FUNCTIONS OF AN EIA-ASSOCIATED CELLULAR PROTEIN

EIA 相关细胞蛋白的功能

• 批准号: 6447218
• 负责人: BAYAR THIMMAPAYA
• 金额: $ 11.69万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6447218
• 关键词: Adenoviridae; DNA binding protein; DNA footprinting; cell growth regulation; complementary DNA; gene expression; genetic promoter element; genetic regulation; genetic transcription; genome; microscopy; molecular cloning; oncogenic virus; open reading frames; polymerase chain reaction; protein protein interaction; protein structure function; regulatory gene; structural genes; tissue /cell culture; transcription factor; virus DNA; virus genetics

This proposal focuses on the transcriptional regulation of an adenovirus (Ad) early promoter (E2-early) which transcribes a region of the viral genome that encodes a 72 kD single stranded DNA binding protein, an 80 kD precursor terminal protein and the 140 kD DNA polymerase all of which are vital for virus growth. Transcriptional regulation of the E2-early promoter involves protein-protein interactions between multiple cellular transcription factors and several virus-encoded transactivators. During the previous grant period, we have defined the cis-acting sequence elements of this promoter within the context of the viral chromosome, identified several cellular transcription factors that bind to these sequence elements and the role of the virus-encoded transactivators that regulate transcription of this promoter. We propose to continue these studies and use genetic and biochemical approaches to elucidate the mechanisms by which this promoter is regulated by the cellular transcription factors and the virus-encoded transactivators. We have the following specific aims: Recent results show that in addition to ElA, the E2 early promoter is transactivated by one of the Ad early region 4 polypeptides (E4 6/7) and the single stranded DNA binding protein. We will use genetic and biochemical approaches to determine the mechanism of transactivation of the E2-early promoter by these gene products. We will attempt to clone the cDNA that encodes the cellular transcription factor E2F which plays a critical role in E2-early promoter transcription. The E2F also plays an important role in cell cycle regulation and binds to retinoblastoma susceptibility gene (RB) product, an RB related protein pl07, cycline A and cdk2 kinase. A cDNA clone of E2F is a valuable reagent for a number of studies which include studies of structure-functional relationship of E2F, and its role in cell cycle regulation. We will dissect the in vivo DNA-protein interactions of the E2-early promoter in basal and virus induced transcription with the ligation mediated PCR amplification approach. In this study, we will use a series of Ad mutants that we previously constructed, which contain mutations in different transcription factor binding sites of the E2-early promoter.

这项建议的重点是对腺病毒的转录调控。 (Ad)转录病毒某一区域的早期启动子(E_2-早期) 编码72kD单链DNA结合蛋白的基因组，80kD 前体末端蛋白和140kD DNA聚合酶，所有这些都是 对病毒的生长至关重要。E_2早期的转录调控 启动子涉及多个细胞之间的蛋白质-蛋白质相互作用 转录因子和几个病毒编码的反式激活因子。在.期间 在前一个授权期内，我们定义了顺式作用序列 该启动子的元件在病毒染色体的上下文中， 确定了几种与这些基因结合的细胞转录因子 序列元件和病毒编码的反式激活子的作用 调节这个启动子的转录。我们建议继续实施这些措施 研究并使用遗传和生化方法来阐明 该启动子受细胞调控的机制 转录因子和病毒编码的反式激活因子。我们有 以下是具体目标： 最近的研究结果表明，除了ELA，E2早期启动子是 被Ad早期区域4多肽之一反式激活(E4 6/7)和 单链DNA结合蛋白。我们将使用基因和 用生物化学方法确定反式激活的机制 通过这些基因产物的E2早期启动子。 我们将尝试克隆编码细胞转录的cDNAs 在E2早期启动子中起关键作用的E2F因子 抄写。E2F在细胞周期中也起着重要作用 调节和结合视网膜母细胞瘤易感基因(Rb)产物， Rb相关蛋白pl07、细胞周期蛋白A和CDK2激酶。猪瘟病毒的一个基因克隆 E2F是一种有价值的试剂，用于许多研究，包括研究 E2F的结构与功能关系及其在细胞周期中的作用 监管。 我们将剖析在体内的DNA-蛋白质相互作用的E2-早期 碱基启动子和病毒诱导的连接转录 介导式聚合酶链式反应扩增法。在这项研究中，我们将使用一系列 我们之前构建的Ad突变体，它包含了 不同转录因子结合部位的E2-早期启动子。