BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

腺病毒早期 RNA 的生物合成

• 批准号: 3166755
• 负责人: ARNOLD J BERK
• 金额: $ 6.82万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3166755
• 关键词: Adenoviridae; HeLa cells; autoradiography; gel electrophoresis; gene mutation; genetic manipulation; genetic mapping; genetic transcription; human tissue; mutant; nucleic acid structure; oncogenic virus; radiotracer; viral carcinogenesis; virus RNA; virus genetics

Our long term goals are to determine the mechanism of viral mRNA synthesis and its regulation during the early phase of Adenovirus infection, and the mechanism of cell transformation by Adenovirus. In the work proposed here, our first specific aim is to determine viral DNA sequence required for initiation of transcription, splicing, polyadenylation, and transport of mRNAs encoded in the transforming region. This will be done by constructing specific mutants with deletions, insertions, and base-pair changes and analyzing the impact of these mutations on these steps in mRNA synthesis. Second, we will determine the requirement of each of the several closely related, overlapping proteins encoded in the transforming region for the process of tranformation by constructing viral mutants with specific base-pair changes in sequences required for splicing specific mRNAs. Because of genetic code degeneracy, base-pair changes can be designed which do not alter the coding capacity of overlapping genes, or make conservative amino acid replacements in the encoded proteins. Thrid, we will analyze the mechanism by which viral proteins encoded in early region IA catalyze the conversion of the viral genome from a poor to an efficient template for transcription. To do this we will search for differences in the nucleoprotein structure of intracellular viral DNA in cells infected with wild-type virus and early region IA mutants by analyzing the products of viral DNA following digestion of isolated nuclei with micrococcal nuclease and DNase I. We will purify early region IA proteins and study their interaction with specific viral DNA and RNA sequences in vitro. To facilitate purification, recombinant viruses will be constructed to greatly overproduce these proteins. Finally, we will isolate mutants with ts lesions in these proteins by selection of ts second site revertants after directed mutagenesis. Studies with such mutants will indicate if these proteins are continuously required for expression of viral mRNAs, and maintenance of the transformed phenotypes of transformed cells.

我们的长期目标是确定病毒 mRNA 合成的机制 及其在腺病毒感染早期的调控，以及 腺病毒转化细胞的机制。 在这里提出的工作中， 我们的第一个具体目标是确定所需的病毒 DNA 序列 转录、剪接、聚腺苷酸化和转运的起始 mRNA 在转化区编码。 这将由 构建具有删除、插入和碱基对的特定突变体 变化并分析这些突变对 mRNA 这些步骤的影响 合成。 其次，我们将确定每个项目的要求 转化过程中编码的几个密切相关、重叠的蛋白质 通过构建病毒突变体进行转化过程的区域 剪接特定序列所需的特定碱基对变化 mRNA。 由于遗传密码的简并性，碱基对的变化可以 设计不改变重叠基因的编码能力，或 在编码的蛋白质中进行保守氨基酸替换。 第三， 我们将分析早期病毒蛋白编码的机制 IA区催化病毒基因组从贫乏基因组转变为贫乏基因组 高效的转录模板。 为此，我们将搜索 细胞内病毒DNA核蛋白结构的差异 感染野生型病毒和早期IA区突变体的细胞 分析分离细胞核消化后的病毒 DNA 产物 用微球菌核酸酶和 DNase I。我们将纯化早期 IA 区域 蛋白质并研究它们与特定病毒 DNA 和 RNA 的相互作用 体外序列。 为了便于纯化，重组病毒将 被构建为大大过量生产这些蛋白质。 最后，我们将 通过选择 ts 第二个来分离这些蛋白质中具有 ts 损伤的突变体 定向诱变后的位点回复突变体。 对此类突变体的研究将 表明这些蛋白质是否持续需要表达 病毒 mRNA 以及转化表型的维持 细胞。