MECHANISM OF INSERTION SEQUENCE TRANSLOCATION

插入序列易位机制

• 批准号: 3275751
• 负责人: NIGEL David GRINDLEY
• 金额: $ 21.07万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-05-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275751
• 关键词: Escherichia coli; bacterial genetics; chromosome translocation; gene expression; genetic manipulation; genetic regulation; mutant

The study of two different types of specialized recombination is proposed. Transpositional recombination, the integration of transposable DNA into a target site, will be investigated using two contrasting transposons as model systems. These are IS903, which generally transposes by a donor-destructive, non-replicative pathway, and GammaDelta which uses a replicative (cointegrate) pathway. Site specific recombination, the recombination between two specific sites by a reciprocal, conservative breakage reunion reaction, will be studied using the resolution of GammaDelta cointegrates by the transposon encoded TnpR protein, resolvase. The following specific projects are proposed. I Transpositional Recombination. 1) Purification and characterization of the transposase proteins of IS903 and GammaDelta, with transposition in vitro as the ultimate goal. 2) Analysis of the interaction of transposon ends and transposase (a) by mutational analysis of the transposon terminal inverted repeats and (b) by identification of DNA binding domains within the transposase protein by isolation of mutants of transposase that show altered sequence specificity. 3) Further analysis of the mechanism of deletion formation by IS903 and IS10. II Site-specific Recombination. 1) Mutational dissection of the cointegrate resolution process, (a) by developing assays for the various steps in recombination and (b)\by isolating and characterizing mutants specifically defective in each step. 2) Detailed analysis of the molecular interactions between (a) the N-terminal domain of resolvase and the crossover site, and (b) the C-terminal domain and the resolvase-binding DNA segment. 3) Analysis of the apparent resolvase-induced bending of res DNA.

提出了两种不同类型的专业重组的研究。 转位重组，将转座性DNA整合到A 目标部位将使用两个对比的转座进行研究 模型系统。 这些是903，通常通过 供体毁灭性，非复制途径和使用gammadelta 复制（协整）途径。 特定地点重组， 通过倒数，保守的两个特定站点之间的重组 破裂团聚反应将使用分辨率进行研究 伽玛达尔塔通过转座子编码的TNPR蛋白Resolvase协整。 提出了以下特定项目。 我转置重组。 1）IS903的转座酶蛋白的纯化和表征 和Gammadelta，在体外换位是最终目标。 2）分析转座子末端和转座酶（a）的相互作用。 转座子末端的突变分析倒重复序列和（b） 通过鉴定转座酶蛋白内的DNA结合结构域 分离转座酶突变体，显示出改变的序列特异性。 3）进一步分析IS903和 IS10。 II地点特异性重组。 1）协整解析过程的突变解剖，（a） 为重组的各种步骤开发测定法，（b）\ 隔离和表征突变体在每个步骤中特异性有缺陷。 2）（a）之间的分子相互作用的详细分析 分辨率和跨界点的N末端结构域，以及（b） C末端结构域和分解结合DNA段。 3）分析分解诱导的RES DNA的弯曲。