IN VITRO TRANSCRIPTION OF CHLOROPLAST DNA GENES

叶绿体 DNA 基因的体外转录

• 批准号: 3279527
• 负责人: KRISHNA K TEWARI
• 金额: $ 17.28万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279527
• 关键词: DNA; DNA directed DNA polymerase; DNA directed RNA polymerase; RNA; chloroplasts; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic recombination; genetic transcription; messenger RNA; molecular cloning; molecular genetics; nucleic acid sequence; organelles; plant genetics; tissue /cell culture; transfer RNA

The long term objective is to study the mechanism of transcription of chloroplast DNA genes using homologous RNA polymerase. We have purified an RNA polymerase preparation from pea chloroplasts that shows strong preference to chloroplast DNA templates and can synthesize in vitro full length rRNA, tRNA, and photogene 32 mRNA transcripts using cloned genes. We now propose to study the following: 1) A detailed study on the nucleotide sequences required (promoter sequences) for recognition by the RNA polymerase for all three types of RNA chains i.e. rRNA, tRNA, and mRNA. These experiments will be carried out by constructing deletion mutants of the sequences that surround transcription initiation sites of genes using Ba131. Identification of the sequences at the 5' end of genes required for the transcription will be followed by experiments where single and multiple point mutations within the control region will be produced and the mutated DNA studied for transcription. These experiments will enable us to precisely identify the nucleotides that constitute specific promoters of each type of gene; 2) An analysis of nucleotide sequences at the 3' end of the genes that might be involved in the temination of transcription; 3) Further purification of the RNA polymerase enzyme using conventional purification procedures to establish whether there is a single RNA polymerase that transcribes different types of chloroplast DNA genes or there are multiple polymerases each specific for ribosomal RNA, transfer RNA, and messenger RNA genes; and 4) Analyze the structure of RNA polymerase(s) using monoclonal antibodies. These studies will, in future, be followed by experiments that would identify polypeptides which control the initiation, elongation and termination of chloroplast RNA transcripts.

长期的目标是研究转录的机制。 利用同源RNA聚合酶扩增叶绿体DNA基因。我们已经净化了一个 豌豆叶绿体RNA聚合酶的制备 偏爱叶绿体DNA模板并能在体外完全合成 使用克隆的基因获得长度为rRNA、tRNA和Photogene 32mRNA的转录本。 我们现在建议进行以下研究：1)详细研究 识别所需的核苷酸序列(启动子序列) 所有三种类型的RNA链的RNA聚合酶，即rRNA、tRNA和 MRNA.这些实验将通过构造删除来进行 转录起始点周围序列的突变体 使用Ba131的基因。基因5‘端序列的鉴定 之后将进行单次实验， 并在控制区内产生多个点突变， 对突变的DNA进行转录研究。这些实验将使 美国精确识别构成特定启动子的核苷酸 2)3‘端核苷酸序列分析 可能参与转录调控的基因；3) 用常规方法进一步纯化RNA聚合酶 确定是否存在单一RNA的纯化程序 转录不同类型的叶绿体DNA基因或 有多个聚合酶，每个聚合酶都专用于核糖体RNA转移 RNA和信使RNA基因；4)分析RNA的结构 聚合酶(S)使用单抗。这些研究将在未来， 之后进行的实验将识别控制 叶绿体RNA转录本的起始、伸长和终止。