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POSTTRANSCRIPTIONAL MECHANISMS OF GENE CONTROL

基因控制的转录后机制

基本信息

批准号：
2178120
负责人：
JOSEPH R NEVINS
金额：
$ 10.78万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-06-01 至 1995-03-31
项目状态：
已结题

项目摘要

The overall aim of the proposed work is to eludidate mechanisms of post transcriptional gene control focusing on the formation of MRNA poly(A) sites. The generation of the polyadenylated messenger RNA 3' end is clearly a crucial event in mRNA biogenesis and several studies demonstrate that it can contribute to the regulation of gene expression. Work in our laboratory, supported by this grant over the past five years, has been successful in identifying sequences in polyadenylation sites that are important for the processing event as well as identifying regulatory pathways involving poly(A) site utilization. More recently, we have used HeLa cell nuclear extracts to isolate factors that support authentic polyadenylation in vitro in a reconstituted system. The analysis of the interaction of these factors with the pre-mRNA has led to an understanding of the relative contributions of the factors in poly(A) site selection, and has provided insight into the manner in which the assembly of factors on an RNA contributes to poly(A) site utilization. We propose to continue these studies with a major focus on the biochemical analysis of polyadenylation factors that have been isolated and purified from HeLa cell nuclear extracts. We propose to complete the purification of these factors to allow detailed biochemical characterizations of the interactions of the factors with the processing complex as well as with the pre-mRNA. Antibodies will be produced against the various activities and genes encoding the polypeptide constituents of the activities will be cloned. We will also begin to explore the role of these factors in the regulation of poly(A) site utilization. These studies will focus on the utilization of the mu immunoglobulin poly A sites and their regulation during B cell differentiation.

拟议工作的总体目标是，转录基因控制，重点是mRNA poly（A）的形成网站. 多聚腺苷酸化信使RNA 3'末端的产生是这显然是mRNA生物发生中的一个关键事件，一些研究表明，它可以有助于基因表达的调节。工作的开展实验室，在过去五年的资助下，成功地鉴定了多聚腺苷酸化位点中的序列，对于处理事件以及识别监管涉及poly（A）位点利用的途径。最近，我们使用 HeLa细胞核提取物，以分离支持真实的在重构系统中体外聚腺苷酸化。的分析这些因子与前mRNA的相互作用导致了对各因素对多聚腺苷酸位点选择的相对贡献，及提供了洞察力的方式，其中组装的因素， RNA有助于多聚腺苷酸位点的利用。我们建议继续这些研究主要集中在生物化学分析，从HeLa细胞中分离纯化的多聚腺苷酸化因子核提取物我们建议完成这些因子的纯化以允许详细的生物化学表征的相互作用，与加工复合物以及与前mRNA的因素。抗体将针对各种活动和基因产生将克隆编码多肽活性成分的基因。我们也将开始探讨这些因素在调控中的作用聚（A）位点的利用率。这些研究将侧重于利用在B细胞增殖过程中，分化