Genetics of Bacteriophage Lambda Red-Mediated Recombination

本研究的目的是进一步发展和分析一个简单的原核系统，该系统模拟了真核生物中同源重组的复杂过程。研究人员已经用λ噬菌体的Red重组系统取代了大肠杆菌的正常重组系统。这种向Red系统的转换使细菌更容易进行涉及单基因大小范围内的线性双链DNA分子的重组事件。重组的DNA将通过用λ噬菌体的修饰版本感染而被递送到表达Red的细菌中。这种噬菌体注入其染色体，该染色体被细菌中存在的限制性内切酶在两个位置切割，释放含有抗生素抗性基因cat的线性双链DNA分子，其侧翼序列与细菌染色体中的lacZ基因的部分相同。线性DNA和环状细菌染色体之间的相互作用产生了一种菌株，其中lacZ被猫取代。与起始菌株不同，这些重组体都具有耐药性，并且不能利用糖乳糖;它们很容易被选择和枚举。实验提出了探索各种重组促进基因的参与红色介导的基因置换。将特别研究细菌基因recF、recO、recR、recA和recQ以及λ基因orf和rap。在第二组实验中，将检查重组事件对线性重组DNA种类的结构特征的依赖性。要解决的问题包括系统是否更有效地响应某些序列比其他人，以及是否各种人工DNA轴承难消化的硫代磷酸酯键是积极的红色介导的重组。

The goal of the research is to further the development and analysis of a simple prokaryotic system which models the complex process of homologous recombination in eukaryotes. The investigators have replaced the normal recombination system of the bacterium Escherichia coli with the Red recombination system of bacteriophage lambda. This switch to the Red system makes the bacterium far more prone to carry out recombination events involving linear double-stranded DNA molecules in the size range of single genes. The recombining DNA will be delivered into the Red-expressing bacteria by infection with a modified version of bacteriophage lambda. This bacteriophage injects its chromosome, which is cut in two places by a restriction endonuclease present in the bacterium, releasing a linear double stranded DNA molecule containing the antibiotic resistance gene cat, flanked by sequences identical to parts of the lacZ gene in the bacterial chromosome. Recombination between the linear DNA and the circular bacterial chromosome produces a strain in which lacZ is replaced by cat. Unlike the starting strain, these recombinants are both antibiotic-resistant and unable to utilize the sugar lactose; they are readily selected and enumerated. Experiments are proposed to explore the involvement of various recombination-promoting genes in Red-mediated gene replacement. The bacterial genes recF, recO, recR, recA, and recQ, and the lambda genes orf and rap will be studied in particular. In a second set of experiments, the dependence of the recombination event upon structural features of the linear recombining DNA species will be examined. Questions to be addressed include whether the system responds more efficiently to some sequences than to others, and whether various artificial DNAs bearing indigestible phosphorothioate linkages are active in Red-mediated recombination.