Visualizing Genetic Activity in Normal and Mutant Yeast

描述(由申请人提供)：拟议研究的长期目标是更好地了解核糖体和信使RNA调节生产的分子机制。在鉴定参与RNA聚合酶I和II转录和RNA加工的蛋白质方面取得了巨大的进展，但关于基本机制仍有许多重要的问题需要回答。我们独特的方法提供了一种通过使用电子显微镜的米勒染色质铺展方法直接显示活性基因来回答其中一些问题的方法。简而言之，使用酿酒酵母作为模型系统，我们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)可视化特定的基因，以确定基因在缺乏的情况下是如何变化的。我们能够辨别(和区分)对转录开始、延伸、终止、新生转录的处理、染色质结构和模板拓扑的影响。此外，对于这些主题中的每一个，我们都能够解析出关于受影响的分子机制的信息模式。我们最近的结果使我们专注于以下目标：(目标1)确定组蛋白和染色质相关的延伸因子在RNA聚合酶I延长中的作用；(目标2)分析在没有拓扑异构酶的情况下出现的rDNA拓扑异常，并询问这些异常是否与这些突变体中发生的重组增加有关；以及(目标3)开发和应用一种系统，用于特定POL II基因的可视化和共转录事件的分析。这些研究通过以下方式与人类健康有关：(A)我们将获得有关核糖体产生调控的信息，这对细胞生长控制很重要，并且在癌症中上调；(B)我们将获得有关转录延长和剪接机制的信息，在这些机制中，突变会导致许多不同的人类疾病；以及(C)我们可以通过研究拓扑异构酶缺陷细胞的这一过程，获得关于基因组不稳定机制的直接信息。

DESCRIPTION (provided by applicant): 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.