ONCOGENE E1A INDUCED TRANSACTIVATION

癌基因 E1A 诱导的反式激活

• 批准号: 3187088
• 负责人: ROBERTO WEINMANN
• 金额: $ 14.49万
• 依托单位: WISTAR INSTITUTE
• 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 1991-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3187088
• 关键词: Adenoviridae; DNA binding protein; DNA footprinting; affinity chromatography; gene expression; genetic transcription; high performance liquid chromatography; nucleic acid sequence; oligonucleotides; oncogenes; protein structure; transforming virus; virus diseases; virus genetics; virus infection mechanism; virus protein

The long term objective of this proposal is to understand the molecular mechanism by which the adenovirus oncogene early region 1a (E1a) induces transcriptional activation. E1a does not bind to DNA directly and may exert its effect by interaction and or modification of a cellular transcriptional factor. Thus, we have chosen to examine and purify the protein factors that are responsible for early region 3 transcription, an adenovirus gene absolutely dependent on E1a for expression. In addition to RNA polymerase II, other general transcription factors like TFII, B, E, D and A, and gene specific factors are required. Some of the latter are sequence specific DNA binding proteins. The experiments described here focus on the isolation and characterization of a protein which binds to a DNA region necessary both for transcription and E1a response, located 85 to 105 nucleotides upstream from the transcription start site. The unique properties that we will exploit for the purification are the partial assay and selectivity provided by binding to specific DNA sequences, by interaction of this protein with E1a protein and the modern methods of protein HPLC. Specific DNA binding columns derived from synthetic oligonucleotides, as well as E1a affinity columns, will be used in combination with FPLC techniques to purify the specific proteins. Footprinting, gel retardation, and in vitro transcription reconstitution assays will be used to follow the activity of this protein. We will investigate the mode of interaction of the purified factor from unifected or infected cells with E3 DNA and with E1a protein in vitro to determine which properties of the E3 transcription factor are modified by E1a during adenovirus infection. Thus, using a partially purified transcription system we will try to reproduce the regulation effected by E1a on the viral gene E3. The elucidation of this regulatory mechanism will clarify the nature of the E1a interaction with viral and cellular genes and the resulting viral induced transformation event.

本提案的长期目标是了解 腺病毒癌基因早期表达的分子机制 区域1a（E1a）诱导转录激活。 E1a不 直接与DNA结合，通过相互作用发挥作用， 或细胞转录因子的修饰。 因此我们 已经选择检查和纯化蛋白质因子， 负责早期区域3转录的腺病毒基因 完全依赖E1a表达。 除了RNA 聚合酶II，其它通用转录因子如TFII，B，E， D和A，以及基因特异性因子是必需的。 一些 后者是序列特异性DNA结合蛋白。 的 这里描述的实验集中在分离和 与DNA区域结合的蛋白质的表征 转录和E1a反应所必需的，位于85至 转录起始位点上游105个核苷酸。 的 我们将用于纯化的独特性质是 通过与特异性DNA结合提供部分测定和选择性 序列，通过这种蛋白质与E1a蛋白质的相互作用， 现代蛋白质HPLC方法。 特异性DNA结合柱 衍生自合成寡核苷酸，以及E1a亲和力 将与FPLC技术结合使用， 纯化特异性蛋白。 足迹，凝胶阻滞， 体外转录重建试验将用于跟踪 这种蛋白质的活性。 我们将研究 来自未感染或感染细胞的纯化因子的相互作用 与E3 DNA和E1a蛋白在体外，以确定 E3转录因子的性质被E1a修饰 在腺病毒感染期间。 因此，使用部分纯化的 转录系统，我们将尝试复制调节 E1a对病毒基因E3的影响。 对这一点的说明 监管机制将澄清E1a的性质 与病毒和细胞基因的相互作用， 诱发转化事件。