UBC Theses and Dissertations
Molecular genetic analysis of the saccharomyces cerevisiae Mat Locus Porter, Susan Dorothy
The MAT∝ locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins responsible for determining the ∝cell type. The MAT∝1 gene encodes ∝1, a positive regulator of ∝cell-specific genes, whereas the MAT∝2 gene encodes a negative regulator of a cell-specific genes (∝2). MAT∝2. (in conjunction with the MATα1 gene) also determines the α/∝ diploid cell type by repressing haploid-specific genes. ∝2 exerts its effect at the transcriptional level in the ∝ cell by binding to a sequence located upstream of α cell-specific genes. The present study undertook to examine, through in vitro genetic manipulation, the structure/function relationship of the MAT∝ regulatory proteins, particularly∝2, in their role as gene regulators. The construction of mutant MAT∝2 genes containing termination codons at various points within the gene, and subsequent transformation of the mutant genes into mat∝2 yeast, indicated that the carboxy-terminal one-third of the gene product was necessary for full repressor activity in the haploid as well as in the diploid. A segment within the carboxy-terminal one-third of ∝2 displays some homology to the higher eukaryote homeo domain as well as to a prokaryotic bihelical DNA-binding structural motif. This region of the gene was subjected to semi-random missense mutagenesis in vitro and the mutant genes were analyzed by transformation into strains containing chimaeric genes that encode β-galactosidase from ∝2 and a1/∝2. repressible promoters. In this manner it was demonstrated that most of those residues in ∝2. which correspond to conserved amino acids in the prokaryotic DNA-binding structure and in the homeo domain are essential for the two repressor activities of ∝2. Several mutations more severely affected the ability of ∝2 to repress α-specific genes than haploid-specific genes. Analysis of the temperature dependence of the activities of some of the mutants was consistent with the existence of a helix-turn-helix structure at this region of the protein. Finally, further analysis of some of these mutants in vitro confirmed that the observed defect correlated with a loss of DNA-binding activity.
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