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?
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