FUNCTIONS OF AN EIA-ASSOCIATED CELLULAR PROTEIN

EIA 相关细胞蛋白的功能

• 批准号: 2468737
• 负责人: BAYAR THIMMAPAYA
• 金额: $ 28万
• 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/2468737
• 关键词: Adenoviridae; DNA binding protein; DNA footprinting; cell growth regulation; complementary DNA; gene expression; genetic promoter element; genetic regulation; genetic transcription; genome; intermolecular interaction; microscopy; molecular cloning; oncogenic virus; open reading frames; polymerase chain reaction; 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.

该提案着重于腺病毒的转录调节 （AD）早期启动子（E2-Early）转录病毒区域 编码72 kD单链DNA结合蛋白的基因组，80 kd 前体末端蛋白和140 kD DNA聚合酶as 对病毒生长至关重要。 e2- e2的转录调节 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剂。 期间 上一个赠款期，我们定义了顺式作用序列 该启动子在病毒染色体的背景下的元素， 确定了几个与这些结合的细胞转录因子 序列元素和病毒编码的反式激活剂的作用 调节该启动子的转录。 我们建议继续这些 研究并使用遗传和生化方法来阐明 该启动子受细胞调节的机制 转录因子和病毒编码的反式激活剂。 我们有 遵循特定目的： 最近的结果表明，除ELA外，E2早期启动子是 由AD早期4个多肽（E4 6/7）和 单链DNA结合蛋白。 我们将使用遗传和 生化方法来确定反式激活的机制 这些基因产物的E2启动子。 我们将尝试克隆编码细胞转录的cDNA E2F因子E2F在E2-Early启动子中起关键作用 转录。 E2F在细胞周期中也起着重要作用 调节并与视网膜母细胞瘤易感基因（RB）产物结合， RB相关的蛋白质PL07，环素A和CDK2激酶。 一个cDNA克隆 E2F是许多研究的宝贵试剂，包括研究 E2F的结构功能关系及其在细胞周期中的作用 规定。 我们将剖析E2-Early的体内DNA-蛋白质相互作用 基底和病毒诱导转录的启动子与连接 介导的PCR扩增方法。 在这项研究中，我们将使用一个系列 我们先前构建的广告突变体的 E2-Early启动子的不同转录因子结合位点。