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调控生产的分子机制。RNA聚合酶I和RNA聚合酶II参与转录和RNA加工的蛋白质鉴定已经取得了巨大进展，但在基本机制方面仍有许多重要问题有待解答。我们独特的方法提供了一种方法，通过使用米勒染色质扩散方法在电子显微镜下直接可视化活性基因来回答这些问题。简而言之，使用酿酒酵母作为模型系统，我们可视化特定基因在关键调控蛋白存在和缺失的情况下（即，在适当的突变体中），以确定基因在缺乏时如何变化。我们能够辨别（和区分）对转录起始、延伸、终止、新生转录本处理、染色质结构和模板拓扑结构的影响。此外，对于每一个主题，我们都能够解析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延伸因子在RNA聚合酶I的延伸中的作用；（目标2）分析在缺乏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以及如何与这些突变体中发生的重组增加相关；（目标3）开发和应用一个系统，用于特定Pol II基因的可视化和共转录事件的分析。这些研究在以下方面与人类健康相关：我们将获得关于核糖体产生调控的信息，核糖体对细胞生长控制很重要，在癌症中被上调；(b)我们将获得转录延伸和剪接机制的信息，这些突变导致许多不同的人类疾病，(c)我们可以通过在拓扑异构酶缺陷细胞中研究这一过程获得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.