REGULATION OF TRANSCRIPTION IN POLYOMA VIRUS

将研究基因转录的机制及其调控。 多瘤病毒(PV)。该病毒的监管区域是 位于两组基因的编码序列之间 与可能重叠的启动子不同地转录 感染的早期和晚期。该计划的主要目标 建议的项目是定义个人的要素 启动子和了解转录的调节 这些基因。后者涉及特定的DNA-蛋白质组和 启动子DNA序列之间的蛋白质相互作用， 细胞RNA聚合酶IL、细胞转录因子与 病毒调节蛋白T抗原。目标将是 通过系统地在启动子中引入突变来实现 区域通过体外程序和通过测试启动子的活性 体内和体外Hela细胞提取物。对于体内试验， 启动子将与氯霉素乙酰转移酶相连 大肠杆菌(CATase)基因在哺乳动物体内生成CATase 通常不存在的单元格。酶的量 合成的产物将被作为启动子活性的衡量标准。 不同的细胞系，如小鼠(3T6)，未分化 胚胎癌(PCC4、F9)和分化细胞(Friend 红白血病细胞)将用于体内表征 变种人。最佳光伏增强剂结构被选为 在这些细胞系中，对细胞因子(S)反应最好的是 被定义。早期的调控机制 转录与T激活晚期转录的关系 抗原将在体内和体外进行研究。两种可能的模式 这将测试切换机制，从“早期- 从“早”到“晚-早”或从“早”到“晚” 转录是a)这种转变是否是T的直接影响 与调节区b)结合的抗原是否为T抗原 由于它在DNA复制中的作用，它起着间接的作用。这个 参与转录的细胞因子(S)将通过 竞争转录和DNA结合试验及其应用 模板上的接触点将由DNA酶确定 体外和体内保护实验，在没有或 T抗原的存在。与年进行的实验平行 将以裸DNA为模板的体外实验将与PV进行 在体外重组的微染色体或从感染病毒中分离出来的微染色体 细胞或成熟的病毒粒子。序列定向的可能性 或者蛋白质诱导的调控区域的弯曲将被测试。

Mechanism of gene transcription and its regulation will be studied in polyoma virus (PV). The regulatory region of the virus is located between the coding sequences of two sets of genes transcribed divergently from presumably overlapping promoters early and late in infection. The primary objectives of the proposed project are to define the elements of individual promoter and to understand the regulation of transcription of these genes. The latter involves specific sets of DNA-protein and protein-protein interactions among promoter DNA sequences, cellular RNA polymerase IL, cellular transcription factors and the viral regulatory protein T antigen. The objectives will be achieved by introducing mutations systematically in the promoter region by in vitro procedures and by testing the promoter activity in vivo and, in vitro in Hela Cell extracts. For in vivo assay, the promoter will be linked to the chloramphenicol acetyltransferase (CATase) gene of E. coli to make the CATase in the mammalian cell where it is normally absent. The amount of the enzyme synthesized will be taken as a measure of the promoter activity. Different cell lines, such as mouse (3T6), undifferentiated embryonal carcinoma (PCC4, F9) and differentiated cells (Friend erythroleukemic cell) will be used for in vivo characterization of the mutants. The "optimal" PV enhancer structure selected as the one best responding to the cellular factor(s) in these cell lines will be defined. The mechanism of regulation of the early transcription and that of the activation of late transcription by T antigen will be studied in vivo and in vitro. Two possible models that will be tested for the switch-over mechanism from "early- early" to the "late-early" or from the "early" to "late" transcription are a) whether this shift is a direct effect of T antigen binding to the regulatory region b) whether T antigen plays an indirect role because of its role in DNA replication. The cellular factor(s) involved in transcription will be identified by competition transcriptional and DNA binding assay and their points of contact on the template, will be determined by DNase protection experiments in vitro and in vivo, in absence or presence of T antigen. Experiments parallel to those conducted in vitro with naked DNA as template will be carried out with PV minichromosome reconstituted in vitro or isolated from infected cells or from mature virions. The possibility of sequence-directed or protein-induced bending of the regulatory region will be tested.