REGULATION OF TRANSCRIPTION IN POLYOMA VIRUS

研究基因转录的机制及其调控 在多瘤病毒（PV）中。 病毒的调控区是 位于两组基因的编码序列之间 从可能重叠的启动子分化转录 感染的早期和晚期。 的主要目标 建议的项目是定义个人的元素 启动子，并了解转录的调控 这些基因。 后者涉及特定的DNA-蛋白质组， 启动子DNA序列之间的蛋白质-蛋白质相互作用， 细胞RNA聚合酶IL，细胞转录因子和 病毒调节蛋白T抗原。 目标是就以下 通过在启动子中系统地引入突变来实现 通过体外程序和测试启动子活性 在体内和体外在Hela细胞提取物中。 对于体内测定， 启动子将与氯霉素乙酰转移酶连接 （CATase）基因。在哺乳动物中产生CATase 细胞通常不存在。 的酶的量 将合成的量作为启动子活性的量度。 不同的细胞系，如小鼠（3 T6），未分化 胚胎癌（PCC 4，F9）和分化细胞（Friend 红白血病细胞）将用于体内表征 变种人 选择的“最佳”PV增强子结构为 对这些细胞系中的细胞因子最好的反应是 被定义。 早期的调节机制 转录和T细胞激活晚期转录的作用 将在体内和体外研究抗原。 两种可能的模式 这将是测试的切换机制，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 抗原结合到调节区B）无论T抗原 由于其在DNA复制中的作用，因此起间接作用。 的 参与转录的细胞因子将通过 竞争转录和DNA结合测定及其应用 模板上的接触点将由DNA酶确定 体外和体内保护实验，在不存在或 T抗原的存在。 与那些在 以裸DNA为模板，用PV进行体外培养 微型染色体在体外重建或从感染的 细胞或来自成熟病毒体。 序列导向的可能性 或蛋白质诱导的调节区弯曲。

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.