The research plan is based upon a molecular approach to understanding the mechanisms by which environmental oxidant stresses affect human cells and ultimately lead to clinical disease. Interestingly, the mechanisms by which eukaryotic cells receive and respond to oxidative stresses through changes in gene expression are not known. We hypothesize that unique oxidant regulatory elements, comprised of novel consensus sequences in the DNA, function to confer oxidant inducibility to certain genes. Preliminary data supports that oxidant regulatory elements exist in the heat shock protein 70A gene promoter. Once these oxidant regulatable elements are defined, their ability to confer oxidant inducibility to gene transfer vectors will be tested.
The specific aims are: 1 Identify the sequences conferring oxidant inducibility to the promoters of the human heat shock protein gene and determine the relationship of these elements to other promoter elements such as heat shock element and TATA elements. 2. Investigate the specificity and mechanisms of transduction of the oxidant signals to the oxidant regulatory elements. 3. Test the feasibility of creating inducible viral vectors using the oxidant regulatory element initially directing expression of the reporter gene beta-galactosidase to determine the timecourse, threshold and strength of inducibility of the oxidant regulation, followed by construction of oxidant inducible recombinant viral vectors directing the expression of the human antioxidant genes (catalase and glutathione peroxidase) to test their efficacy in protection against oxidant stresses.
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