Elucidation of the Histone 3' End Formation Mechanism

组蛋白 3 末端形成机制的阐明

• 批准号: 6924691
• 负责人: ERIC J WAGNER
• 金额: $ 4.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6924691
• 关键词: Drosophilidae; HeLa cells; gene mutation; green fluorescent proteins; histones; messenger RNA; nucleic acid metabolism; postdoctoral investigator; posttranscriptional RNA processing; protein binding; protein protein interaction; protein purification; small interfering RNA; transcription factor

DESCRIPTION (provided by applicant): The broad long-term objectives to this proposal are to identify new factors involved in histone mRNA 3' end formation both biochemically and genetically and to characterize those proteins in vivo and in vitro. Specific Aim 1 is designed to use a combination of classical and innovative biochemical techniques to identify and purify proteins binding in a functionally relevant manner to histone pre-mRNA in HeLa nuclear extract. Specific Aim 2 will involve the use of a novel GFP based genetic screen to be performed in Drosophila melanogaster aimed at identifying loss of function mutants in the process. This screen will be performed to near saturating levels with the aid of an embryo sorter. Specific Aim 3 is to develop an in vivo system to study the function relevance of identified factors as well as to characterize them. This in vivo assay may also be applied in a novel siRNA-based genetic screen in mammalian cells. An in vitro system will also be developed to study histone mRNA 3' end formation in the context of active transcription. Histone mRNA 3' end formation is intimately related to the cell cycle and DNA replication, thus understanding this process may lead to the production of more effective chemotherapeutic agents.

描述（由申请人提供）： 该提案的长期目标是在生化和遗传上确定组蛋白mRNA 3'末端形成的新因素，并在体内和体外表征这些蛋白质。特定的目标1旨在结合经典和创新的生化技术的组合，以功能相关的方式与HELA核提取物中的组蛋白pre-mRNA识别和纯化蛋白质结合。具体的目标2将涉及使用新型基于GFP的遗传筛选，以在果蝇中进行，旨在识别该过程中功能突变体的丧失。该屏幕将借助胚胎分散者进行到接近饱和的水平。特定目的3是开发一个体内系统，以研究确定的因素的功能相关性以及对其进行表征。该体内测定也可以应用于哺乳动物细胞中新型的基于siRNA的遗传筛选中。还将开发一个体外系统，以研究活跃转录的背景下的组蛋白mRNA 3'末端形成。组蛋白mRNA 3'末端的形成与细胞周期和DNA复制密切相关，因此理解该过程可能会导致产生更有效的化学治疗剂。