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Environmental Stress-Responsive Gene Expression Mediated Through Heat Shock Transcription Factor
EPA Grant Number: GF9500923Title: Environmental Stress-Responsive Gene Expression Mediated Through Heat Shock Transcription Factor
Investigators: Santoro, Nicholas
Institution: University of Michigan
EPA Project Officer: Broadway, Virginia
Project Period: September 1, 1995 through January 1, 2000
Project Amount: $27,732
RFA: STAR Graduate Fellowships (1995)
Research Category: Fellowship - Health , Health Effects , Academic Fellowships
Description:
Objective:This proposed research project seeks to understand environmental stress-responsive gene expression mediated through Heat Shock Transcription Factor (HSF). This project will involve the following three components: 1) a study of the DNA-binding properties of HSF to different stress-responsive promoters using the electrophoretic mobility shift assay to determine the relative binding affinity, and chemical cross linking reagents to determine the multimerization state of HSF; 2) map the sites of phosphorylation on HCF in response to different cellular stress; 3) determine the importance of the phosphorylation of HCF in regulating stress-responsive gene expression in vivo by constructing site directed mutations in the HCF phosphorylation sites and replacing the endogenous copy of HCF with each mutated HCF. Cells expressing the mutationally altered HCF molecules will be tested for their ability to activate CUP transcription in response to heat and oxidative stress. For the first research goal, DNA binding studies will be performed using DNA sequences that are naturally found in stress responsive promoters. CUP and SSA promoter DIAS in the gel shift analyses will be compared in these DNA binding studies. For the second goal, yeast cells will be treated with heat or oxidative stress both of which are known to activate CUP, and cell extracts prepared. These extracts will be used for in vitro phosphorylation assays with the purified HCF, and the sites of phosphorylation will be mapped and compared to control extracts which were not exposed to stress. The third goal will be undertaken once the sites of phosphorylation are mapped. Then the project will construct site-directed mutations in each site and determine the importance of phosphorylation of HCF on the activation of gene expression in response to environmental stress.
Supplemental Keywords:Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, RFA, Ecosystem/Assessment/Indicators, Molecular Biology/Genetics, Biology, exploratory research environmental biology, Ecology, Microbiology, Analytical Chemistry, Ecological Risk Assessment, Chemistry, Ecological Indicators, Biochemistry, Environmental Microbiology, Ecological Effects - Human Health, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Ecology and Ecosystems, Genetics, ecosystem response , heat schock transcription factor (HSF), ecosystem assessment, environmental stress, phosphorylation of HCF, environmental stress-responsive gene expression