CONTROL OF MRNA SYNTHESIS IN MAMMALIAN CELLS

这项研究的长期目标是了解这些机制。 在哺乳动物细胞中合成mRNAs，并了解这是如何实现的 合成是受调控的。特别是，我们将专注于基因 来自两种细胞研究的DNA肿瘤病毒，SV40和腺病毒。 在这项研究中，我们将集中在几个方面的mrna。 综合。首先，我们将利用我们最近的观察，以外在的方式 添加的信使核糖核酸前体可以准确高效地在 HeLa全细胞裂解物。我们将详细研究遗传学和 这一重要反应的生物化学。第二，我们最近展示了 两个克隆的病毒启动子(AD2晚期和SV40早期)有很大的 不同的核苷酸序列要求才能在两个 不同的人类细胞系HeLa和293。我们将确定基因 这些作用的基础，以及研究生化机制 负责任。第三，我们将深入分析一系列删除和 我们在AD2晚期启动子中构建的点突变 更好地理解转录起始和转录的分子基础 监管。第四，我们将研究和推广一系列插入 倡导和规范。四是研究推广一系列 我们构建的插入突变体，其中AD2晚期启动子具有 已在SV40中的不同位置插入。这些插入物为我们提供了 对SV40中的SV40站点有重要的见解。这些插入是 为我们提供了对SV40基因表达和 一般情况下，转录控制。最后，使用我们准备的抗体 针对HeLa拓扑异构酶I，我们将研究DNA拓扑结构对 转录启动。

The long term goal of this research effort is to understand the mechanisms by which mRNAs are synthesized in mammalian cells, and to learn how this synthesis is regulated. In particular, we will be concentrating on genes from two cell-studied DNA tumor viruses, SV40 and adenovirus. In this research effort, we will concentrate on several on aspects mRNA synthesis. First, we will exploit our recent observation that exogenously added mRNA precursors can be accurately and efficiently polyadenylated in a HeLa whole-cell lysate. We will study in detail both the genetics and biochemistry of this important reaction. Second, we showed recently that two cloned viral promoters (Ad2 late and SV40 early) have drastically different nucleotide sequence requirements in order to be expressed in two different human cell lines, HeLa and 293. We will determine the genetic basis for these effects, as well as study the biochemical mechanism responsible. Third, we will analyze in depth a series of deletion and point mutations that we constructed in the Ad2 late promoter in order to better understand the molecular basis of transcription initiation and regulation. Fourth, we will study and extend a series of insertion initiation and regulation. Fourth, we will study and extend a series of insertion mutants that we constructed in which the Ad2 late promoter has been inserted at various sites in SV40. These insertions are providing us with important insights into SV40 sites in SV40. These insertions are providing us with important insights into SV40 gene expression and transcriptional control in general. Finally, using antibodies we prepared against HeLa Topoisomerase I, we will study the effects of DNA topology on transcription initiation.