Specificity Determinants in Cre/Lox Recombination

Cre/Lox 重组中的特异性决定因素

• 批准号: 6540803
• 负责人: ARTHUR LANDY
• 金额: $ 4.03万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540803
• 关键词: Escherichia coli; Europe; bacteriophage P1; enzyme activity; gene mutation; genetic recombination; nucleotides; oligonucleotides; plasmids; recombinase; site directed mutagenesis; virus genetics; virus infection mechanism; virus integration; virus protein; virus replication

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.

拟议项目的总体目标是了解 位点特异性重组系统的特异性在 进化Cre蛋白及其重组靶点，即Lox位点 噬菌体P1将作为实验模型。新的起点上建设更加富裕 多用途实验系统，我们计划确定具体的 lox位点内的核苷酸和Cre蛋白中的特定氨基酸， 负责具体性。为了实现这一目标，我们将进行系统的 突变的区域怀疑有助于特异性，无论是在 位点和重组酶中，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， 系统通过组合在lox位点和在 蛋白如果成功的话，这将回答一个问题， 进化机制。作为一项实际成果，它将提供新的 具有不同特异性的Cre/lox系统的品种，增加其 操纵真核细胞用于研究目的。