REGULATION OF TRANSCRIPTION BY A TUMOR PROMOTER

这项工作的长期目标是了解 分子水平上肿瘤促进剂，特别是佛波醇 酯12十四酰基佛波醇13乙酸酯（TPA），相互作用 与细胞和病毒基因一起产生转化表型， 暴露在致癌刺激物中的细胞。 这项建议 继续测试一般假设，TPA刺激 - 通过改变活性来转录特定基因，和/或 RNA聚合酶II的特异性，并检查其机制 这种效果。 拟议的实验将试图确定 调节途径的组分以及靶点 与转录过程直接相关。 成功 实验的结论将提供一种体外试验， 在体内调节响应TPA的克隆细胞序列，和 将为体外完全重建 生物学上重要的跨膜信号通路。 拟议的实验将描述以下要求： 5型人的体外转录的刺激 腺病毒（Ad 5）立即早期基因（E1 A） 内源性蛋白激酶C的全细胞提取物和纯化 使用纯化RNA的重构反应中的激酶 聚合酶II加上含有部分纯化的 转录因子 实验将试图确定一个 粗提物中的C激酶的磷蛋白靶; 刺激转录活性的纯化级分;分离 从Ad 5感染的细胞和从体外 转录反应;和从TPA处理的染色质 细胞 来自标记的转录复合物的磷蛋白将 以二维聚丙烯酰胺凝胶为特征 电泳和放射自显影。 C激酶对 将测试其它TPA诱导基因的体外转录。

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.