BIOCHEMICAL FUNCTIONS OF ADENOVIRUS ONCOGENES

描述（改编自申请人的摘要）：腺病毒E1 A 243 R 癌蛋白是一种多功能的转录调节因子， 在多个域中编码的不同功能。 特别感兴趣的是 转录抑制功能，映射到E1 A N-末端蛋白 对诱导细胞周期进程和细胞增殖重要的序列 转型 我们的主要目标是了解分子机制， E1 A转录抑制的细胞靶点。 我们的长期目标是 了解E1 A抑制如何调节细胞周期的进展。 我们已经开发了一种体外转录抑制系统，其中E1 A 1-80是含有N-末端80个氨基酸的重组蛋白， 用作原型阻遏物，特异性抑制 E1 A阻遏基因。 抑制需要E1 A内的两个小区域 N-末端，并且似乎不需要启动子元件上游的启动子元件。 塔塔盒子。 第一个具体目标是将这些发现扩展到其他领域。 E1 A阻遏型启动子，包括那些具有医学意义的 erbB 2原癌基因和HIV-1。 第二个具体目标是继续做好 发达的研究强烈暗示TBP（TFIID）作为一个直接的细胞 E1 A阻遏靶向，并显示E1 A 1-80可阻断相互作用 TBP和TFIIB之间。 我们将：㈠进一步界定信托基金的作用， TFIIB在E1 A阻遏中通过分析E1 A阻遏之间的相互作用 使用E1 A和TBP丙氨酸扫描突变体的集合， (ii)探讨E1 A在阻断TBP与 TATA盒DNA和TFIIB;（iii）使用纯化的预起始复合物 （太平洋岛屿国家）询问E1 A是否抑制TBP、TFIIB或其他GTF的募集 （iv）分析TBP和TFIIB克服启动子的能力， 体内E1 A抑制。 第三个具体目标是继续研究 细胞蛋白p300在E1 A阻遏中的作用。 我们将询问： (i)纯化的p300可以克服体外E1 A阻遏;（ii）E1 A 通过使用E1 A，可以在功能上将阻遏与p300结合分开 丙氨酸扫描突变体;和（iii）E1 A阻遏可以完全逆转， 过表达或细胞显微注射p300和TBP（或TFIID）。 的 第四个具体目的是使用重构的体外转录系统 为了更详细地研究E1 A抑制与 结构域及其细胞靶点TFIID、TFIIB和p300。 该系统允许 实验阐明E1 A阻遏和 非阻遏型启动子 最后，作为长期目标，我们建议使用 差减cDNA文库和mRNA差异显示， 表达后早期表达受到调节的细胞基因 E1 A静止细胞中的N-末端结构域。 功能测定将用于 鉴定静止维持基因，即潜在的肿瘤抑制基因 可能是E1 A抑制的靶点，和（ii）上调的基因， 潜在的总开关在增长周期。

DESCRIPTION (Adapted from Applicant's Abstract): The adenovirus E1A 243R oncoprotein is a multifunctional transcriptional regulator that expresses diverse functions encoded in multiple domains. Of special interest is the transcriptional repression function that maps to E1A N-terminal protein sequences important for induction of cell cycle progression and cellular transformation. Our primary goal is to understand molecular mechanism and cellular targets of E1A transcriptional repression. Our long term goal is to understand how E1A repression regulates progression of the cell cycle. We have developed an in vitro transcription-repression system in which E1A 1-80, a recombinant protein containing the N-terminal 80 amino acids, is used as a prototype repressor to specifically repress transcription of E1A-repressible genes. Repression requires two small regions within the E1A N-terminus and does not appear to require promoter elements upstream of the TATA box. The first specific aim is to extend these findings to other E1A-repressible promoters, including those of the medically significant erbB2 protooncogene and HIV-1. The second specific aim is to continue well developed studies strongly implicating TBP (TFIID) as a direct cellular target of E1A repression and showing that E1A 1-80 can block interaction between TBP and TFIIB. We will: (i) define further the role of TFIID and TFIIB in E1A repression by analyzing interaction between the E1A repression domain and TBP using a collection of E1A and TBP alanine scanning mutants; (ii) probe the dual function of E1A in blocking TBP interaction with TATA-box DNA and with TFIIB; (iii) use purified preinitiation complexes (PICs) to ask whether E1A inhibits recruitment of TBP, TFIIB, or other GTFs to the promoter, and (iv) analyze the ability of TBP and TFIIB to overcome E1A repression in vivo. The third specific aim is to continue studies on the role of cellular protein p300 in E1A repression. We will ask whether: (i) E1A repression in vitro can be overcome by purified p300; (ii) E1A repression can be functionally separated from p300 binding by use of E1A alanine scanning mutants; and (iii) E1A repression can be fully reversed by overexpression or cell microinjection of p300 and TBP (or TFIID). The fourth specific aim is to use a reconstituted in vitro transcription system to study in greater detail the interactions between the E1A repression domain and its cellular targets TFIID, TFIIB, and p300. This system permits experiments to elucidate differences between E1A repressible and nonrepressible promoters. Finally, as long term goal, we propose to use subtractive cDNA libraries and mRNA Differential Display to identify cellular genes whose expression is modulated early after expression of the E1A N-terminal domain in quiescent cells. Functional assays will be used to identify Quiescence maintaining genes, i.e. potential tumor suppressor genes that may be targets for E1A repression, and (ii) up-regulated genes that are potential master switches in the growth cycle.