We propose to study the structure and regulation of th alcohol dehydrogenase (ADH) genes in the baker's yeast, Saccharomyces cerevisiae, by genetic and biochemical techniques. The wild-type gene for both major isozymes has been isolated, as well as the genes from several mutants. More mutant genes coding for the glucose-repressed ADHII isozyme will be cloned by recombinant DNA techniques. Most of the mutant genes have cis-acting regulatory mutations that are due to insertion of the yeast transposable element, ty. The exact site of the insertion in te 7 mutants will be determined by DNA sequence analysis. The site of mutation in two putative point mutants will be determined in the same manner. Transcriptional regulation of the two homologous isozyme genes will be studied by constructing mutant ADH genes containing insertions of phage T4 DNA. These insertions will allow unique mRNA species from the two related genes to be distinguished and quantitated by hybridization. Both major isozyme genes will be modified by in vitro mutagenesis and used to identify DNA sequences necessary for initiation of transcription, regulation by the specific regulatory locus for ADR2, ADR1, and regulation by the catabolite repression system. The modified ADH genes will be studied by transformation of appropriate yeast mutant strains followed by enzyme and mRNA analysis. The regulatory gene ADR1 will be isolated by transformation of yeast and complementation of function. The cloned ADR1 gene will be used to determine whether the ADR1 locus is transcriptionally controlled by glucose and/or the carbon catabolite system, specifically by CCR1 and related genes regulating catrabolite repressed enzymes. The cloned ADR1 gene will also be used to identify and characterize the ADR1 protein. The DNAse sensitivity of the structural genes in chromatin, and the location of hypersensitive sites near the ADH structural genes will be determined in strains containing normal and mutant alleles of the regulatory loci ADR3, ADR1, and CCR1.
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