INVOLVEMENT OF T ANTIGEN IN SV40 LATE GENE EXPRESSION

拟议研究的长期目标是了解 在分子水平上探讨了大T抗原在晚期基因调控中的作用 SV 40的表达。 在此期间，我们将测试的有效性， 关于这个普遍问题的一些假设。 我们特别 将：（i）确定晚链是否需要大T抗原 通过在感染了一种突变的 SV 40不编码该蛋白质;（ii）确定晚链是否 RNA合成可以发生在病毒DNA复制的情况下， 用适当的限制性内切核酸酶， 在猴细胞和非洲爪蟾卵母细胞中合成这种RNA的突变体， 真正不能进行甚至一轮病毒DNA复制;（iii） 确定大T抗原是否直接增强晚期 通过与启动子调控区序列相互作用 通过检测各种T抗原缺陷和 启动子-调节区突变体的能力，使晚链 （iv）通过在WT-1中制备的RNA的5'末端的S1作图来确定。 和tsA 58感染的细胞中是否发生T抗原诱导的变化， 用于晚链RNA的转录起始位点中的裂解周期 合成;和（v）确定晚链转录 SV 40中存在“衰减剂”，通过S1映射寻找 （a）WT和突变体感染的猴子中具有适当3'末端的小RNA （B）在感染后不同时间注射的非洲爪蟾卵母细胞 WT和突变型SV 40 DNA，并在随后的实验中， 病毒编码的蛋白质 这些研究应该可以帮助我们了解 肿瘤抗原改变基因表达的机制， 有时可能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.