DNAA PROTEIN AND REPLICATION INITIATION IN MYCOBACTERIA

分枝杆菌中的 DNAA 蛋白和复制起始

• 批准号: 2457822
• 负责人: MALINI RAJAGOPALAN
• 金额: $ 8.99万
• 依托单位: UNIVERSITY OF TEXAS HLTH CTR AT TYLER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457822
• 关键词: DNA footprinting; DNA replication; DNA replication origin; Escherichia coli; Mycobacterium tuberculosis; bacterial DNA; bacterial proteins; gene deletion mutation; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; plasmids; protein purification

M. tuberculosis, the causative agent of tuberculosis is a well known pathogen that has reestablished itself as a significant world wide public health hazard. While there is a great deal of published information in the physiology and pathogenesis of M. tuberculosis, there is very little information available on genetic control of DNA synthesis and related metabolism governing DNA replication. Replication of bacterial chromosomes occurs at unique sequences called 'oriC' and is thought to be regulated at the level of initiation. Studies on replication initiation in other bacteria suggest that both DnaA, the initiator protein and replication origin sequences are structurally conserved. The replication origin is AT rich and contains repeats of nine nucleotide long DnaA protein recognition sequences called the DnaA boxes and repeats of 13 - 16 nucleotide long AT rich elements. The number and position of some of the DnaA boxes is variable in each bacteria. The dnaA gene flanking region in many bacteria supports autonomous replication activity. The genetic and biochemical aspects of replication initiation in mycobacteria are not known. Our research proposal focuses on identifying the key elements needed for replication initiation in M. tuberculosis. During the granting period M. tuberculosis dnaA gene will be cloned and its nucleotide sequence will be determined. The nucleotide sequence of the dnaA flanking region will be determined and putative replication origin sequence features, if any, will be identified. The ability of the dnaA gene flanking sequences to support autonomous replication when present in nonreplicative plasmids will be determined. Sequential deletions from both the 5' and the 3' end of the flanking regions will be carried out to identify the minimal size DNA fragment that supports autonomous replication. If dnaA gene flanking region is not the 'oriC', then shot gun cloning approach will be used to identify the replication origin sequences. Once M. tuberculosis oriC is identified, then oligonucleotide primers specific to oriC will be synthesized and used to amplify related replication origin sequences from the genomic DNA preparations of other species of mycobacteria. The nucleotide sequence of these replication origins will be determined and compared to determine similarities and dissimilarities in the organization of 'oriC' in mycobacteria. M. tuberculosis DnaA protein will be overproduced in E. coli and the protein thus overproduced will be purified. The interactions of DnaA protein with the DnaA boxes of the replication origin will be established in vitro by gel retardation, nitrocellulose and DNasel foot printing assays. Thus, by identifying the DNA fragments that support autonomous replication and investigating the interaction of DnaA protein with the autonomous replication sequences, this study will establish the early events in initiation of DNA replication in M. tuberculosis.

M.结核病，结核病的病原体是众所周知的 病原体已经重新确立了自己作为一个重要的世界范围内的公众 健康危害。 虽然有大量的公开信息， 对M.肺结核， 关于DNA合成遗传控制和相关 控制DNA复制的代谢。 细菌复制 染色体发生在称为“oriC”的独特序列上， 在初始阶段进行规范。 关于复制的研究 在其他细菌中的启动表明，启动子DnaA 蛋白质和复制起点序列在结构上是保守的。 的 复制起点富含AT并含有9个核苷酸的重复 长的DnaA蛋白识别序列称为DnaA盒和重复序列 13 - 16个核苷酸长的AT富集元件。 的数量和位置 每种细菌的某些DNA盒是可变的。 dnaA基因 许多细菌中的侧翼区支持自主复制 活动 复制起始的遗传和生物化学方面 在分枝杆菌中是未知的。 我们的研究计划集中在 确定在M中复制启动所需的关键元件。 结核 在授予期间，M。结核病dnaA基因将 克隆并测定其核苷酸序列。 的核苷酸 将确定dnaA侧翼区的序列，并推定 将鉴定复制起点序列特征（如果有的话）。 的 dnaA基因侧翼序列的能力，以支持自主 当存在于非复制型质粒中时，将测定复制。 从侧翼序列的5'和3'端的连续缺失 将进行区域鉴定，以确定最小大小的DNA片段 支持自主复制。 如果dnaA基因侧翼区是 如果不是“oriC”，则将使用鸟枪克隆的方法来识别 复制起点序列 一旦M.结核病oriC是 鉴定，然后将对oriC特异性的寡核苷酸引物 合成并用于扩增相关的复制起点序列， 其他分枝杆菌的基因组DNA制备。 的 将确定这些复制起点的核苷酸序列， 比较，以确定相似性和不同之处， 分枝杆菌中的“oriC”组织。M.结核DnaA蛋白将 在E.大肠杆菌和蛋白质，因此过度生产将是 提纯 DnaA蛋白与细胞DNA盒的相互作用 复制起点将通过凝胶阻滞在体外建立， 硝酸纤维素和DNA酶1足迹测定。 因此，通过识别 支持自主复制的DNA片段， DnaA蛋白与自主复制序列的相互作用， 这项研究将确定DNA启动的早期事件， 在M.结核