REGULATION OF TRANSCRIPTION BY A TUMOR PROMOTER

拟议研究的长期目标是了解 分子水平肿瘤促进剂，尤其是佛波酯 肿瘤促进剂12-o-十四酰-佛波酯(TPA)与 在细胞中产生转化表型的细胞和病毒基因 暴露在开始致癌的刺激下。这项提议将检验 TPA通过以下途径刺激特定基因转录的假说 改变RNA聚合酶II的活性或特异性，并将检查 这种效应的机制。圆满结束拟议中的 实验将为尝试识别和克隆提供理论基础 哺乳动物基因组中包含对病毒有反应的基因 TPA在体内的应用，并将为克隆的 调控序列。 克隆的病毒和细胞基因将在体外通过可溶性转录 全细胞提取物和部分纯化的RNA聚合酶II产生 特定启动子启动的“径流”产物 序列。经TPA处理的HeLa细胞和对照细胞的提取物将 比较人5型腺病毒和人腺病毒的转录活性 SV40早期和晚期基因，单纯疱疹病毒胸苷激酶基因， 禽类和小鼠乳腺肿瘤病毒长末端重复序列， 以及人类干扰素和β-珠蛋白基因。动力学分析 转录将包括总DNA和底物DNA的影响 浓度和不同的蛋白质浓度。混合实验 将评估整个细胞中是否存在可溶的调节因子 萃取物和粗提物的再分级将尝试 隔离这些因素。TPA作用与早期TPA的关系 对腺病毒Ela区编码的调节产物将进行研究 评估TPA抑制各种表型缺失的能力 突变体和它们的返回体，在埃拉地区。这些表型包括 病毒在Hela细胞中早期转录的刺激和转化 培养中的大鼠胚胎细胞(CREF)。

The long-term objective of the proposed study is to understand at the molecular level how tumor promoters, and in particular the phorbol ester tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA), interact with cellular and viral genes to produce a transformed phenotype in cells exposed to an initiating carcinogenic stimulus. This proposal will test the hypothesis that TPA stimulates the transcription of specific genes by altering the activity or specificity of RNA polymerase II, and will examine the mechanism of this effect. Successful conclusion of the proposed experiments will provide the rationale for attempting to identify and clone regions of the mammalian genome that contain genes that are responsive to TPA in vivo, and will also provide an in vitro test for the cloned regulatory sequences. Cloned viral and cellular genes will be transcribed in vitro by soluble whole cell extracts and partially-purified RNA polymerase II to yield "runoff" products characteristic of initiation at specific promoter sequences. Extracts from TPA-treated and control HeLa cells will be compared for transcriptional activity using human adenovirus type 5 and SV40 early and late genes, the thymidine kinase gene of herpes simplex, avian and mouse mammary tumor virus long terminal repeat (LTR) sequences, and human interferon and beta-globin genes. Kinetic analysis of transcription will include the effect of total- and substrate-DNA concentration and of varying protein concentration. Mixing experiments will assess the presence of soluble regulatory factors in the whole cell extracts, and subfractionation of the crude extracts will be attempted to isolate such factors. The relationship of TPA action to that of an early regulatory product encoded by the adenovirus Ela region will be studied by assessing the ability of TPA to suppress various phenotypes of deletion mutants, and their revertants, in the Ela region. These phenotypes include stimulation of early viral transcription in Hela cells, and transformation of rat embryo cells (CREF) in culture.