Elucidation of the Histone 3' End Formation Mechanism

描述(由申请人提供)： 这项建议的长期目标是从生物化学和遗传两个方面确定与组蛋白mRNA3‘端形成有关的新因素，并在体内和体外对这些蛋白进行表征。特定目的1旨在结合经典和创新的生化技术来鉴定和纯化HeLa核提取液中与组蛋白前mRNA功能相关的结合蛋白。具体目标2将涉及使用一种新的基于绿色荧光蛋白的遗传筛查在果蝇中进行，目的是识别在这一过程中功能突变的丢失。这一筛查将在胚胎分选机的帮助下进行到接近饱和的水平。具体目标3是开发一个体内系统，研究已识别的因子的功能相关性，并对它们进行表征。这种体内试验也可能应用于哺乳动物细胞中基于siRNA的新的遗传筛选。还将开发一个体外系统，在活性转录的背景下研究组蛋白mRNA3‘末端的形成。组蛋白mRNA3‘末端的形成与细胞周期和DNA复制密切相关，因此了解这一过程可能会导致更有效的化疗药物的生产。

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.