PRODUCTS OF THE TRANSFORMING GENES OF POLYOMA VIRUS

该提案涉及多瘤病毒所需的基因产物 病毒的生长和转化。我们特别关注的是如何 合成后修饰调节蛋白质功能。因此，我们 集中在大T和中T，这两种都是磷蛋白。我们的 目标是了解这些蛋白质如何发挥作用以及如何磷酸化。 有助于实现这一功能。我们的方法既是生化的，也是遗传的。 中间T与pp60c-src结合，pp60c-src是转化的细胞同源物 劳斯肉瘤病毒的蛋白质。很可能中T的动作 这种相互作用导致了转变。我们将直接对此进行测试 通过创建pp60c-src突变体来阻断中T介导的 转型。我们将探索这两种蛋白质之间的相互作用。 含有pp60c-src和pp60v-src序列的杂化分子 Pp60v-src突变体。生化证据显示中T丝氨酸 与pp60c-src结合需要磷酸化。这将是 直接测试。丝氨酸磷酸化位点将被确定。 然后，寡核苷酸突变将被用来创造缺乏 磷酸化位点。这些突变体的生物学和生化将 可以与野生型相比较。中间的T/PP-60C-SrC络合物具有 酪氨酸激酶活性；细胞蛋白的酪氨酸磷酸化 已在RSV转化的细胞中得到证实。然而，这些综合体也 具有磷脂酰肌醇激酶活性。涉及到的实验 钙的释放和蛋白激酶C的激活将进行 确定体内磷脂酰肌醇代谢的变化是否 对转型很重要。 大T抗原，对DNA复制和RNA很重要 转录，是研究非组蛋白染色体蛋白的一种有价值的模式。 使用ts-a和hr-t突变体，我们已经获得了一些证据表明 功能可能需要磷酸化。这些地点的地图和 突变将被用来检验这一假设。磷酸化模式 将在不同的细胞和不同的亚细胞位置进行检查 试图评估大T的其他磷酸化的重要性。 在这些分析中，我们将使用野生型病毒和大量的 大型T突变体现已上市。

This proposal concerns the gene products of polyoma virus required for virus growth and transformation. We are particularly concerned with how post-synthetic modifications regulate protein function. We therefore concentrate on large T and middle T, which are both phosphoproteins. Our goal is to learn how these proteins function and how phosphorylation contributes to that function. Our approach is both biochemical and genetic. Middle T associates with pp60c-src, the cell homologue of the transforming protein of Rous sarcoma virus. It is likely that the action of middle T in transformation results from this interaction. We will test this directly by creating mutants of pp60c-src designed to block middle T mediated transformation. We will probe the interaction between these two proteins with hybrid molecules containing pp60c-src and pp60v-src sequences and with pp60v-src mutants. Biochemical evidence suggests middle T serine phosphorylation is required for association with pp60c-src. This will be tested directly. The serine phosphorylation sites will be identified. Oligonucleotide mutagenesis will then be used to create mutants lacking the phosphorylation sites. The biology and biochemistry of these mutants will be compared to wild type. The middle T/pp-60c-src complex possesses tyrosine kinase activity; tyrosine phosphorylation of cell proteins has been demonstrated in RSV-transformed cells. However, the complexes also possess phosphatidylinositol kinase activity. Experiments involving calcium release and protein kinase C activation will be carried out to decide whether in vivo alterations in phosphatidylinositol metabolism are important for transformation. Large T antigen, which is important for DNA replication and RNA transcription, is a valuable model for non-histone chromosomal proteins. Using ts-a and hr-t mutants, we have obtained evidence that some of the phosphorylations may be required for function. Mapping of the sites and mutagenesis will be used to test this hypothesis. Phosphorylation patterns will be examined in different cells and different subcellular locations to to try to assess the importance of the other phosphorylations of large T. In these analyses, we will use both wild type virus and the large number of large T mutants now available.