REGULATION OF TRANSCRIPTION BY A TUMOR PROMOTER

这项工作的长期目标是在 分子水平肿瘤促进剂尤其是佛波醇 十二烷基膦-13-乙酸酯(TPA)，相互作用 与细胞和病毒基因一起产生转化的表型 暴露在启动致癌刺激下的细胞。这项建议 继续检验TPA刺激 通过改变活性和/或改变特定基因的转录 RNA聚合酶II的特异性，并探讨其作用机制 这种效果。拟议中的实验将试图确定 调控途径的组成部分以及靶点 与转录过程直接相关的。成功 实验的结论将为体外试验提供实验依据 体内对TPA反应的克隆细胞序列的调节，以及 将为完成体外重建奠定基础 具有重要生物学意义的跨膜信号通路。 拟议的实验将表征以下方面的要求 对人5型基因体外转录的刺激 腺病毒(Ad5)即刻早期基因(E1a)激活 全细胞提取液和纯化后的内源性蛋白激酶C 利用纯化的RNA进行重组反应中的肌动蛋白 含有部分纯化的聚合酶II+细胞组分 转录因子。实验将尝试识别一种 粗提物中Ckinin的磷酸蛋白靶标； 刺激转录活性的纯化组分；在分离物上 Ad5感染细胞和体外转录复合体的研究 转录反应；以及从TPA处理后的色素体 细胞。来自标记转录复合体的磷蛋白将 用二维聚丙烯酰胺凝胶进行表征 电泳法和放射自显影术。肌酸激酶对血管紧张素转换酶的影响 其他TPA诱导基因的体外转录将被测试。

The long term objective of the work is to understand at the molecular level how tumor promoters in particular the phorbol ester 12otetradecanoylphorbol13acetate (TPA), interact with cell and virus genes to produce a transformed phenotype in cells exposed to an initiating carcinogenic stimulus. This proposal continues to test the general hypothesis that TPA stimulates the transcription of specific genes by altering the activity and/or specificity of RNA polymerase II, and to examine the mechanism of this effect. The proposed experiments will attempt to identify components of the regulatory pathway as well as targets associated directly with the process of transcription. Successful conclusion of the experiments will provide an in vitro test for the regulation of cloned cell sequences responsive to TPA in vivo, and will lay the groundwork for complete in vitro reconstruction of a biologically important transmembrane signaling pathway. The proposed experiments will characterize the requirements for stimulation of in vitro transcription of the type 5 human adenovirus (Ad5) immediate early gene (E1A) by activated endogenous protein kinase C in whole cell extracts and by purified Ckinase in reconstructed reactions using purified RNA polymerase II plus cell fractions containing partially purified transcription factors. Experiments will attempt to identify a phosphoprotein target for Ckinase in in crude extracts; in purified fractions that stimulate transcription activity; on isolate transcription complexes from Ad5infected cells and from in vitro transcription reactions; and from the chromain of TPAtreated cells. Phosphoproteins from labeled transcription complexes will be characterized by two dimensional polyacrylamide gel electrophoresis and autoradiography. The effect of Ckinase on in vitro transcription of other TPAinducible genes will be tested.