PROTEIN INTERACTIONS AND REPRESSION FUNCTION OF THE E1A

这项建议的长期目标是了解生物化学 将正常静息细胞转化为恶性细胞的步骤 特点。这项建议的重点是 腺病毒E1a癌基因可诱导静止的原代细胞增殖。 最近的证据表明，这一过程与E1a结合有关。 未知的300 kDa蜂窝产物。这个项目的具体目标是 通过(1)提纯和提纯对300 kDa的产物进行表征 抗体；(2)克隆和高效表达；(3)鉴定其特性 翻译后修饰，以及(4)确定其内在修饰性 生物活动。 P300的鉴定和特性将主要集中在 获得识别该产品的特定抗体。当抗体出现时 或获得一些序列后，对文库进行筛选以鉴定 P300的克隆。P300克隆分离和表达将允许 更广泛的表征，包括体外诱变，以研究其 生物活性。该菌的生化和生物学特性 300 kDa蛋白质的特征将特别强调性质 与细胞生长控制和基因表达调控有关。这个 300 kDa产物是一种磷酸蛋白，它似乎经历了 有丝分裂期间的过度磷酸化(初步结果)。这个 将绘制间期和有丝分裂p300的磷酸化模式 使用磷酸多肽分析等技术。最终， 磷酸化事件将与生物活性相关。这个 本提案的总体目标是确定什么是生物 P300的活性使其成为E1A细胞生长调节的明显靶点 活动。获得纯化的p300将使我们能够测试其内在 生物活性。例如，我们将确定它是否是DNA 结合蛋白。我们将确定p300的亚细胞定位， 以及在各种正常和异常组织中的蛋白表达水平 受调控的细胞。P300与其他细胞间可能的相互作用 将对产品进行调查，以确定可能影响 P300功能。 最近的发现(1989年在格林杂志上回顾)强调了 E1a转化功能与人类肿瘤的关系这个 腺病毒的转化机制与病毒的转化机制密切相关 人类乳头瘤病毒，与人类恶性肿瘤有关。E1a协会 与视网膜母细胞瘤蛋白的关系提供了进一步的证据 与E1a相关的300kodalton产品将促进我们对 人类肿瘤发生学。

The long term objective of this proposal is to understand the biochemical steps which transform a normal resting cell to one with malignant characteristics. This proposal focuses on the mechanism by which the Adenovirus E1A oncogene induces proliferation of quiescent primary cells. Recent evidence indicates that this process is related to E1A binding of an unidentified 300kDa cellular product. The specific aims of this project are to characterize the 300kDa product by (1) purifying and raising antibodies to it; (2) cloning and overexpressing it; (3) characterizing its posttranslational modifications, and (4) determining its intrinsic biological activities. The identification and characterization of p300 will focus largely on obtaining specific antibodies that recognize this product. When antibodies or some sequence are obtained, cDNA libraries will be screened to identify a p300 clone. The isolation and expression of a p300 clone will permit more extensive characterization including in vitro mutagenesis to study its biological activity. The biochemical and biological properties of the 300kDa protein will be characterized with particular emphasis on properties related to cell growth control and regulation of gene expression. The 300kDa product is a phosphoprotein which appears to undergo specific hyperphosphorylation during mitosis (preliminary results). The phosphorylation patterns of interphase and mitotic p300 will be mapped using techniques such as phosphopeptide analysis. Ultimately, phosphorylation events will be correlated with biological activity. The overall goal of the present proposal is to determine what biological activity of p300 makes it an apparent target for E1A cell growth regulating activity. Obtaining purified p300 will allow us to test its intrinsic biological activity. We will determine, for example, whether it is a DNA binding protein. We will determine the subcellular localization of p300, and the level of protein expression in various normal and aberrantly regulated cells. Possible interactions between p300 and other cellular products will be probed to identify cellular factors that may influence p300 function. Recent discoveries (reviewed in Green, 1989) have emphasized the relationship between E1A transforming functions and human cancer. The transforming mechanisms of adenoviruses are closely related to those of the human papillomaviruses, implicated in human malignancies. E1A association with the retinoblastoma protein provides further evidence that study of the E1A-associated 300-kilodalton product will promote our understanding of human tumorigenesis.