Specificity Determinants in Cre/Lox Recombination

拟议项目的总体目标是了解通过什么机制 特定部位重组系统的特异性在 进化论。Cre蛋白及其重组靶点、Cre蛋白的LOX位点 噬菌体P1将作为实验模型。我们将建立一个 多用途实验系统，我们计划通过它来识别特定的 Lox位内的核苷酸和Cre蛋白中的特定氨基酸， 对专一性负责。为实现这一目标，我们将进行系统的 怀疑有助于特异性的区域的突变，无论是在 并将测量它们的重组特性。 基于这些信息，我们打算改变Cre/lox的特异性 通过组合lox位点和lox位点中相互兼容的突变来构建系统 蛋白。如果成功，这将回答我们在多大程度上理解 进化机制。作为一个实际结果，它将提供新的 具有不同特性的CRE/LOX系统的品种，增加了其 为了研究目的操纵真核细胞的实用程序。

The general aim of the proposed project is to understand the mechanism by which the specificity of the site-specific recombination systems changes during evolution. The Cre protein and its recombinational target, the lox sites of bacteriophage P1 will serve as an experimental model. We will build a multipurpose experimental system by which we plan to identify specific nucleotides within the loxsites and specific amino acids in the Cre protein, responsible for specificity. To achieve this goal we will carry out systematic mutagenesis of the regions suspected to contribute to specificity, both in the sites and in the recombinase and will measure their recombinational properties. Based on this information we intend to change the specificity of the Cre/lox system by combining mutually compatible mutations in the lox sites and in the protein. If successful, this will answer the question how well we understood the evolutionary mechanisms. As a practical outcome, it will provide new varieties of the Cre/lox system with different specificities, increasing its utility to manipulate eukaryotic cells for research purposes.