REGULATORY RNA IN HELA AND OTHER CELLS

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后启动转录的进一步研究 细胞提取液及其未受感染的对应物将进行 特别强调RNA的特定亚群的起源 最近在HeLa亚细胞提取物中发现的聚合酶II产物。 这些可能是第一个细胞转录本的例子，其 合成是通过添加能够发挥作用的带帽RNA来刺激的 流感病毒转录系统中的引子。两种控制 试验将与上述研究同时进行：(一) 流感病毒RNA在新的HeLa中启动转录的机制 本实验室最近研制出的细胞提取系统将继续 (B)开展一系列合作研究，以确定 另一个细胞RNA启动转录的真实案例-- 在锥虫系统中--将继续提供另一个例子 这样一个系统。(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，它 调节它自己的转录，我们最近为它制定了 一种将RNA结构和功能联系起来的检测系统；这一检测将是 应用于5S rRNA与其转录产物的特异性络合物 含因子IIIA的控制复合体。(B)RNA加工的RNA亚单位 酶，RNaseP，专注于这种RNA(在 HeLa细胞和其他细胞)可以促进一整套的特定切割 不同的tRNA前体。 使用的方法包括RNA和DNA模板的体外转录； 用亲和和杂交法纯化RNA群体； 二维凝胶电泳法RNA结构表征； 形成特定的紫外线诱导的RNA分子的衍生物，其中 在生物活性部位引入单一的、特定的交联物 以及传统的RNA和DNA指纹图谱和 测序方法。因此，RNA信号在基因调控中的作用 被进一步分析，方法和途径的扩展首先奏效 病毒系统中的Out将扩展到细胞群体，两者都是正常的 并改变了自己。

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.