INVOLVEMENT OF T ANTIGEN IN SV40 LATE GENE EXPRESSION

拟议研究的长期目标是了解 分子水平上大T抗原在晚期基因调控中的作用 SV40的表达。在这笔赠款期间，我们将测试 与此一般问题相关的假设数量。具体来说，我们 将：(I)确定晚期链是否需要大T抗原 通过在感染突变体的细胞中寻找这种RNA来合成RNA 不编码该蛋白的SV40；(Ii)确定晚期链是否 通过检测，在没有病毒DNA复制的情况下，RNA合成可以发生 有适当限制的核酸内切酶是否存在复制缺陷 在猴子细胞和非洲爪哇卵母细胞中合成这种RNA的突变体是 真的无法进行一轮病毒DNA复制；(Iii) 确定大T抗原是否直接促进晚期细胞的合成 链RNA通过其与启动子调节区序列的相互作用 通过检测各种T抗原缺陷和 启动子-调控区突变体制造晚期链的能力 RNA；(Iv)通过S1作图确定在WT中制造的RNA的5‘端- 以及tsA58感染的细胞在T抗原诱导的变化过程中是否发生 晚期链RNA转录起始点的裂解循环 合成；以及(V)确定晚期链是否转录 在SV40中，通过S1映射查找是否存在 (A)WT和突变感染的猴子体内含有适当3‘端的小RNA 感染后不同时间的细胞；和(B)注射非洲爪哇卵母细胞 用WT和突变的SV40DNA，在后来的实验中，准备 病毒编码的蛋白质。这些研究应该有助于我们了解 肿瘤抗原通过以下方式改变基因表达的机制 有时可能会导致细胞转化为致癌状态。

The long-term objective of the proposed research is to understand at the molecular level the roles played by large T antigen in regulating late gene expression of SV40. During this grant, we will test the validity of a number of hypotheses relating to this general problem. Specifically, we will: (i) determine whether large T antigen is required for late strand RNA synthesis by looking for this RNA in cells infected with a mutant of SV40 that does not encode this protein; (ii) determine whether late strand RNA synthesis can occur in the absence of viral DNA replication by testing with appropriate restriction endonucleases whether replication-defective mutants that synthesize this RNA in monkey cells and Xenopus oocytes are truly unable to undergo even a single round of viral DNA replication; (iii) determine whether large T antigen directly enhances synthesis of late strand RNA via its interactions with promoter-regulatory region sequences on the viral genome by examining a variety of T antigen-defective and promoter-regulatory region mutants for their ability to make late strand RNA; (iv) determine by S1 mapping of the 5' ends of the RNAs made in WT- and tsA58-infected cells whether T antigen-induced changes occur during the lytic cycle in the transcription initiation sites used for late strand RNA synthesis; and (v) determine whether a late strand transcriptional "attenuator" exists in SV40 by looking by S1 mapping for the presence of small RNAs with appropriate 3' ends in (a) WT- and mutant-infected monkey cells at various times after infection; and (b) Xenopus oocytes injected with WT and mutant SV40 DNAs and, in later experiments, preparations of virus-coded proteins. These studies should help us to learn about mechanisms by which tumor antigens can alter gene expression in ways that may on occasion lead to the transformation of cells to an oncogenic state.