The goal of proposed research is to elucidate the mechanism by which determinants of cell-type, such as specific DNA binding proteins or transcription factors, affect enhancer function. Investigations toward this goal will exploit the three cell-types of Saccharomyces cerevisiae along with the CYC7-H2 regulatory mutation. The CYC7-H2 mutation was caused by insertion of the yeast transposable element, Tyl, in the control region of the CYC7 locus. Enhancer regions in Tyl cause a 20-fold overproducton of the CYC7 gene product, iso-2-cytochrome c, in both a and Alpha cell-types of yeast. Ty-mediated activation requires trans-actin factors identified by ste mutations that cause a nonmating phenotype in yeast and concomitantly reduce CYC7-H2 gene expression. In the a/Alpha cell-type CYC7-H2 expression is repressed approximately 10-fold. Cell-type control of Ty-mediated effects may represent a general mechanism for tissue and host specificity observed for viral and cellular enhancers as well as for determination in higher eukaryotic organisms. Site directed mutagenesis methods will be applied to regions of the CYC7-H2 Tyl proven to be necessary for its activating function. Methods will include linkerscanning, oligonucleotide directed and base-analogue induced mutagenesis. The various mutant genes will be introduced into a cytochrome c deficient yeast strain by transformation. Gene expression will be compared by quantitative determination of iso-2-cytochrome c mRNA and protein product. These methods will identify the base pair composition of the Tyl enhancer and indicate critical residues for function. Conditional revertants of CYC7-H2 ste strains will be isolated and characterized. One class of revertants will be extragenic suppressors of the Tyl trans-acting regulators. Mutants comprising this class will define the genetic loci of an interacting regulatory network that includes the putative cell specificity determinants of enhancer function. CYC7-H2 chromatin structure in different cell-types and in strains with various regulatory mutant backgrounds will be compared. These comparisons will identify chromatin features such a nuclease hypersensitive sites and protected regions that correspond to active and repressed states of the Tyl enhancer. Any specific features that may be discerned will be used as guides for isolation of Tyl specific binding proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030619-06
Application #
3278422
Study Section
Genetics Study Section (GEN)
Project Start
1982-07-01
Project End
1988-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
6
Fiscal Year
1987
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

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