REGULATION OF SV40 LATE TRANSCRIPTION BY LARGE T-ANTIGEN

大 T 抗原对 SV40 晚期转录的调控

• 批准号: 3963503
• 负责人: J BRADY
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963503
• 关键词: HeLa cells; gel electrophoresis; gene expression; genetic manipulation; genetic transcription; mutant; neoplasm /cancer immunology; plasmids; simian virus 40; transforming virus; viral carcinogenesis; virus DNA; virus antigen; virus genetics

In previous experiments, we have demonstrated that the SV40 late promoter could be trans-activated by simian virus 40 (SV40) T-antigen in the absence of DNA replication. Activation could be achieved by either cotransfection of the late promoter with a plasmid coding for T-antigen or by transfection of the late promoter into COS-1 cells which constitutively express T-antigen. In the latter case, it was not clear whether expression of the endogenous T-antigen was continuously required or whether a set of cellular transcription factors alone led to the activation of the late promoter. To test these alternatives, we have performed transfection experiments in ts2 COS cells, which express the ts 1609 SV40 T-antigen. Transfection at the non-permissive temperature (40 degrees C) resulted in 5- to 10-fold reduction in SV40 late promoter activity compared to the permissive temperature (32 degrees C). An in vitro transcription system has been developed in order to study the mechanism of late promoter activation. Manley whole cell extracts were prepared from the trans-activation-positive COS-1 cell line. Preincubation of an SV40 DNA template with the COS-1 extract preferentially increases transcription from the major late start site at m.p. 325. Activation of the late promoter required optimization of the DNA template concentration ratio of COS-1 and HeLa extracts, and the length and temperature of preincubation. Under these conditions, increased transcription from the other SV40 late initiation sites or from the adeno major late promoter was not observed. The promoter sequence requirements for activation of the SV40 late promoter and the requirement for SV40 T-antigen in the preincubation COS-1 extract are presently being analyzed.

在以前的实验中，我们已经证明SV 40晚期启动子 可以被猿猴病毒40（SV 40）T抗原反式激活， DNA复制。 激活可以通过共转染或 晚期启动子与编码T抗原的质粒或通过转染 COS-1细胞中的晚期启动子， T抗原 在后一种情况下，不清楚是否表达了 内源性T抗原是持续需要的，或者是否有一组细胞 转录因子单独导致晚期启动子的激活。 到 为了测试这些替代方案，我们在TS 2中进行了转染实验， COS细胞，其表达ts 1609 SV 40 T抗原。 转染在 非允许温度（40摄氏度）导致5至10倍 SV 40晚期启动子活性的降低 温度（32摄氏度）。 已经开发了一种体外转录系统，以研究 晚期启动子激活机制。 Manley全细胞提取物 由反式激活阳性COS-1细胞系制备。 预孵 与COS-1提取物的SV 40 DNA模板的结合优先增加 从m.p.325的主要晚期起始位点转录。 激活 晚期启动子需要优化DNA模板浓度 COS-1和HeLa提取物的比例，以及 预孵育 在这些条件下，增加转录从 其他SV 40晚期起始位点或来自腺主要晚期启动子的启动子， 未观察到。 启动子序列的激活要求， SV 40晚期启动子和SV 40 T抗原的需要， 目前正在分析预孵育COS-1提取物。