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.

古细菌与细菌和真核生物不同。尽管在三个王国中的基本生活过程相似，但分子特征和各种功能机制的细节，古细菌是独特的，要么类似于一个或另一个剩下的两个王国。古细菌有两个主要分区：（i）甲烷激素 - 嗜热蛋白，（ii）硫化的嗜热剂。后一个分区比真核生物更像以前的。因此，除了具有比较系统发育重要性也可以视为一个更简单（原核）水平的真核生物样系统，它可能具有演变成更复杂的（真核）水平。这项研究的长期目标是了解结构和转移RNA的功能以及tRNA基因的组织和表达在考古细菌中。该提议的具体目的是隔离和Sulfolobus sp的测序。 trnas，修改的表征核苷中的细菌trNA中的核苷，测定的序列火山片tRNA基因及其侧翼区域，确定古细菌基因中的RNA聚合酶结合位点的鉴定启动子区域，以及开发用于体外RNA处理系统考古细菌基因转录本。 TRNA将通过二维纸的结合进行测序均匀32p标记的TRNA和凝胶的RNase消化的电泳使用最终标记的TRNA进行测序方法。修饰的核苷将是通过各种电泳，色谱，质谱法鉴定和NMR技术。 tRNA基因将在质粒载体中克隆和通过二氧基终止和化学方法测序。 RNA聚合酶含有启动子DNA中的结合位点将由DNase I确定 “脚印”技术。试点研究将进行识别RNA 使用任何一种天然前体的粗细细胞提取物加工活性或体外SP6或T7聚合酶转录的RNA，来自考古细菌tRNA 基因。然后将尝试定义和表征该处理系统涉及的组件。