REGULATORY RNA IN HELA CELLS

HELA 细胞中的调节性 RNA

• 批准号: 3275588
• 负责人: HUGH D ROBERTSON
• 金额: $ 12.67万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 1986-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275588
• 关键词: DNA directed RNA polymerase; HeLa cells; RNA; affinity chromatography; cell free system; chemical cleavage; chemical fingerprinting; eukaryote; gel electrophoresis; gene expression; genetic registry /resource /referral center; genetic transcription; heterogeneous nuclear RNA; messenger RNA; molecular cloning; nucleic acid sequence; radiotracer; tissue /cell culture; virus DNA; virus RNA

The specific aims of this proposal are (1) to study the involvement of HeLa cell components in primed influenza mRNA transcription--these studies will include characterization of capped cellular RNA segments transferred onto flu NS mRNA during cell-free synthesis; characterization of the 5 feet terminal RNA segments cleaved by the flu-associated endonuclease in vitro from HeLa cell mRNA and hnRNA populations; seeking activities capable of cleaving capped RNA's to form flu mRNA primers in HeLa nuclear and cytoplasmic subcellular fractions prepared from normal and flu-infected cells; and to carry out flu mRNA transcription in a HeLa cell extract capable of cellular mRNA synthesis and processing. (2) To seek primed transcription by HeLa cell RNA polymerase II, using various cloned cellular DNA genes and adenovirus, using cleaved RNA segments active in the flu system as primers; mixed HeLA cell DNA templates will also be utilized, and the extent of uptake and specificity of any primer RNA populations ascertained. (3) To study RNA signals for specific RNA processing, including characterizing the RNA subunit of HeLA cell RNase P; isolating RNA's from HeLa subcellular fractions capable of specific flu mRNA processing; characterizing the two RNA co-factors involved in removing Alu family transcripts from HeLa cell hnRNA: 2 feet 5 feet oligo(A) and double-stranded RNA regions of the hnRNA substrate; and to study RNA ligation and circularization to seek both protein-level and RNA-level reactions. Methods to be used include in vitro transcription and oligo (dT) column and DNA cellulose affinity column chromatography of viral and cellular mRNA transcripts; 32P in vivo labeling and 125I-labeling in vitro; RNA fingerprinting and sequence analysis of labeled RNA primer segments; and cellulose CF11 and polyacrylamide gel electrophoretic isolation of specific RNA's; E. coli RNase III and specific subcellular fractions of HeLa cells capable of RNA processing reactions will also be employed. These studies support our long term goal of assessing the role of RNA signals in gene regulation in two ways: first, the specific reactions of the primed flu mRNA transcription system allow us to develop assays to find out once and for all whether such reactions occur in the cell; and second, RNA subunits and co-factors inspecific RNA processing reactions comprise and unprecedented opportunity to study gene regulation at this level.

该提案的具体目的是（1）研究HELA的参与 启动流感mRNA转录中的细胞成分 - 这些研究将 包括转移到 无细胞合成过程中的流感mRNA； 5英尺的特征 末端RNA片段在体外被流感相关的内切酶裂解 来自HeLa细胞mRNA和HNRNA种群；寻求能够的活动 切割盖的RNA以在Hela核和 由正常和流感感染的细胞质亚细胞级分 细胞；并在HeLa细胞提取物中进行流感mRNA转录 能够细胞mRNA合成和加工。 （2）寻求底漆 使用各种克隆的细胞进行HeLa细胞RNA聚合酶II的转录 DNA基因和腺病毒，使用活性在流感中的裂解RNA片段 系统作为底漆；混合HeLa细胞DNA模板也将被使用，并且 任何引物RNA群体的摄取程度和特异性程度 确定。 （3）研究特定RNA处理的RNA信号， 包括表征HeLa细胞RNase P的RNA亚基；隔离 来自HeLa亚细胞级分的RNA能够特异性流感mRNA 加工;表征涉及删除Alu的两个RNA辅助因素 HELA细胞HnRNA的家庭成绩单：2英尺5英尺的寡核（A）和 HNRNA底物的双链RNA区域；并研究RNA 连接和循环以寻求蛋白质级和RNA级别 反应。 要使用的方法包括体外转录和寡头 （DT）病毒和DNA纤维素亲和力柱色谱柱和 细胞mRNA转录本； 32p体内标记和125i标记的体外标记； 标记的RNA底漆段的RNA指纹和序列分析； 和纤维素CF11和聚丙烯酰胺凝胶电泳分离 特定的RNA；大肠杆菌III和特定的亚细胞分数 还将采用能够进行RNA加工反应的HeLa细胞。 这些研究支持我们评估RNA作用的长期目标 基因调控中的信号以两种方式：首先，特定反应 底漆流感mRNA转录系统使我们能够开发测定 一劳永逸地出现这种反应是否发生在细胞中。第二， RNA亚基和辅助因子特定的RNA加工反应包括 以及在此级别研究基因调节的前所未有的机会。