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TRANSFER RNAS AND THEIR GENES IN THE ARCHAEBACTERIA

古细菌中的 RNA 转移及其基因

基本信息

批准号：
3289612
负责人：
Ramesh Gupta
金额：
$ 9.77万
依托单位：
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-08-01 至 1990-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3289612
关键词：
DNA directed RNA polymerase Halobacteriaceae Methanobacteriaceae bacterial genetics gene expression genetic manipulation genetic transcription microorganism culture nucleic acid sequence nucleic acid structure sulfur metabolizing bacteria thermophilic organism transfer RNA

项目摘要

The archaebacteria are distinct from the eubacteria and the eukaryotes. Though the basic life processes are similar in the three kingdoms, in details of molecular features and various functional mechanisms, the archaebacteria are either unique or resemble one or the other of the remaining two kingdoms. There are two main divisions of archaebacteria: (i) methanogen-extreme halophiles, and (ii) sulfur-utilizing thermophiles. The latter division more closely resembles eukaryotes than does the former. Therefore, the study of archaebacterial systems, besides having a comparative phylogenetic importance, could also be considered as a study of a eukaryote-like system at a simpler (prokaryotic) level, which might have evolved to a more complex (eukaryotic) level. The long term goals of this study are to understand the structure and function of transfer RNAs and the organization and expression of tRNA genes in archaebacteria. The specific aims of this proposal are the isolation and sequencing of Sulfolobus sp. tRNAs, characterization of modified nucleosides in archaebacterial tRNAs, determination of the sequences of Halobacterium volcanii tRNA genes and their flanking regions, determination of RNA polymerase binding sites in archaebacterial genes to identify promotor regions, and development of an in vitro RNA processing system for archaebacterial gene transcripts. The tRNAs will be sequenced by a combination of two-dimensional paper electrophoresis of RNAse digests of uniformly 32P-labeled tRNAs and gel sequencing methods using end-labeled tRNAs. Modified nucleosides will be identified by various electrophoretic, chromatographic, mass spectrometric and NMR techniques. The tRNA genes will be cloned in plasmid vectors and sequenced by dideoxy termination and chemical methods. RNA polymerase binding sites in promotor-containing DNA will be determined by the DNAse I "foot printing" technique. Pilot studies will be done to identify an RNA processing activity in crude cell extracts using either natural precursors or in vitro SP6 or T7 polymerase-transcribed RNAs from archaebacterial tRNA genes. Then attempts will be made to define and characterize the components involved in this processing system.

古细菌不同于真细菌和真核生物。虽然三国的基本生命过程相似，但在分子特征和各种作用机制的详细信息，古细菌要么是独一无二的，要么类似于剩下的两个王国。古细菌有两个主要的分类： (I)产甲烷极端嗜盐菌，以及(Ii)利用硫磺的嗜热菌。后者更接近于真核生物，而不是以前的。因此，对考古细菌系统的研究，除了具有相对系统发育的重要性，也可以被认为是对在更简单的(原核生物)水平上的类真核生物系统，这可能有进化到更复杂的(真核)水平。这项研究的长期目标是理解结构和转移RNA的功能与tRNA基因的组织和表达在考古细菌中。这项提议的具体目的是孤立和Sulfolobus sp.的序列分析。TRNAs，修饰的特性古细菌tRNAs中核苷序列的测定火山嗜盐杆菌tRNA基因及其侧翼区的测定古细菌基因中RNA聚合酶结合位点的鉴定启动子区域的构建及体外RNA处理系统的建立古细菌基因转录本。 TRNAs将通过结合二维纸片进行测序 32P均一标记tRNAs的核糖核酸酶酶切及凝胶电泳法使用末端标记的tRNA的测序方法。修饰后的核苷将是通过各种电泳法、层析法、质谱法进行鉴定和核磁共振技术。TRNA基因将被克隆到质粒载体中并通过双脱氧终止和化学方法进行测序。RNA聚合酶含有启动子的DNA中的结合部位将由DNA酶I决定 “脚印”技术。将进行初步研究，以确定一种RNA 使用任何一种天然前体的粗细胞提取物中的加工活性或体外从古细菌tRNA中获得SP6或T7聚合酶转录的RNA 基因。然后，将尝试定义和表征此处理系统中涉及的组件。