This competing renewal Program-Project Research Grant application will provide a vehicle for a highly integrated series of investigations designed to test theories of aging that emphasize a major role for oxidative damage to DNA. The major approach is to utilize transgenic mice constitutively overexpressing human cDNAs for enzymes that scavenge reactive oxygen species (organelle-targeted catalase; superoxide dismutase-1 plus catalase) in order to decrease the rate of DNA damage, retard pathology (particularly neoplasms), preserve cognitive performance and extend life span. The constructs are driven by a strong promoter/enhancer (chick beta actin/CMV) known to provide high expression of a transgene in all major tissues. Characterizations will include documentation of the phenotype of resistance to oxidative stress in cultured somatic cells; determinations of accumulation of chromosomal mutations, deletions and point mutations (APRT and HPRT) and putative """"""""signature"""""""" mutations of oxidative damage (CC to TT) in renal tubular epithelial cells; assays of free radical """"""""scavenger"""""""" enzymes, hydroxyl ion formation, DNA adduct formation, mitochondrial DNA mutation, and of DNA strand break repair in various tissues. A second approach will document preliminary evidence of unusual resistance of somatic cells from long- lived avian species to oxidative stress and will begin a program of research designed to isolate avian genes responsible for that phenotype. A cohort of aging parakeets (Melopsittacus undulatus) will provide tissues for DNA adduct, mt DNA mutation, and DNA repair studies. Avian tissues will also be provided to other gerontologists. Finally, our original finding of an exponential rise in prevalence of HPRT mutations in aging human renal tubular epithelium will be extended to provide evidence for a molecular spectrum of mutations consistent with a major contribution via oxidative damage.
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