MECHANISM OF EUCARYOTIC GENE REGULATION

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We are attempting to elucidate basic molecular mechanisms underlying the regulation of phosphorus metabolism in Saccharomyces cerevisiae. Our approach is a coordinate one utilizing both biochemical and genetic techniques to study the transcriptional control of the acid phosphatase (APase) genes. We anticipate that results from these studies will provide new insights into a variety of interrelated aspects of relevance to gene regulation in higher eucaryotes, and will furher develop our understanding of the molecular biology of the phosphorus system, particularly with respect to mechanisms of regulatory gene actin and transcriptional regulation. The phosphorus metabolism system in yeast consists of a dispersed gene family of positive and negative regulators of numerous structural genes involved in phosphorus metabolism. In previous studies we cloned and characterized the expression of the APase genes (PHO5, PH03, PHO10, and PH011), members of this family. The PH05 and PH03 genes are tightly linked as a tandem duplication, and are reciprocally regulated: PH05 is expressed only in low-Pi grown cells, and PH03 only when PH05 is repressed. We have begun to physically isolated and characterize the regulatory genes and their products by recombinant DNA and genetics strategies. Positive regulators of PH05, PH04, and PH02, have been cloned and their products identified and subjected to investigation. The PH081, PH085, and PH080 regulatory genes are currently being isolated and analyzed by similar methods. We plan to continue these studies by addressing several specific issues. We will evaluate the structure and function of the protien domains of PH04 by site-specific mutagenesis. This will include identificatin of the cellular components responsible for transcriptional control which interact with PH04. We will also define components that interact and participate in PH05 regulation by isolation and characterization of second-site suppressors of UAS rgulatory matations. This will involve chemically synthesizing a minimal UAS sequence and then introducing base substitution and deletions into the sequence. Finally, by subjecting PH05 and PH03 to metagenic analysis, we will analyze the PH05 mediated reciprocal regulation of PH03, genetically identifying and characterizing other trans-acting PH03 regulators factors involved.