MOLECULAR MECHANISM OF DNA TRANSPOSITION

该计划的重点是持续的styd的分子 转座因子IS 1和IS 50（和 由元件介导的其它重组事件）。 这些元素 参与形成复合转座子如Tn 9（IS 1）和Tn 5 （IS50）。 已经开发了一个模型系统并用于研究换位， 共整合形成（质粒融合）和其他重组事件 由元素介导。 该系统使用F衍生的质粒，其含有 没有常驻转座因子作为一个组分， 非接合的pBR 322衍生质粒作为另一个元件。 系统 是高度可操作的，容易移植到不同的遗传背景中， 并允许对重组产物进行精确分析。 研究 到目前为止，该计划已经确定协整函数是 而不是Tn 9或Tn 5转座的中间体，与之相反， Tn 3样元件转座的主要途径。 的精确 然而，分子途径的性质尚不清楚。 的目标 拟议的计划将是：1）了解分子基础的 通过外部转录强烈调节转座活性， 我们已经发现，2）了解不同的定量影响， 活性的化合物转座子的侧翼元件上的 重组产物的光谱（我们已经开发了一个简单的定量 可以使用我们的系统进行测试的模型），3）了解 我们观察到强靶向特异性和定向效应 对于IS 1，4）了解IS 1的结构组件在 转座过程（克隆的末端序列，和六个最大的， 重叠元件内的开放阅读框）， 定点诱变方法，5）分析性质（体内 和体外）的蛋白质，和/或由元件编码的RNA（一个 IS 1蛋白已在微细胞中被鉴定， 启动）。 另一个最近观察到的效应是IS 1可以激活 通过提供“杂合”启动子的-35区域来改变基因的表达， 研究了 这一效应可能对人类的进化产生重要影响。 理解复合转座子的形成和稳定性。

The focus of the proposed program is the continuing styd of the molecular mechanisms for transposition of the transposable elements IS1 and IS50 (and other recombination events mediated by the elements). These elements participate in the formation of compund transposons like Tn9 (IS1) and Tn5 (IS50). A model system has been developed and used to study transposition, cointegrate formation (plasmid fusion) and other recombination events mediated by the elements. This system uses an F-derived plasmid containing no resident transposable elements as one component, and various non-conjugative, pBR322-derived plasmids as the other element. The system is highly manipulable, easily portable into different genetic backgrounds, and permits precise analysis of the recombination products. The studies carried out so far in this program have established that cointegrates are not intermediates in the transposition of Tn9 or Tn5, in contrast to the major pathway for transposition of the Tn3-like elements. The precise nature of the molecular pathways are not yet clear, however. The goals of the proposed program will be: 1) to understand the molecular basis of the strong modulation of transposition activity by external transcription that we have discovered, 2) to understand the quantitative effects of differing activities for the flanking elements of the compound transposons on the spectrum of recombination products (we have developed a simple quantitative model that can be tested using our system), 3) to understand the source of the strong target specificity and orientation effect that we have observed for IS1, 4) to understand the roles of the structural components of IS1 in the transposition process (the cloned end sequences, and the six largest, overlapping open reading frames within the element) by cloning and site-directed mutagenesis methods, 5) to analyze the properties (in vivo and in vitro) of the proteins, and/or RNA's coded for by the element (one IS1 protein has been identified in minicells, and a model-building study initiated). Another, recently-observed effect by which IS1 can activate the expression of a gene by providing the -35 region of a "hybrid" promoter will be investigated. This effect may have important implications for the understanding of the formation and stability of compound transposons.