The goal of the proposed research is to determine the molecular mechanism of genetic regulation in a eucaryote. As a simple model system we are studying induction of beta-galactosidase in the yeast Kluyveromyces lactis. Our experimental approach is to probe the mechanism of induction by standard genetic techniques and by recombinant DNA and biochemical procedures. In previous studies we have shown that induction of beta-galactosidase is regulated at the level of transcription. We now wish to determine the molecular details of this control process.
Our specific aims are to (1) construct a genetic regulatory model and test predictions of the model, (2) determine the nucleotide sequence of the promoter and terminator regions of the cloned beta-galactosidase gene, (3) introduce site-specific mutations into the promotor and terminator regions of this gene by in vitro procedures, and examine the effect of the mutation on gene expression following transformation of the mutant gene into K. lactis, (4) determine the nucleotide sequence of those mutations which perturb expression of the beta-galactosidase gene, (5) isolate other lactose-inducible genes including galactokinase, transferase, and epimerase and compare the structure of their promotor to the beta-galactosidase promotor, and (6) isolate regulatory genes and examine their function. The later project is our most important aim. The results of these studies should lead to a better understanding of genetic regulation in relation to organismal development, environmental adaptation, and disease states such as cancer.
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