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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022749-10
Application #
3271297
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-04-01
Project End
1987-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
10
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Czyz, M; Nagiec, M M; Dickson, R C (1993) Autoregulation of GAL4 transcription is essential for rapid growth of Kluyveromyces lactis on lactose and galactose. Nucleic Acids Res 21:4378-82
Kuzhandaivelu, N; Jones, W K; Martin, A K et al. (1992) The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene. Mol Cell Biol 12:1924-31
Mylin, L M; Gerardot, C J; Hopper, J E et al. (1991) Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains. Nucleic Acids Res 19:5345-50
Halvorsen, Y D; Nandabalan, K; Dickson, R C (1991) Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger. Mol Cell Biol 11:1777-84
Pan, T; Halvorsen, Y D; Dickson, R C et al. (1990) The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster. J Biol Chem 265:21427-9
Witte, M M; Dickson, R C (1990) The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1. Mol Cell Biol 10:5128-37
Dickson, R C; Gerardot, C J; Martin, A K (1990) Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9. Nucleic Acids Res 18:5213-7
Halvorsen, Y C; Nandabalan, K; Dickson, R C (1990) LAC9 DNA-binding domain coordinates two zinc atoms per monomer and contacts DNA as a dimer. J Biol Chem 265:13283-9
Webster, T D; Dickson, R C (1988) The organization and transcription of the galactose gene cluster of Kluyveromyces lactis. Nucleic Acids Res 16:8011-28
Witte, M M; Dickson, R C (1988) Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis. Mol Cell Biol 8:3726-33

Showing the most recent 10 out of 17 publications