates expression of alpha- specific genes. We have focused on a particular alpha-specific gene, STE3, whose transcription is subject to two inputs in addition to the requirement for the activator alphal. In particular, the products of five others genes (STE4, STE5, STE7, STE11, and STE12) are required for efficient transcription and part of the response to the a-factor peptide pheromone secreted by a cells is increased transcription of STE3. We have delimited a segment of STE3 (called its UAS) that is necessary and sufficient to impart alpha-specific expression. We have shown that alphal protein, in conjunction with an as yet uncharacterized protein, binds to the STE3 UAS element. We will use gel mobility shift assays and DNAase footprinting assays to study the formation of this protein-DNA complex. Specifically we will determine which nucleotides within the UAS are essential, and whether the STE4/12 genes or response to a-factor affect the formation of the complex. We will determine whether alphal makes specific contacts with the UAS DNA and also identify the other protein(s) that is part of the complex. Our views is that this other protein is a general transcription factor required for expression of many genes, and that it can interact with the STE3 UAS only with the help of alphal. A second goal of this proposal is to understand the mechanism by which a-factor pheromone alters the physiology of alpha cells, for instance to cause arrest of the cell division cycle. We have shown that STE3 encodes the cell surface receptor for a-factor. We will use in vitro mutagenesis of the STE3 gene in an effort to identify the part of the STE3 protein that is involved in production of the intracellular second-messenger. Isolation of phenotype suppressors of STE3 mutations will identify genes that play a role in the normal response to a-factor.