CHROMATIN EFFECTS ON RNA POLYMERASE II TRANSCRIPTION

一套精确的基因表达程序控制着许多 复杂的生物过程，如细胞生长，分化， 发展先行者的要求 阐明的机制， 正常对照可以中止，如在各种肿瘤和其他肿瘤中。 因此，需要对疾病有基本的了解， 基因表达的调控。 关键的一步是 调节可以发生在基因的转录水平， 大多数基因由RNA聚合酶II（pol II）介导。 染色质似乎在决定哪些细胞 基因由pol II转录。 本建议的目的是阐明一些 染色质控制转录的分子基础。 的 一种研究猿猴转录起始的方法 无细胞转录系统中的病毒（SV 40）微型染色体， 其中转录因子和模板（都是DNA） 序列和染色质组分）可以独立地改变 并受到监控。 SV40是一个理想的研究体系， 染色质对RNA聚合酶II转录的影响。 微型染色体可以从以下生物化学量中分离： 感染和转染的细胞，用于分析和改变， 体外 此外，SV40基因表达的一些调控 很可能取决于微型染色体的结构 （由增强子和可能的T抗原调节）。 等 监管尚未准确复制， 脱蛋白DNA作为体外转录反应的载体。 研究转录从头起始的可行性 SV40微型染色体上的SV40早期和晚期启动子 已经证明了使用HeLa全细胞 细胞提取物或部分纯化的 转录因子 这项建议的具体目标是： 1)为了表征SV 40微型染色体的群体， 有能力在体外启动转录，2）确定 所述细胞转录因子稳定地与 分离的微型染色体，以及这些复合物是否形成 稳定的转录前起始复合物，3）以确定 SV40增强子序列对启动 在微型染色体上的SV40启动子处转录，以及4） 为了检测SV40 T抗原是否可以刺激 在微型染色体上的晚期SV40启动子处转录。

A set of precise programs of gene expression control many complex biological processes, such as cell growth, differentiation, and development. An elucidation of the mechanisms by which the normal controls can be aborted, as in various neoplasms and other diseases, will therefore require a basic understanding of the regulation of gene expression. One pivotal step at which regulation can occur is at the level of transcription of the gene, mediated for most genes by RNA polymerase II (pol II). Chromatin appears to play a major role in determining which genes are transcribed by pol II. The objective of this proposal is to elucidate some of the molecular bases for control of transcription by chromatin. The approach is to study the initiation of transcription from Simian Virus (SV40) minichromosomes in a cell-free transcription system, where the transcription factors and the template (both DNA sequence and chromatin components) can be independently altered and monitored. SV40 is an ideal system with which to study the effects of chromatin on RNA polymerase II transcription. Minichromosomes can be isolated in biochemical quantities from both infected and transfected cells, for analysis and alteration in vitro. In addition, some of the regulation of SV40 gene expression is likely to be dependent on the structure of the minichromosomes (regulation by the enhancer, and possibly T antigen). Such regulation has not yet been accurately reproduced with deproteinized DNA as a for in vitro transcription reactions. The feasibility of studying de novo initiation of transcription from both the SV40 early and late promoters on SV40 minichromosome templates in vitro has been demonstrated using either HeLa whole cell extracts or a reconstituted system of partially purified transcription factors. The specific objectives of this proposal are: 1) to characterize the population of SV40 minichromosomes which are competent to initiate transcription in vitro, 2) to determine which cellular transcription factors are stably complexed with the isolated minichromosomes, and whether these complexes form stable preinitiation complexes for transcription, 3) to determine the effect of the SV40 enhancer sequences on initiation of transcription at the SV40 promoters on minichromosomes, and 4) to examine whether the SV40 T antigen can stimulate initiation of transcription at the late SV40 promoter on minichromosomes.