BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

腺病毒2(AD2)左端编码蛋白 名为E1a的转录单位的基因组极大地刺激 转录自至少9个已知AD2启动子中的7个 由RNA聚合酶II转录。类似 病毒蛋白的“反式激活”功能已被描述 在感染后立即在一些DNA病毒中表达 系统。E1a功能也是该过程的核心 AD2的致癌转化，是一种 癌基因产品类别，包括多发性肿瘤和SV40 T- 抗原，myc，N-myc和p53，所有核蛋白都可以 协同c-Ha-ras-1癌基因彻底转化 初级胚胎细胞。 这里提出的研究的主要目标是 了解E1a蛋白的分子机制 从两个AD2启动子E1B和IX刺激转录 推动者。E1B启动子在启动前后都是活跃的 病毒DNA在允许细胞和转化细胞中的复制 细胞。我们未发表的关于突变的研究结果 在E1B启动子区域构建表明E1B 启动子异常简单，只有一个结合 Sp1转录因子(Tf)和TATA盒的位置。不是 E1a似乎需要特定的序列 激活。来自IX启动子的转录只是 观察病毒DNA在允许细胞和 在转化的细胞中没有观察到。然而，IX的顺序 启动子表明，它也包含一个关闭的Sp1位点 离塔塔箱很近。这就提出了一个问题： 导致这两种AD2时间调节不同的原因 推动者。我们建议完成对该基因的突变分析 E1B启动子并对IX进行类似的分析 推动者。我们将检测TFS和其他物质的浓度 序列特异的DNA结合蛋白，与 通过突变研究确定的启动子元件，他们的 启动子序列的亲和力及其比活性 用于体外转录感染HeLa细胞的提取物 带有AD2和E1a-突变体。与这些启动子结合的蛋白质 体内元素将在感染AD2，E1a-的细胞中进行检测 突变，以及E1B和IX启动子突变。这些研究 应该为转录的机制提供一些洞察力 在这些启动子的启动，它的刺激由E1a蛋白， 以及IX启动子的时间调控。最终的具体目标 是设计和构建温度敏感型E1a蛋白。 这些E1a蛋白将成为研究人类免疫缺陷的有用工具。 E1a蛋白的反式激活和转化。

Proteins encoded at the left end of the adenovirus 2 (Ad2) genome in a transcription unit called E1A greatly stimulate transcription from at least seven of nine known Ad2 promoters for transcription by RNA polymerase II. Similar "transactivating" function have been described for viral proteins expressed immediately after infection in a number of DNA virus systems. E1A functions are also central to the process of oncogenic transformation by Ad2, and are representative of a class of oncogene products including polyoma and SV40 T- antigens, myc, N-myc and p53, all nuclear proteins which can cooperate with the c-Ha-ras 1 oncogene to completely transform primary embryo cells. The principal goals of the research proposed here are to understand the molecular mechanisms by which E1A proteins stimulate transcription from two Ad2 promoters, the E1B and IX promoters. The E1B promoter is active both before and after viral DNA replication in permissive cells and in transformed cells. Results of our unpublished studies on mutations constructed in the E1B promoter region suggest that the E1B promoter is unusually simple, being comprised of a single binding site for the Sp1 transcription factor (TF) and a TATA-box. No specific sequences appear to be required for E1A transactivation. Transcription from the IX promoter is only observed following viral DNA replication in permissive cells and is not observed in transformed cells. Yet the sequence of the IX promoter suggests that it too contains a single Sp1 site in close proximity to a TATA-box. This raises the question of what causes the difference in temporal regulation of these two Ad2 promoters. We propose to complete a mutational analysis of the E1B promoter and to perform a similar analysis of the IX promoter. We will assay the concentrations of TFs and other sequence specific DNA binding proteins which interact with the promoter elements identified by the mutational studies, their affinities for promoter sequences, and their specific activities for in vitro transcription in extracts from HeLa cells infected with Ad2 and E1A-mutants. Protein binding to these promoter elements in vivo will be assayed in cells infected with Ad2, E1A- mutants, and E1B and IX promoter mutants. Theses studies should provide some insight into the mechanism of transcription initiation at these promoters, its stimulation by E1A proteins, and temporal regulation of the IX promoter. A final specific aim is to design and construct temperature sensitive E1A proteins. Such ts E1A proteins would be useful tools in the study of transactivation and transformation by E1A proteins.