STUDIES ON MECHANISMS OF IS2 TRANSPOSITION

这项研究的长期目标是了解在一个机械 水平转座如何在插入序列IS 2中发生。 IS 2是一个 1.3kb的转座因子，属于IS 3插入家族 序列的 目前，还不知道如何机械细节， 在这组转座因子中实现转座。 这 家族在核苷酸上与其他TE有几个共同的特征， 序列水平;此外，IS 2和其他IS 3样TE产生转座 它能催化转座， 整合酶蛋白（IN）的结构和功能相似性 逆转录病毒 理解转置的机制 因此，催化转座可能有助于整体 以及对整合酶的机制和功能的理解。 的 本研究的具体目的是：（i）开发体内转座 分子间和分子内转座事件可通过该测定法 研究了 这将涉及制备质粒构建体， IS 2的改变版本，其以高频率转置并携带 抗生素耐药性标记;有了这些结构， 可以使用突变方法容易地研究转座。 （二） 为了鉴定转座酶结合的DNA序列， 元件和在优先插入位点（例如，将E. coli）。 这将通过凝胶阻滞和DNA足迹来实现 问题研究 (iii)剖析转座酶介导的 重组事件;这些包括其切割的目标和性质 或加工反应，链转移反应的性质， 生成双金属复合产物，并形成更高阶的 DNA-蛋白质复合物，其中切割和连接反应是 以为发生。 这将使用体外转座测定来完成， 其反应产物将用标记底物在 聚丙烯酰胺凝胶和Southern杂交技术，使用琼脂糖 凝胶。

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.