POSTTRANSCRIPTIONAL MECHANISMS OF GENE CONTROL

拟议工作的总体目标是找出员额机制。 转录基因调控侧重于mRNA聚(A)的形成 网站。多腺化信使RNA3‘端的产生是 显然，mRNA生物发生中的一个关键事件和几项研究表明 它可以帮助调节基因的表达。在我们的 实验室，在过去的五年里得到了这笔赠款的支持， 成功鉴定多聚腺苷酸化位点上的序列 对于处理事件以及识别监管机构来说很重要 涉及Poly(A)位点利用的途径。最近，我们使用了 HeLa细胞核提取液分离支持正品的因子 重组体系中的体外多腺苷基化反应。对中国传统文化的分析与思考 这些因子与前信使核糖核酸的相互作用导致了一种理解 各因素对保利(A)选址的相对贡献为何；及 提供了对因素集合的方式的洞察 RNA有助于Poly(A)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上 从HeLa细胞中分离纯化的多聚腺苷酸化因子 核萃取物。我们建议完成这些因子的纯化 以允许详细的生物化学特征的相互作用 与加工复合体以及与前信使核糖核酸有关的因素。 将产生针对各种活动和基因的抗体 编码活性的多肽成分将被克隆。 我们还将开始探索这些因素在监管中的作用 Poly(A)站点利用率的百分比。这些研究将集中在利用 MU免疫球蛋白聚A位点及其在B细胞中的调节 差异化。

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.