The long range goal is to contribute to an understanding of how regulation of nuclear gene activity occurs in a typical eukaryotic cell. The system under study is the galactose/melibiose regulon of S. cerevisiae. In this system transcriptional expression of five enzyme coding genes is induced in the presence of galactose and repressed in the absence of galactose or the presence of glucose. The overall question we are addressing is: how in response to small molecule signals do two positive regulatory proteins (GAL4 and GAL3) and one negative regulatory protein (GAL80) regulate transcription of the five individually transcribed target genes (GAL1, GAL2, GAL7, GAL10, and MEL1)? Three interrelated lines of research are proposed. One major line is to identify, purify, and obtain antibodies to the GAL4, GAL80, and GAL3 proteins. The purified proteins and the antibodies will be used to determine any physical associations and activities of these regulatory proteins. Major interests here are to: 1) determine if the GAL4 protein binds specifically to target gene DNA sites, 2) determine if GAL80 protein binds directly to the GAL4 protein or/and to target gene DNA sites, 3) determine if the GAL3 protein binds galactose and converts galactose to an inducer molecule which binds to the GAL80 protein. A second major line is to determine the identity of proteins and small molecule signals involved in modulating transcription of GAL3. A major question here is whether GAL3 gene transcription itself is under GAL4 and GAL80 control. If it is, the GAL3 gene control regions responsive to regulation by GAL4 and GAL80 proteins will be compared, by DNA sequencing and by GAL4 and GAL80 protein binding assays, to MEL1 and GAL1 control regions. If the GAL3 gene transcription is not under GAL4 and/or GAL80 control, genes coding for transacting regulators of GAL3 will be identified and cloned as a prelude to the isolation of the active gene products. A third major line is to isolate by in vitro mutagenesis cis-acting target gene mutations which allow up-expression of the MEL1 gene in the absence of normal GAL4 protein function. The DNA base changes in such mutations will be identified by DNA sequencing. We anticipate that the proposed lines of research will generate a variety of significant and useful information regarding regulatory circuitry and mechanisms, and provide tools for further, more highly detailed, analysis of this system.
Showing the most recent 10 out of 27 publications
