RNA-DIRECTED DNA POLYMERASE & 70S RNA OF ONCORNAVIRUSES

我们建议继续分析该组织的结构和功能 禽流感逆转录病毒逆转录酶。我们在任期间的学习 先前申请表明，这样的分析不适用于 只是为了证实逆转录病毒DNA合成的细节，但 描绘各种酶活性的新功能(例如，核糖核酸酶 与逆转录病毒逆转录酶相关)，从而 扩展我们之前对这些特殊角色的概念 活动。为了完整地阐明 其中逆转录酶相关的核糖核酸酶H、DNA内切酶和 解离活动参与VDNA的合成，我们建议 继续我们的体外研究，使用包含以下内容的重建反应 严格纯化的逆转录酶和定义的模拟底物 据称是体内逆转录酶的天然底物。 此外，我们计划勾勒出这些逆转的组织 病毒DNA中转录酶相关活性及其功能 利用定点突变技术进行体内外合成 我们已经从分子上克隆了Poll基因，并在大肠杆菌中表达。 最后，对缺乏的禽类细胞进行低严格性杂交分析 内源性逆转录病毒已揭示与该病毒相关的核苷酸序列 逆转录病毒Poll基因。因为这些可能的影响 初步研究，我们建议确定：1)这些病毒的性质 鸟类和哺乳动物细胞中与Polo基因相关的序列；2)它们的 在各种类型的细胞中表达，包括发育中的胚胎；3) 与该细胞酶相关的酶活性和它们的 与病毒逆转录酶的关系；以及4)它们在 假基因的产生。将在这些项目中采用的方法 研究包括各种物理化学和酶学方法。 目前在我们实验室可用于分析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.