Reduction of dioxygen, an initial product of which is the superoxide radical 02-, generates free radicals that have been implicated in the genesis of oxygen toxicity reperfusion injury, radiation injury, and in the action of certain xenobiotics. The superoxide dismutases play a critical role in the scavenging of superoxide. Delineation of their molecular biology will provide a basis for the understanding of their role in these pathologies. The primary goal of this proposal is to examine the molecular mechanisms which mediate the expression of both the rat and Mn and Cu/Zn superoxide dismutase (SOD) genes in vivo. We plan to isolate the cDNA clones for both of these genes via the screening of a rat liver gt11 expression library with polyclonal antibodies to each of these enzymes. Oligonucleotide probes derived from conserved regions of published human enzyme amino acid sequence will serve as direct verification of the recombinant clones. The isolated cDNA clones will be utilized as probes in the isolation of genomic clones and in the evaluation of mRNA levels from primary cultures of microvascular rat lung endothelial cells. mRNA levels from these endothelial cells and control cell lines will also be examined in response to stimuli demonstrated to effect SOD activity, specifically, oxygen tension, bacterial endotoxin and paraquat will be tested. In the same context, we plan experiments directed at understanding whether fluctuations in SOD mRNA levels in response to such stimuli are a consequence of transcriptional regulation or the inherent stability of the mRNA. With a basic understanding of transcriptional levels, we then plan to delineate the boundaries of DNA sequences required for cell specific control using studies on the expression of promoter deletion mutants and a detailed analysis of SOD chromatin structure. The extension of these goals of the nucleotide level using genomic sequencing will provide a """"""""fingerprint"""""""" unique to each regulatory protein involved in SOD repression and/or induction. This fingerprint will serve as the assay for future isolation and characterization of regulatory proteins, their genes, and their regulatory response to superoxide levels.
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