The overall objective of proposed research is to learn how interactions between DNA regulatory elements and DNA binding proteins are controlled during the establishment and maintenance of cell-type specific gene expression. The principles of this regulation are central to global control mechanisms for differentiation and cell specialization. The medical relevance of these mechanisms can be appreciated by considering the result of their disruption in the transformed state where cells have lost aspects of their differentiated phenotype and proliferate outside of normal regulatory constraints. Research will focus on cell-type specific activation of adjacent gene expression caused by the transposable element, Ty, in Saccharomyces cerevisiae. The following specific questions will be addressed. (a) Are observed protein binding interactions critical for control of gene expression? Oligonucleotide mutagenesis methods will be applied to generate mutations in identified regulatory sequences. Defined mutations will be analyzed to correlate function of these sequences in cell-type specific activation of gene expression measured in vivo with their protein binding function measured in vitro. (b) What are the properties of the proteins that bind to identified DNA regulatory sequences? DNA affinity chromatography will be used to purify proteins that bind to specific regulatory sequences. Purified proteins will be characterized. (c) What are the genes that encode the binding proteins? An oligonucleotide screening method based on partial amino acid sequence information obtained from purified DNA binding proteins and a selection method based on complementation of a functional deficiency in site-specific DNA binding proteins are proposed as alternative methods for cloning these genes. (d) What is the role of known regulators of cell-type in the control of specific protein-DNA complexes? Genetic and biochemical experiments are proposed to test some hypotheses. For example, is one of binding proteins we detect the STE12 gene product? Does protein phosphorylation account for the STE7 protein kinase control of protein complex formation at certain of the regulatory sites?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM030619-07
Application #
3278420
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1993-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Baur, M; Esch, R K; Errede, B (1997) Cooperative binding interactions required for function of the Ty1 sterile responsive element. Mol Cell Biol 17:4330-7
Doi, K; Gartner, A; Ammerer, G et al. (1994) MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae. EMBO J 13:61-70
Errede, B (1993) MCM1 binds to a transcriptional control element in Ty1. Mol Cell Biol 13:57-62
Zhou, Z; Gartner, A; Cade, R et al. (1993) Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases. Mol Cell Biol 13:2069-80
Rhodes, N; Connell, L; Errede, B (1990) STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast. Genes Dev 4:1862-74
Rhodes, N; Company, M; Errede, B (1990) A yeast-Escherichia coli shuttle vector containing the M13 origin of replication. Plasmid 23:159-62
Errede, B; Ammerer, G (1989) STE12, a protein involved in cell-type-specific transcription and signal transduction in yeast, is part of protein-DNA complexes. Genes Dev 3:1349-61
Company, M; Adler, C; Errede, B (1988) Identification of a Ty1 regulatory sequence responsive to STE7 and STE12. Mol Cell Biol 8:2545-54
Company, M; Errede, B (1988) A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor. Mol Cell Biol 8:5299-309
Company, M; Errede, B (1987) Cell-type-dependent gene activation by yeast transposon Ty1 involves multiple regulatory determinants. Mol Cell Biol 7:3205-11

Showing the most recent 10 out of 16 publications