MOLECULAR MECHANISM OF DNA TRANSPOSITION

提出的方案的重点是分子的延续风格 转座元件IS1和IS50的转座机制(和 由元素介导的其他重组事件)。这些元素 参与Tn9(IS1)和Tn5等复合转座子的形成 (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 在这些因素的调停下。该系统使用的是F衍生的含有 没有常驻转座元件作为一个组件，并且各种 非接合的，pBR322衍生的质粒作为另一个元件。系统 是高度可操控的，很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。这些研究 到目前为止在这个计划中进行的工作已经建立了协同整合是 不是Tn9或Tn5转座的中间体，与之相反 类Tn3转位的主要途径。精准的 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 外部转录对转座活性的强烈调控 我们已经发现，2)理解不同的 复合转座子侧翼元件在分子水平上的活性 重组产物的光谱(我们已经开发出一种简单的定量 可以使用我们的系统进行测试的模型)，3)了解 我们观察到的强烈的靶标特异性和定向效应 对于IS1，4)了解IS1的结构组件在 转座过程(克隆的末端序列，和六个最大的， 元件内重叠的开放阅读框)通过克隆和 定点突变方法，5)分析(活体)性质 和/或由元件(1)编码的RNA 已经在微细胞中发现了IS1蛋白，并进行了一项建模研究 启动)。 最近观察到的另一个效应是IS1可以激活该表达 通过提供一个“杂合”启动子的-35区域 调查过了。这一效应可能会对 了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.