PRODUCTS OF THE TRANSFORMING GENES OF POLYOMA VIRUS

该提案涉及多瘤病毒所需的基因产物 病毒生长和肿瘤转化。我们的目标是了解 大T和中T抗原在这两个过程中都起作用。这包括 它们与其他细胞通路的元件相互作用， 调节细胞生长。我们强调磷酸化，因为 磷酸化似乎是它们功能的中心。我们的方法是既 生化和遗传。 大T抗原，对DNA复制和RNA很重要 转录，有一个复杂的磷酸化模式，突变体 分析与功能有关。我们将继续确认 磷酸化位点。随着每个地点的确定，它将受到 到寡核苷酸突变。我们将表达和研究这些性质 该蛋白的C-末端结构域。我们将探索该体系结构 大T分子。将进行标签实验，以 确定N-端结构域和C-端结构域之间的关键接触点， 在低聚物中的大T之间以及大T与DNA之间。第二站点 回复变种将被寻求作为一种基因方式来确定重要的 互动。对于生化研究，我们将表达重要的 使用杆状病毒载体的突变体和C-末端结构域。 对于中期T，我们将完成对 丝氨酸/苏氨酸磷酸化位点。磷脂酰肌醇-3-激酶是 T中的目标与转型联系最紧密。它有很宽的 一般意义，因为它与其他癌基因和 增长因素。我们将继续对这种酶进行分析。此外 制定出它与中T的联系细节，我们将评估 这种酶是否足以调节细胞生长。因为一个 NPXY序列中与定位相关的突变 涂布的凹坑使中T不转化，内体实验 将对中T进行目标定位。中T对儿童心理健康的影响 还将对内化和内体贩运进行测试。 多发性肿瘤T抗原与其他途径的相互作用 对细胞生长的调控也将受到考验。看起来像是一个 中间T转化需要发挥作用的ras途径，因此 我们将研究这条途径的要素。野生型P53为阴性 SV40转化失活的细胞生长调节因子；我们将 确定细胞的多瘤转化是否受P53和 P53是否受多瘤病毒的影响。

This proposal concerns the gene products of polyoma virus required for viral growth and neoplastic transformation. Our goal is to understand how the large T and middle T antigens act in both processes. This includes their interactions with elements of other cellular pathways which are, regulatory for cell growth. We emphasize phosphorylation, because phosphorylation appears central to their function. Our approach is both biochemical and genetic. Large T antigen, which is important for DNA replication and RNA transcription, has a complicated pattern of phosphorylation that mutant analysis relates to function. We will continue identification of the phosphorylation sites. As each site is identified, it will be subjected to oligonucleotide mutagenesis. We will express and study the properties of the C-terminal domain of the protein. We will probe the architecture of the large T molecule. Labeling experiments will be carried out to determine the key contact points between the N- and C-terminal domains, between large T in oligomers and between large T and DNA. Second-site revertants will be sought as a genetic way to determine important interactions. For the biochemical studies, we will express important mutants and the C-terminal domains using baculovirus vectors. For middle T we will complete the analysis and mutagenesis of the serine/threonine phosphorylation sites. Phosphatidylinositol-3-kinase is the target of middle T most closely linked with transformation. It has wide general significance because of its association with other oncogenes and growth factors. Our analysis of this enzyme will continue. Besides working out the details of its association with middle T, we will assess whether this enzyme is sufficient to regulate cell growth. Because a mutation in an NPXY sequence that has been associated with localization to coated pits renders middle T non-transforming, experiments on the endosomal targeting of middle T will be carried out. The effects of middle T on internalization and endosomal trafficking will also be tested. The interactions of polyoma T antigens with other pathways which are regulatory for cell growth win also be tested. It appears that a functioning ras pathway is required for middle T transformation, so elements of that pathway will be examined. Wild type p53 is a negative regulator of cell growth inactivated by SV40 transformation; we will determine whether polyoma transformation of cells is affected by p53 and whether p53 is in turn affected by polyoma virus.