The regulatory circuit that acts upon the GAL genes of the yeast Saccharomyces cerevisiae, which has been well defined genetically, is governed by both positive and negative regulatory elements. Further genetic analysis of GAL structural and regulatory genes is psroposed to gain insight into the details of the molecular machinery that regulates their expression.
The specific aims are: 1) To define genetically the regulatory sites for selected GAL genes, including (a) promoter sequences, (b) sites recognized by specific GAL regulatory proteins, and (c) sequences responsible for catabolite repression of the GAL genes. (2) To identify other genes involved in GAL regulation, including (a) genes responsible for catabolite repression of the GAL genes, and (b) genes specifically involved in galactose induction of GAL gene expression. (3) To study induction of GAL gene expression, by (a) identifying the in vivo inducer and (b) characterizing a gene (GAL3) involved in the induction process. The genetic flexibility provided by plasmids carrying GAL promoters fused to genes whose expression can be monitored and selected for will be exploited to isolate and characterize several kinds of GAL regulatory mutants. The advantages this approach provides include 1) the ability to uncouple GAL regulation from galactose metabolism thereby 2) enabling recovery of mutants altered in expression from a single GAL promoter. 3) The fusions will enable selection of mutants with subtle alterations in GAL regulation and 4) will also enable simple in vivo assessment of the relative levels of GAL expression in various mutants. 5) Finally, certain genes fused to GAL promoters will enable selections both for and against GAL expression. Knowledge gained from analysis of mutations in GAL gene regulatory sites will not only broaden understanding of mechanisms of GAL regulation, but should also be useful for the future study of GAL regulation in vitro. One long term goal is to study the process of catabolite repression in yeast; experiments designed to isolate mutants defective in that process are proposed here.
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