ONCOGENE E1A INDUCED TRANSACTIVATION

这项建议的长期目标是了解 腺病毒癌基因早期表达的分子机制 1a区(E1a)可诱导转录激活。E1a并非如此 直接与DNA结合，并可能通过相互作用和 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，这些因子是 负责早期区域3转录，这是一种腺病毒基因 完全依赖于E1a的表达。除了RNA之外 聚合酶II，其他通用转录因子如TFII，B，E， D和A，以及基因特定的因子是必需的。其中一些 后者是序列特异的DNA结合蛋白。这个 这里描述的实验主要集中在分离和 一种与DNA区域结合的蛋白质的特性 转录和E1a反应都是必需的，位于85到 转录起始点上游105个核苷酸。这个 我们将用于纯化的独特属性是 通过与特定DNA结合提供部分检测和选择性 序列，通过该蛋白与E1a蛋白和 蛋白质高效液相色谱的现代方法。特定DNA结合柱 源自合成的寡核苷酸，以及E1a亲和力 列，将与FPLC技术结合使用以 提纯特定的蛋白质。足迹、凝胶延迟和 将使用体外转录重建试验来跟踪 这种蛋白质的活性。我们将调查这一模式 单细胞和感染细胞中纯化因子的相互作用 用E3DNA和E1a蛋白在体外确定 E1a修饰E3转录因子的性质 在腺病毒感染期间。因此，使用部分纯化的 转录系统，我们将尝试复制该调控 E1a对病毒基因E3的影响。对这一点的澄清 监管机制将澄清E1A的性质 与病毒和细胞基因的相互作用以及由此产生的病毒 诱发相变事件。

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.