RNA-DIRECTED DNA POLYMERASE & 70S RNA OF ONCORNAVIRUSES

我们建议继续分析 禽逆转录病毒逆转录酶。 我们在任期内的研究 先前申请已经提出，这样的分析不 只是为了证实逆转录病毒DNA合成的细节， 描述了各种酶活性的新功能（例如，rna酶 H，解旋）与逆转录病毒逆转录酶相关，从而 扩展了我们以前对这些特定的角色的概念， 活动 为了完全阐明 其中逆转录酶相关的RNase H、DNA内切酶和 解旋活动参与合成的vDNA，我们建议， 继续我们的研究在体外，采用重建的反应， 严格纯化的逆转录酶和确定的底物， 体内逆转录酶的天然底物。 此外，我们计划描绘这些反向组织 病毒DNA中转录酶相关活性及其功能 通过使用位点特异性诱变在体外和体内合成 pol基因，我们已经分子克隆到E.杆菌 最后，对缺乏DNA的禽类细胞进行低严格性杂交分析。 内源性逆转录病毒已经揭示了与 逆转录病毒pol基因 由于这些可能的影响， 初步研究，我们建议确定：1）这些性质 鸟类和哺乳动物细胞中的细胞pol基因相关序列; 2）它们的 在多种细胞类型中表达，包括发育中的胚胎; 3） 与这种细胞酶相关的酶活性及其 与病毒逆转录酶的关系;以及4）它们在 伪基因的产生 在这方面采用的方法 研究包括各种物理化学和酶学程序 目前在我们实验室可以分析DNA，RNA和蛋白质。

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.