PRODUCTS OF THE TRANSFORMING GENES OF POLYOMA VIRUS

本提案涉及所需的多瘤病毒基因产物。 对病毒生长和恶性细胞转化都有作用。我们将寻求 定义体内合成后修饰的作用，如 调节这些病毒基因产物功能的磷酸化。 这种方法将既是生化的，也是遗传的。目标将是 定义特定的修饰位点，以准备缺乏个体的突变体 并比较了突变和野生型蛋白的差异。 从生物学和生化的角度来看。具体目标包括：1) 大T蛋白和大T蛋白体内磷酸化位点的鉴定 中T抗原，2)可使用的免疫试剂的制备 检测每个修饰位点，并可用于分离变性T细胞 抗原，3)大T和中T的磷酸化分析 不同的细胞和不同的亚细胞位置，4)创建 缺乏单独修饰位点的病毒突变体使用技术 定点突变；5)突变体与野生型的比较 蛋白质的生物学功能(生长和转化) 和生化性质(酶活性、DNA结合、膜 本地化)。

This proposal concerns the products of the genes of polyoma virus required both for virus growth and malignant cell transformation. We shall seek to define the role of post-synthetic in vivo modifications such as phosphorylation in regulating the function of these viral gene products. The approach will be both biochemical and genetic. The goal will be to define specific modification sites, to prepare mutants that lack individual sites, and to compare the mutant and wild type proteins from both a biological and a biochemical point of view. Specific aims include: 1) identification of the sites of in vivo phosphorylation of large T and middle T antigen, 2) preparation of immunologic reagents that can be used to detect each modification site and can be used to isolate denatured T antigens, 3) analysis of phosphorylation of large T and middle T in different cells and in different subcellular locations, 4) creation of viral mutants which lack individual modification sites using techniques of site specific mutagenesis, and 5) comparison of the mutant and wild type proteins in terms of both biological function (growth and transformation) and biochemical properties (enzymatic activities, DNA-binding, membrane localization).