MRNA SPLICING MECHANISMS USING TUMOR NUCLEAR PROTEINS

这个项目的目标是定义单个的生化步骤 参与真核生物信使RNA的转录和加工。 实验将包括以下内容： 1.一种可剪接mRNA的无细胞抽提系统的建立 本文介绍 该系统利用浓缩的HeLa细胞提取物， 含有腺病毒-2DNA片段的重组pBR 322衍生物 其编码EIII区mRNA前体，该前体含有两个天然的 内含子 提取系统能够合成mRNA前体 并去除内含子。 实验程序描述如下： 探测前体RNA转录的确切位点 开始和结束，以及内含子切除的位点，B.提高 通过用一个或多个核苷酸取代质粒来修饰质粒的转录效率 强启动子对弱启动子，c.体外测试剪接 在体内制备的用于30 K的核mRNA前体的底物质量 腺病毒-SV 40杂合蛋白和d.研究抑制剂的作用， 转录和处理。 2.与此同时，正在研究体内特定基因的剪接 使用真核病毒克隆载体。 嵌合基因，具有 基因组中调控元件的缺失、取代或重排 EIII区，将插入病毒。 这些病毒将被用于 感染培养中的人类细胞，以及调控序列的作用 将测定mRNA转录和翻译的变化。 特别强调的是mRNA在3'端的加工。 特米尼

The goal of this project is to define the individual biochemical steps involved in the transcription and processing of eukaryotic messenger RNA. Experiments will include the following: 1. The development of a cell-free extract system capable of splicing mRNA is described. This system utilizes concentrated HeLa cell extracts, and a recombinant pBR322 derivative containing a segment of adenovirus-2 DNA which codes for an EIII region mRNA precursor containing two natural introns. The extract system is capable of synthesizing the mRNA precursor and removing the intron. Experimental procedures are described for: a. probing the exact sites at which the transcription of the precursor RNA begins and ends, and the sites of intron excision, b. improving the efficiency of transcription by modifying the plasmid by substitution of a strong promotor for the weak promotor, c. testing in vitro the splicing substrate quality of nuclear mRNA precursor made in vivo for the 30K adeno-SV40 hybrid protein and d. investigating the effects of inhibitors of transcription and processing. 2. Simultaneously, splicing of specific genes in vivo is being investigated using a eukaryotic viral cloning vehicle. Chimeric genes, having deletions, substitutions or rearrangements in regulatory elements in the EIII region, will be inserted into the virus. Such viruses will be used to infect human cells in culture, and the effects of the regulatory sequence changes on mRNA transcription and translation will be determined. Particular emphasis will be placed on the processing of mRNA at the 3' termini.