E1A ONCOGENE MEDIATED GENE REGULATION

E1A癌基因介导的基因调控

• 批准号: 2091488
• 负责人: ROBERTO WEINMANN
• 金额: $ 13.48万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2091488
• 关键词: Adenoviridae; DNA binding protein; HeLa cells; affinity chromatography; chemical binding; gel mobility shift assay; gene deletion mutation; genetic library; genetic promoter element; genetic transcription; molecular cloning; mutant; northern blottings; nucleic acid sequence; oncogenes; oncoproteins; point mutation; protein structure function; recombinant proteins; site directed mutagenesis; transcription factor; transfection; virus genetics; virus protein

The adenovirus E1A oncogene encodes two major proteins of 289 and 243 aa involved in cell immortalization, transformation in cooperation with ras, and transcriptional activation and repression on both viral and cellular genes. The E1A protein binds specifically to different gene products, including the tumor suppressor gene product of the retinoblastoma, a related 107 kDa protein, a 300 kDa polypeptide, and a 60 kDa cyclin a subunit of the cdc2 type kinase. Since E1A is by itself unable to bind DNA and no consensus activator or repressor site has been detected, we investigated interactions between E1A and cellular proteins required for transcription. We have thus described a novel interaction between adenovirus E1A and the cellular TATA-box binding protein TBP. A region close to the basic domain of TBP and the Zn finger domain are involved in this interaction. Moreover, we have established that ATF2, a cellular DNA binding protein also is able to interact with TBP, probably via a third protein. To establish the functional relevance of these interactions, we propose to perform in vitro and in vivo assays using E1A and TBP mutants, as well as ATF2 recombinants on several viral promoters. In vitro assays will focus on the rate, efficiency and stability of transcription complex formation and transcription stimulation. In vivo assays will either alter the levels of these basal components or assay transdominance effects (i.e., E1A or TBP mutants which interfere with normal transactivation) in promoters that have been shown to respond to E1A via the TATA-box sequence. We will use protein-protein screening of expression libraries to look for additional sequences encoding proteins which may be, at least in part, responsible for the pleiotropic effects of the E1A oncogene. The role of these proteins in normal cellular processes in the absence of E1A will also be determined. These experiments will clarify the mechanism by which E1A stimulates or represses transcription, an important part of its oncogenic potential.

腺病毒E1a癌基因编码289和243氨基酸两种主要蛋白质。 参与细胞永生化，与RAS合作转化， 以及对病毒和细胞的转录激活和抑制 基因。 E1a蛋白与不同的基因产物特异结合，包括 视网膜母细胞瘤的肿瘤抑制基因产物，相关的107 KDA蛋白，一个300 kDa的多肽和一个60 kDa的Cyclin A亚基。 Cdc2类型的激酶。由于E1a本身不能结合DNA，所以没有 已检测到共识激活子或抑制子位点，我们调查了 E1a与细胞内蛋白质的相互作用 抄写。因此，我们描述了一种新的相互作用 腺病毒E1a和细胞内TATA盒结合蛋白TBP。一个地区 TBP的近碱性结构域和锌指结构域都参与了 这种互动。此外，我们已经确定ATF2，一种细胞 DNA结合蛋白也能够与TBP相互作用，可能是通过一种 第三种蛋白质。 为了确定这些交互作用的功能相关性，我们建议 使用E1a和TBP突变体进行体外和体内检测 作为ATF2重组体在几个病毒启动子上。体外分析将会 关注转录复合体的速度、效率和稳定性 形成和转录刺激。活体化验将会 改变这些基本成分的水平或分析反式优势 影响(即干扰正常的E1a或TBP突变体 反式激活)的启动子，已被证明通过 塔塔-BOX序列。 我们将使用蛋白质-蛋白质筛选表达文库来寻找 编码蛋白质的额外序列，至少部分地， 对E1a癌基因的多效性负责。的作用 在没有E1a的情况下，这些蛋白质在正常的细胞过程中将 也要有决心。 这些实验将阐明E1a刺激或 抑制转录，这是其致癌潜力的重要组成部分。