We propose to continue our analysis of the structure and function of the avian retrovirus reverse transcriptase. Our studies during the tenure of the previous application have suggested that such an analysis serves not only to substantiate the details of retrovirus DNA synthesis but to delineate novel functions for the various enzymatic activities (e.g., RNase H, unwinding) associated with the retrovirus reverse transcriptase, thereby expanding our previous conceptions of the roles of these particular activities. In an effort to completely elucidate the precise details in which the reverse transcriptase-associated RNase H, DNA endoculease, and unwinding activities participate in the synthesis of vDNA, we propose to continue our studies in vitro, employing reconstructed reactions containing rigorously purified reverse transcriptase and defined substrates that mimic the purported natural substrates of reverse transcriptase in vivo. Moreover, we plan to delineate the organization of these reverse transcriptase-assocated activities and their functions in viral DNA synthesis both in vitro and in vivo by employing site-specific mutagenesis of the pol gene that we have molecularly cloned to expression in E. coli. Finally, low stringency hybridization analysis of avian cells that lack endogenous retrovirus have revealed nucleotide sequences related to the retrovirus pol gene. Because of the possible implications of these preliminary studies, we propose to determine: 1) the nature of these cellular pol gene-related sequences in avian and mammalian cells; 2) their expression in a variety of cell types, including developing embryos; 3) the enzymatic activities associated with this cellular enzyme and their relationship to viral reverse transcriptase; and 4) their possible role in the generation of pseudogenes. The methodologies to be employed in these studies include a variety of physico-chemical and enzymological procedures presently available in our laboratory to analyze DNA, RNA and protein.
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