The proposed studies are designed to investigate the molecular mechanism(s) controlling the expression of the structural genes involved in maltose (and starch) metabolism in yeast. We are particularly interested in characterizing the cis and trans regulatory components of the MAL locus involved in induction by maltose and carbon catabolite repression by glucose. Effort is directed at the structure and function of the maltase and maltose permease promoter elements and of the trans-acting regulatory protein and its site(s) of action. 1. Complete the sequencing of the MAL6 locus, characterize the MALR repeated sequence (MALRR) and sequence the MAL1 intergenic (IG) region. In these experiments, the sequencing of the entire MAL6 locus and of a repeated sequence (MAL6RR) homologous to MAL6R will be completed. Preliminary studies on the properties of MAL6RR will be initiated. 2. Additional studies on the promoter elements in the MAL6 IG region. The UASM sequence will be studied in greater detail with respect to its boundaries, whether its orientation and distance from the TATA sequence are important and for its possible role in regulating the divergently transcribed permease gene. The role of the T-rich sequence in glucose repression will be studied further and examined to determine whether specific proteins bind to it. 3. Studies on the MAL6R encoded regulatory protein. The regulatory protein will be expressed in E. coli and synthesized in vitro in order to make antibodies to it. The regulatory protein and some of its mutationally altered derivatives will be studied for interaction with the maltase promoter region. 4. Search for maltase constitutive and glucose repression insensitive mutants. Constitutive mutants will be generated by mutagenizing the regulatory gene and the IG region. They will be checked for their repression by glucose. 5. Duplication of the MAL1 locus. Diploids will be constructed which will allow one to analyze random spores for duplications of the MAL1 locus. 6. Studies on yeast glucoamylase. Sequencing of the STA2 gene will be completed, the STA10 gene cloned, the STA genes mapped and the regulation of STA genes by MATa/MATalpha investigated.
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