BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

腺病毒早期 RNA 的生物合成

• 批准号: 3482017
• 负责人: ARNOLD J BERK
• 金额: $ 29.63万
• 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 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3482017
• 关键词: Adenoviridae; DNA binding protein; HeLa cells; chemical binding; gel electrophoresis; gene deletion mutation; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic promoter element; genetic transcription; human tissue; laboratory mouse; laboratory rabbit; laboratory rat; methylation; molecular cloning; nucleic acid hybridization; nucleic acid sequence; oncogenic virus; radiotracer; structural genes; temperature sensitive mutant; transcription factor; transfection; viral carcinogenesis; virus DNA; virus RNA; virus antigen; virus genetics; virus infection mechanism; virus protein; virus replication

Proteins encoded at the left end of the adenovirus 2 (Ad2) genome in a transcription unit called E1A greatly stimulate transcription from at least seven of nine known Ad2 promoters for transcription by RNA polymerase II. Similar "transactivating" function have been described for viral proteins expressed immediately after infection in a number of DNA virus systems. E1A functions are also central to the process of oncogenic transformation by Ad2, and are representative of a class of oncogene products including polyoma and SV40 T- antigens, myc, N-myc and p53, all nuclear proteins which can cooperate with the c-Ha-ras 1 oncogene to completely transform primary embryo cells. The principal goals of the research proposed here are to understand the molecular mechanisms by which E1A proteins stimulate transcription from two Ad2 promoters, the E1B and IX promoters. The E1B promoter is active both before and after viral DNA replication in permissive cells and in transformed cells. Results of our unpublished studies on mutations constructed in the E1B promoter region suggest that the E1B promoter is unusually simple, being comprised of a single binding site for the Sp1 transcription factor (TF) and a TATA-box. No specific sequences appear to be required for E1A transactivation. Transcription from the IX promoter is only observed following viral DNA replication in permissive cells and is not observed in transformed cells. Yet the sequence of the IX promoter suggests that it too contains a single Sp1 site in close proximity to a TATA-box. This raises the question of what causes the difference in temporal regulation of these two Ad2 promoters. We propose to complete a mutational analysis of the E1B promoter and to perform a similar analysis of the IX promoter. We will assay the concentrations of TFs and other sequence specific DNA binding proteins which interact with the promoter elements identified by the mutational studies, their affinities for promoter sequences, and their specific activities for in vitro transcription in extracts from HeLa cells infected with Ad2 and E1A-mutants. Protein binding to these promoter elements in vivo will be assayed in cells infected with Ad2, E1A- mutants, and E1B and IX promoter mutants. Theses studies should provide some insight into the mechanism of transcription initiation at these promoters, its stimulation by E1A proteins, and temporal regulation of the IX promoter. A final specific aim is to design and construct temperature sensitive E1A proteins. Such ts E1A proteins would be useful tools in the study of transactivation and transformation by E1A proteins.

腺病毒2（Ad 2）左端编码的蛋白质 一个叫做E1 A的转录单位的基因组极大地刺激了 从九个已知的Ad 2启动子中的至少七个转录 用于RNA聚合酶II的转录。 类似 已经描述了病毒蛋白的“反式激活”功能 在许多DNA病毒感染后立即表达 系统. E1 A功能也是 Ad 2的致癌转化，并代表了一种 一类癌基因产物，包括多瘤和SV 40 T- 抗原，myc，N-myc和p53，所有的核蛋白， 与c-Ha-ras 1癌基因协同作用， 初级胚胎细胞 本研究的主要目标是 了解E1 A蛋白的分子机制 刺激两个Ad 2启动子E1 B和IX的转录 发起人。 E1 B启动子在治疗前和治疗后都有活性。 病毒DNA在允许细胞和转化细胞中的复制 细胞 我们未发表的突变研究结果 在E1 B启动子区构建的基因表明，E1 B 启动子是异常简单的，由一个单一的结合 Sp1转录因子（TF）和TATA盒的位点。 没有 E1 A似乎需要特定的序列 反式激活 从IX启动子的转录仅 在病毒DNA复制后在允许细胞中观察到， 在转化细胞中未观察到。 然而第九章的顺序 启动子表明它也包含一个单一的Sp1位点， 靠近一个塔塔盒子 这就提出了一个问题， 导致这两种Ad 2的时间调节差异 发起人。 我们建议完成一个突变分析， E1 B启动子，并对IX启动子进行类似的分析。 启动子 我们将分析TF和其他 序列特异性DNA结合蛋白，其与 通过突变研究鉴定的启动子元件，其 启动子序列的亲和力及其特异性活性 用于在感染的HeLa细胞提取物中的体外转录 Ad 2和E1 A突变体。 与这些启动子结合的蛋白质 将在用Ad 2、E1 A- 突变体，以及E1 B和IX启动子突变体。 论文研究 应该能提供一些关于转录机制的信息 在这些启动子处的起始，其由E1 A蛋白的刺激， 和IX启动子的时间调节。最后一个具体目标 是设计和构建温度敏感的E1 A蛋白。 这种ts E1 A蛋白将是研究 通过E1 A蛋白的反式激活和转化。