权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

CONTROL OF ADENOVIRUS TRANSCRIPTION

腺病毒转录的控制

基本信息

批准号：
3125417
负责人：
ROBERTO WEINMANN
金额：
$ 16.9万
依托单位：
WISTAR INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1976
资助国家：
美国
起止时间：
1976-06-30 至 1989-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3125417
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

项目摘要

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.

致癌细胞基因的激活参与转化和至少在某些情况下，许多其他基因的水平似乎处于转录。该提案的重点是分析启动 RNA聚合酶II和III在体外系统中的转录忠实地再现体内启动的位点和要求转录。我们将使用直接生化的组合采用遗传方法的分馏方法利用了模板和转录机制突变体以及核苷酸类似物。 RNA 聚合酶 II 的工作将重点关注参与的蛋白质通过详细分析生化（带有辅因子的酶）来启动以及所需的遗传（即模板序列中）特征转录起始复合物的形成。我们将使用 HeLa 细胞和模板的转录提取物包括插入细菌质粒或噬菌体的致癌腺病毒 DNA。小说起始过程的特征，例如 DNA 的解旋双螺旋或转录抑制剂的作用机制启动，DRB，将是一些部分过程在转录起始复合物中进行详细分析。放松测定也将用于 RNA 聚合酶转录的 VAI RNA 基因三．转录复合物的形成，区分在转录的起始阶段和延伸阶段之间，揭示了引发事件的摩尔过量。这种过量可能与通过增加延伸量来激活基因的机制因素或可能是体外系统的人为因素。点突变体 DNA 模板将通过 M-13 上的位点特异性诱变生成允许对突变体进行快速序列分析的克隆载体以重组 DNA 的复制形式进行体外转录噬菌体。位点特异性点突变可以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和转录元件机器，细胞RNA聚合酶IIα-鹅膏蕈碱抗性突变体和将分析转录抗性 DRB 抗性突变体。 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机制转录起始抑制剂 DRB 的作用和生化这些细胞突变体的基础。