E1A ONCOGENE MEDIATED GENE REGULATION

腺病毒E1 A癌基因编码289和243个氨基酸的两种主要蛋白质 参与细胞永生化，与ras合作转化， 以及病毒和细胞上的转录激活和抑制 基因. E1 A蛋白特异性结合不同的基因产物，包括 视网膜母细胞瘤的肿瘤抑制基因产物，一个相关的107 在一些实施方案中，细胞周期蛋白包括300 kDa蛋白、300 kDa多肽和60 kDa细胞周期蛋白a亚基。 cdc 2型激酶。 由于E1 A本身不能结合DNA， 一致激活或阻遏位点已被检测到，我们调查 E1 A和细胞蛋白之间的相互作用 转录。 因此，我们描述了一种新的相互作用， 腺病毒E1 A和细胞TATA盒结合蛋白TBP。 的区域 与TBP的基本结构域和Zn指结构域接近， 这种互动。 此外，我们已经确定，ATF 2，一个细胞， DNA结合蛋白也能够与TBP相互作用，可能是通过 第三种蛋白质 为了建立这些相互作用的功能相关性，我们建议 使用E1 A和TBP突变体进行体外和体内试验，以及 作为几种病毒启动子上的ATF 2重组体。 体外试验将 重点关注转录复合体的速率、效率和稳定性 形成和转录刺激。 体内试验将 改变这些基础成分的水平或测定转显性 效果（即，E1 A或TBP突变体干扰正常 反式激活）的启动子，已显示响应E1 A通过 TATA盒序列 我们将使用蛋白质-蛋白质筛选表达文库来寻找 编码蛋白质的其它序列，至少部分， 负责E1 A癌基因的多效性效应。 的作用 这些蛋白质在正常细胞过程中，在缺乏E1 A的情况下， 也要坚决。 这些实验将阐明E1 A刺激或 抑制转录，这是其致癌潜力的重要部分。

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.