Factors Involed in Chloroplast RNA Editing

描述(由申请人提供) 该项目的长期目标是定义RNA：RNA和RNA：蛋白质 参与RNA编辑的相互作用。在人类中，编辑对RNA至关重要 几个疾病相关基因的表达过程中的处理，包括 Wilm？S肿瘤易感基因、谷氨酸受体B亚单位和 载脂蛋白B这项研究将阐明指导胞苷的因素 尿苷编辑，增加我们对细胞器编辑的理解 机械装置。 在团契期间，转基因和生化技术将 用于识别与叶绿体rpoB-I编辑相互作用的分子 地点。为了研究RNA二级结构在编辑中的作用，rpoB-I将被 融合成反义序列并在转基因烟草叶绿体中表达。 将进行电泳迁移率变化分析来鉴定叶绿体 体外转录的rpoB-I编辑具有结合特异性的蛋白质 网站。RNA：蛋白质相互作用的性质将用 修改后的编辑序列。推定化合物的提纯及性质 编辑蛋白质将通过亲和力方法得到便利。功能界别 筛选将用于分离编码编辑因子的基因。

DESCRIPTION (provided by applicant) The long-term objective of this project is to define RNA:RNA and RNA:protein interactions involved in RNA editing. In humans, editing is critical for RNA processing in the expression of several disease-related genes including the Wilm?s tumor susceptibility gene, glutamate receptor subunit B, and apolipoprotein B. This research will elucidate the factors that direct cytidine to uridine editing and increase our understanding of organellar editing mechanisms. During the fellowship period, transgenic and biochemical techniques will be used to identify molecules that interact with the chloroplast rpoB-I editing site. To study the role of RNA secondary structure in editing, rpoB-I will be fused to antisense sequences and expressed in transgenic tobacco chloroplasts. Electrophoretic mobility-shift assays will be performed to identify chloroplast proteins with binding specificity for in vitro transcribed rpoB-I editing sites. The nature of the RNA:protein interaction will be examined using modified editing sequences. The purification and characterization of putative editing proteins will be facilitated by an affinity approach. A functional screen will be utilized for the isolation of genes encoding editing factors.