CONTROL OF ADENOVIRUS TRANSCRIPTION

腺病毒转录的控制

• 批准号: 3125414
• 负责人: ROBERTO WEINMANN
• 金额: $ 16.09万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-06-30 至 1988-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3125414
• 关键词: Adenoviridae; chromatography; electron microscopy; gel electrophoresis; gene expression; genetic manipulation; genetic recombination; genetic transcription; mutant; nucleic acid sequence; oncogenic virus; point mutation; radiotracer; tissue /cell culture; virus genetics

The activation of oncogenic cellular genes involved in transformation and of many other genes seems to be, at least in some cases, at the level of transcription. This proposal centers on the analysis of initiation of transcription by RNA polymerases II and III in vitro systems which faithfully reproduce the site and requirements for in vivo initiation of transcription. We will use a combination of straight biochemical fractionation approaches with a genetic approach which takes advantage of template and transcriptional machinery mutants as well as nucleotide analogs. The RNA polymerase II work will focus on the proteins involved in initiation by analyzing in detail the biochemical (enzyme with cofactors) as well as genetic (i.e., in the template sequence) features required for the formation of the transcription initiation complex. We will use transcriptional extracts from HeLa cells and templates consisting of oncogenic adenovirus DNA inserted in bacterial plasmids or phages. Novel features of the initiation process, like for example, unwinding of the DNA double helix or the mechanism of action of the inhibitor of transcription initiation, DRB, will be some of the partial processes that will be analyzed in detail in transcriptional initiation complexes. The unwinding assay will also be used for the VAI RNA genes transcribed by RNA polymerase III. The formation of a transcriptional complex, which distinguishes between the initiation and elongation phases of transcription, revealed a large molar excess of initiation events. This excess could be related to a mechanism of gene activation by an increase in the amounts of elongation factors or might be an artifact of the in vitro system. Point mutants in the DNA template will be generated by site-specific mutagenesis on M-13 cloning vectors which allow rapid sequence analysis of the mutants and in vitro transcription with the replicative form of the recombinant DNA phage. Site-specific point mutants allow to better establish the role of each element of the promoter sequence, since the environment around the mutation remains constant. The interactions between the mutant or wild-type promoter sequences and the elements of the transcriptional machinery, cellular RNA polymerase II Alpha-amanitin resistant mutants and transcriptionally resistant DRB resistant mutants will be analyzed. Mutations in the RNA polymerase or factors should produce some abnormal interactions. We will continue our efforts in establishing the mechanism of action of the transcription initiation inhibitor DRB and the biochemical basis for these cell mutants.

参与转化和转移的致癌细胞基因的激活 许多其他基因似乎，至少在某些情况下，处于 抄写。这一建议的核心是对发起 RNA聚合酶II和III在体外系统中的转录 忠实地复制体内启动的位置和要求 抄写。我们将使用一种直接的生化组合 利用遗传方法进行分馏的方法 模板和转录机制突变体以及核苷酸 类比。RNA聚合酶II的工作将集中在参与 通过详细分析生化(辅酶)引发 以及所需的遗传(即在模板序列中)特征 转录起始复合体的形成。我们将使用 HeLa细胞的转录提取物和由以下组成的模板 插入到细菌质粒或噬菌体中的致癌腺病毒DNA。小说 启动过程的特征，例如，DNA的解离 双螺旋或转录抑制物的作用机制 启动，即DRB，将是将被 在转录起始复合体中进行了详细分析。《解开》 还将对由RNA聚合酶转录的VAI rna基因进行检测 三、转录复合体的形成，这区分了 在转录的起始和延伸阶段之间，揭示了 大摩尔过量的引发事件。这种过量可能与 通过增加伸长量来激活基因的机制 因素或可能是体外系统的人工制品。中的点突变 DNA模板将通过对M-13进行定点突变而产生 克隆载体，允许快速分析突变体和 复制形式的重组DNA体外转录 噬菌体。定点突变体允许更好地确定 启动子序列的每个元件，因为周围的环境 突变保持不变。突变体或突变体之间的相互作用 野生型启动子序列及其转录元件 机械，细胞RNA聚合酶II，抗α-Amanitin突变体和 将对转录抗性DRB抗性突变体进行分析。 RNA聚合酶或因子的突变应该会产生一些异常 互动。我们将继续努力建立这一机制 转录起始抑制物DRB的作用与生化 这些细胞突变体的基础。