The goal is to understand the molecular mechanism of transcriptional regulation in a eucaryote. As a model system we are studying the inducible lactose-galactose regulon in the yeast Kluyveromyces lactis. We have isolated LAC9, a trans-acting positive regulatory gene, and shown that it has both similarities and differences to the GAL4 regulatory gene of S. cerevisiae. This relationship provides a unique opportunity to understand how the LAC9 protein functions, how it connects to the induction signal, and how it connects to global regulatory circuits including glucose repression.
Our specific aims are to (1) determine which amino acids in the LAC9 protein specify binding to Upstream Activator Sites (UAS), (2) determine which domains of LAC9 protein sense the induction signal, mediate glucose repression, and activated transcription, (3) purify the LAC9 protein and characterize its interaction with UAS, (4) use purified LAC9 protein to identify and characterize interactions with other proteins, (5) determine if there are cis-acting promoter elements besides UAS and how they function, (6) determine which bases in the 17 bp UAS specify binding of LAC9 protein, (7) examine the hypothesis that LAC10 protein acts as a negative regulator by binding directly to LAC9 protein. Recombinant DNA techniques will be used to isolate genes and change their base sequence, and biochemical techniques will used to study DNA-protein and protein-protein interactions. The information obtained from these studies will aid our understanding of more complex regulatory phenomena including cellular differentiation, environmental adaptation, and disease states such as cancer.
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