Visualizing Genetic Activity in Normal and Mutant Yeast

这项拟议研究的长期目标是更好地了解受调控的 核糖体和信使RNA的产生。在鉴定和鉴定 参与RNA聚合酶I和II转录和RNA加工的蛋白质，但有许多 关于基本机制需要回答的重要问题。我们独特的方法提供了一种方法来 通过使用米勒染色质扩散直接显示活性基因来回答其中一些问题 电子显微镜检查方法。简而言之，以酿酒酵母为模型系统，我们可视化地 在存在和不存在关键调控蛋白的情况下(即在适当的突变体中)特定基因 确定基因在缺失的情况下是如何改变的。我们能够察觉(和区分)对……的影响 转录的起始、延伸、终止、新生转录本的加工、染色质结构 模板拓扑。此外，对于这些主题中的每一个，我们都能够解析提供信息的模式 关于受影响的分子机制。我们最近的成果使我们专注于以下目标： (目的1)确定组蛋白和染色质相关的延伸因子在RNA延伸中的作用 聚合酶I；(目的2)分析rDNA在缺乏DNA时出现的拓扑异常 拓扑异构酶，并询问它们是否以及如何与在这些异构酶中发生的增加的重组相关 突变体；以及(目标3)开发和应用一个用于特定POL II基因可视化和分析的系统 共同转录事件。这些研究在以下方面与人类健康相关：我们将获得 核糖体产生调控的信息，这对细胞生长控制很重要，并正在上升- 在癌症中调控；(B)我们将获得关于转录延长和 剪接，其中的突变导致许多不同的人类疾病，以及(C)我们可以获得直接的信息 通过研究拓扑异构酶缺陷细胞的这一过程来探讨基因组不稳定性的机制。

The long-term goal of the proposed research is to better understand molecular mechanisms in the regulated production of ribosomal and messenger RNA. Huge advances have been made in the identification of proteins involved in transcription and RNA processing for RNA polymerase I and II, yet there are many important questions to be answered concerning basic mechanisms. Our unique approach offers a means to answer some of these questions by direct visualization of active genes using the Miller chromatin spreading method for electron microscopy. In brief, using Saccharomyces cerevisiae as a model system, we visualize specific genes in the presence and absence of key regulatory proteins (i.e., in appropriate mutants) to determine how the gene is changed in its absence. We are able to discern (and distinguish) effects on transcription initiation, elongation, termination, processing of nascent transcripts, chromatin structure, and template topology. Furthermore, for each of these topics, we are able to resolve patterns that are informative regarding the molecular mechanism affected. Our recent results have led us to focus on the following Aims: (Aim 1) Determine the role of histones and chromatin-associated elongation factors in elongation by RNA polymerase I; (Aim 2) Analyze topological abnormalities in rDNA that appear in the absence of topoisomerases and ask if and how these correlate with the increased recombination that occurs in these mutants; and (Aim 3) Develop and apply a system for the visualization of specific Pol II genes and analysis of co-transcriptional events. These studies are relevant to human health in the following ways: we will gain information on the regulation of ribosome production, which is important to cell growth control and is up- regulated in cancer; (b) we will gain information about the mechanisms of transcription elongation and splicing, mutations in which lead to many different human diseases, and (c) we may gain direct information on mechanisms of genomic instability by studying this process in topoisomerase-deficient cells.