REGULATORY RNA IN HELA AND OTHER CELLS

HELA 和其他细胞中的调节 RNA

• 批准号: 3275584
• 负责人: HUGH D ROBERTSON
• 金额: $ 13.33万
• 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 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275584
• 关键词: DNA directed RNA polymerase; HeLa cells; RNA; affinity chromatography; cell free system; 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; regulatory gene; tissue /cell culture; virus DNA; virus RNA; virus genetics; virus infection mechanism

Our specific aims are (1) to study the possibility that RNA-primed transcription is of general significance in eukaryotic cells and viruses. Further studies on primed transcription in influenza virus infected HeLA cell extracts and their uninfected counterpartts will be conducted, with particular emphasis on the origin of a particular sub-population of RNA polymerase II products recently discovered in HeLa subcellular extracts. These may represent the first example of cellular transcripts whose synthesis is stimulated by addition of capped RNA's capable of serving as primers in the influenza virus transcription system. Two sorts of control experiments will be carried out in parallel to the above studies: (a) the mechanics of influenza viral RNA primed transcription in the novel HeLa cell extract system recently worked out in this laboratory will continue to be ascertained; and (b) a series of collaborative studies on what may be another authentic case of cellular RNA-primed transcription--that occurring in the trypanosome system--will be continued to provide another example of such a system. (2) A second aim is to study regulatory RNA in HeLa cells by focusing on two RNA's which carry out such a role--(a) 5S RNA, which regulates its own transcription and for which we have recently worked out an assay system correlating RNA structure and function; this assay will be applied to specific complexes between 5S rRNA and its transcriptional control complex with factor IIIA. (b) The RNa subunit of an RNA processing enzyme, RNase P, concentrating on the specific way in which this RNA (in HeLa cells and elsewhere) can promote the specific cleavage of a whole set of differing tRNA precursors. Methods to be used include in vitro transcription of RNA and DNA templates; purification of RNA populations by affinity and hybridization methods; two-dimensional gel electrophoretic RNA structural characterization; formation of specific UV-induced derivatives of RNA molecules in which a single, specific crosslink is introduced at a site of biologically active local tertiary structure; and conventional RNA and DNA fingerprinting and sequencing methods. The role of RNA signals in gene regulation will thus be further analyzed, and the extension of methods and pathways first worked out in viral systems will be extended to cellular populations, both normal and transformed.

我们的具体目标是 (1) 研究 RNA 引发的可能性 转录在真核细胞和病毒中具有普遍意义。 流感病毒感染 HeLA 引物转录的进一步研究 将进行细胞提取物及其未感染的对应物， 特别强调特定 RNA 亚群的起源 最近在 HeLa 亚细胞提取物中发现了聚合酶 II 产品。 这些可能代表了细胞转录本的第一个例子，其 通过添加加帽 RNA 来刺激合成，该 RNA 能够充当 流感病毒转录系统中的引物。 两种控制方式 实验将与上述研究同时进行：(a) 新型 HeLa 中流感病毒 RNA 引发转录的机制 本实验室最近研制的细胞提取系统将继续 被确定； (b) 一系列关于可能的合作研究 细胞 RNA 启动转录的另一个真实案例——正在发生 在锥虫系统中——将继续提供另一个例子 这样的系统。 (2)第二个目标是研究HeLa细胞中的调控RNA 通过关注两个发挥这种作用的RNA——(a) 5S RNA， 调节其自身的转录，我们最近已经解决了这个问题 关联 RNA 结构和功能的测定系统；该测定将是 应用于 5S rRNA 及其转录之间的特定复合物 与因子 IIIA 的对照复合物。 (b) RNA 加工的 RNA 亚基 RNase P 酶，专注于该 RNA（在 HeLa 细胞和其他细胞）可以促进整组细胞的特异性裂解 不同的 tRNA 前体。 使用的方法包括 RNA 和 DNA 模板的体外转录； 通过亲和和杂交方法纯化 RNA 群体； 二维凝胶电泳RNA结构表征； 形成特定的 UV 诱导的 RNA 分子衍生物，其中 在生物活性位点引入单一的、特定的交联 局部三级结构；以及常规的 RNA 和 DNA 指纹识别 测序方法。 RNA信号在基因调控中的作用将因此 进一步分析，方法和路径的拓展首先发挥作用 病毒系统中的病毒将扩展到细胞群，无论是正常的还是 并转变。