REGULATION OF SV40 LATE TRANSCRIPTION BY LARGE T-ANTIGEN

在以前的实验中，我们已经证明了SV40晚期启动子 可在没有的情况下被猴病毒40(SV40)T抗原反式激活 DNA复制。激活可以通过两种方法中的一种来实现 晚期启动子与编码T抗原的质粒组的比较 将晚期启动子导入COS-1细胞，构件性表达 T抗原。在后一种情况下，尚不清楚 内源性T抗原是持续需要的，还是一组细胞 转录因子单独导致晚期启动子的激活。至 测试这些替代方案，我们已经在TS2中进行了转基因实验 COS细胞，表达TS1609 SV40T抗原。基因转染法 不允许的温度(40摄氏度)导致5到10倍 SV40晚期启动子活性与允许的相比降低 温度(32摄氏度)。 我们已经建立了体外转录系统来研究该基因 晚期启动子激活的机制。曼利全细胞提取液 从反式激活阳性的COS-1细胞系制备。预孵化 具有COS-1提取物的SV40 DNA模板的比例优先增加 转录自M.P.325的主要晚期起始点。激活 晚期启动子需要优化DNA模板浓度 COS-1和HeLa萃取物的比例，以及长度和温度 预孵化。在这些条件下，从 其他SV40晚期起始点或来自腺主要晚期启动子的是 没有被观察到。激活该基因的启动子序列要求 SV40晚期启动子与猪对SV40T抗原的需求 目前正在分析预孵化的COS-1提取物。

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.