STUDIES ON MECHANISMS OF IS2 TRANSPOSITION

IS2转座机制的研究

• 批准号: 6217805
• 负责人: LESLIE A LEWIS
• 金额: $ 20.71万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6217805
• 关键词: DNA binding protein; DNA footprinting; SDS polyacrylamide gel electrophoresis; cell free system; chemical binding; gel mobility shift assay; gene expression; genetic recombination; genetic regulation; intermolecular interaction; molecular biology; nucleic acid sequence; polymerase chain reaction; radionuclides; restriction endonucleases; southern blotting; transposon /insertion element

The long term objective of this study is to understand at a mechanistic level how transposition occurs in the insertion sequence IS2. IS2 is a 1.3kb transposable element which is a member of the IS3 family of insertion sequences. Presently, nothing is known of the mechanistic details of how transposition is achieved in this group of transposable elements. This family has several features in common with other TEs at the nucleotide sequence level; in addition IS2 and other IS3-like TEs produce a transposes which catalyzes transposition and which is thought to bear striking structural and functional similarities to the integrase protein (IN) of the retroviruses. An understanding of the mechanisms by which the transposes catalyzes transposition could, therefore, contribute to an overall mechanistic and functional understanding of integrases as well. The specific aims of this study are; (i) to develop an in vivo transpositional assay through which inter and intramolecular transposition events can be studied. This will involve the preparation of plasmid constructs with altered versions of IS2 which transpose at high frequencies and carry antibiotic resistance markers; with these constructs the genetic criteria for transposition can be readily studied using a mutational approach. (ii) to identify the DNA sequences to which transposase binds, both within the element and at preferential insertion sites (e.g., the hemB gene of E. coli). This will be achieved through gel retardation and DNA footprinting studies. (iii) to dissect the mechanistic details of transposase-mediated recombination events; these include the target and nature of its cleavage or processing reactions, the nature of strand transfer reactions which generate athe recombination product(s), and the formation of higher order DNA-protein complexes, within which the cleavage and joining reactions are thought to occur. This will be done using an in vitro transposition assay, whose reaction products will be analyzed with labelled substrates on polyacrylamide gels and by Southern hybridization techniques using agarose gels.

这项研究的长期目标是从机制上理解 级别如何在插入序列 IS2 中发生转座。 IS2 是一个 1.3kb 转座元件，IS3 插入家族成员 序列。 目前，我们对如何实现这一机制的机制细节一无所知。 转座是在这组转座元件中实现的。 这 该家族在核苷酸方面与其他 TE 有几个共同特征 序列级别；此外，IS2 和其他类似 IS3 的 TE 会产生转置 它催化转位并被认为具有惊人的作用 与整合酶蛋白（IN）的结构和功能相似 逆转录病毒。 了解转置机制 因此，催化转座可能有助于整体 对整合的机制和功能的理解。 这 这项研究的具体目标是； (i) 开发体内转座 分子间和分子内转座事件可以通过测定 研究过。 这将涉及质粒构建体的制备 IS2 的修改版本可以高频移调并携带 抗生素抗性标记；有了这些构建体的遗传标准 使用突变方法可以很容易地研究转座。 (二) 鉴定转座酶结合的 DNA 序列，均位于 元件和优先插入位点（例如大肠杆菌的 hemB 基因） 大肠杆菌）。 这将通过凝胶阻滞和 DNA 足迹来实现 研究。 (iii) 剖析转座酶介导的机制细节 重组事件；这些包括其裂解的目标和性质 或加工反应，链转移反应的性质 产生重组产物，并形成更高阶 DNA-蛋白质复合物，其中发生裂解和连接反应 认为发生。 这将使用体外转座测定来完成， 其反应产物将用标记底物进行分析 聚丙烯酰胺凝胶和使用琼脂糖的 Southern 杂交技术 凝胶。