This research project is designed to identify and characterize possible regulatory control of the transcription of genetic information for the cytochrome P-450-dependent monooxygenase system. Nucleic acid extracts of lung, kidney, and liver from fetal, neonatal, adult, and aged rats and rabbits will be studied. A cDNA clone, designated PB-8, representing molecular information for a major phenobarbital-inducible species of cytochrome P-450 from rat liver, will be employed as a hybridization proble. Genomic blot hybridization experiments will be performed, under conditions of both high and low hybridization stringency, with the PB-8 probe and with restriction endonuclease-digested DNA isolated from tissues of the rat and rabbit. Possible DNA sequence homologies between the rat and rabbit preparations will be identified. Highly homologous and partially homologous polyA+ RNA molecules will be characterized and isolated by Northern blot hybridization procedures. The isolated polyA+ RNA fractions will be cloned as cDNA derivatives and compared with the techniques of restriction endonuclease mapping and DNA sequencing. Quantitation of the expression of polyA+ RNA molecules, homologous to the PB-8 probe, will be performed and compared in the rat and rabbit lung, kidney and liver. Differences in the expression and the capacity for expression of polyA+ RNA in these tissues will be evaluated as a function of animal development and phenobarbital pretreatment. Methylation patterns and nuclease-digestion sensitivities of PB-8 homologous genomic DNA will also be assessed in animal tissue extracts exhibiting basal levels and induced levels of expression. The data obtained from these studies should enhance our understanding of the molecular mechanisms of P-450 gene activation. In addition, the results of these investigations should provide direct comparisons of the molecular similarities and heterogeneities that exist between related forms of cytochrome P-450 expressed during animal development and in different tissues of the rat and rabbit. Such information should contribute to our understanding not only of gene expression, but also of cytochrome P-450 gene evolution, and may help determine which organs or organisms are most susceptible to the toxicologic effects of environmental chemicals that require activation.
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