We are attempting to elucidate basic molecular control mechanisms underlying regulation of eucaryotic gene expression. Our approach is a coordinate one utilizing both biochemical and genetic techniques to study the phosphorus metabolizing system of Saccharomyces cerevisiae. The phosphatases are a major constituent of degradation and salvage pathways in yeast, and a major determinant of vacuolar and cellular homeostatis. They provide an opportunity to investigate a system of regulatory gene control which may have broad application to general housekeeping systems in other eucaryotes. They may also help to elucidate mechanisms by which cells arrest in G1 upon starvation. A distinctive property of this system is the action of two classes of regulatory genes. A group of one positive (PHO4) and two negative (PHO80 and PHO85) genes which function in a concerted fashion, mediated by the PHO81 gene, in the regulation of a dispersed cellular family of phosphatases. A second class of positive regulatory genes are specific for individual members of this family. We are interested in understanding funcional aspects of regulatory gene action within this system. We are proposing to determine by direct molecular procedures, the nature of the positive-negative interplay between the PHO4, PHO80 and PHO85 proteins. Specifically, we aim to isolate the PHO4 and PHO80 genes and characterize them by recombinant procedures and DNA sequence analysis. We will determine the constitutivity of their expression and the phenotypes of over-producing and null mutants generated by in vitro modification, using gene disruption procedures. We will prepare antibody probes from synthetic peptides and hybrid proteins to identify the in vivo proteins encoded by these genes, and will determine their cellular location, their constitutivity and their ability to bind specific sequences flanking their target structural genes.
