Genetics of Bacteriophage Lambda Red-Mediated Recombination

噬菌体 Lambda Red 介导的重组的遗传学

• 批准号: 0234991
• 负责人: Anthony Poteete
• 金额: $ 36万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0234991&HistoricalAwards=false
• 关键词: Genetics; Bacteriophage; Lambda; Red; Mediated

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.

这项研究的目标是进一步开发和分析一种简单的原核系统，该系统模拟真核生物中复杂的同源重组过程。研究人员用噬菌体Lambda的红色重组系统取代了正常的大肠杆菌重组系统。这种向Red系统的切换使细菌更容易进行重组事件，涉及单基因大小范围内的线性双链DNA分子。重组的DNA将通过感染改良的Lambda噬菌体转移到红色表达细菌中。这种噬菌体将其染色体注入细菌中存在的限制性内切酶切割成两部分的染色体，释放出含有抗生素耐药性基因CAT的线性双链DNA分子，两侧是与细菌染色体中LacZ基因部分相同的序列。线性DNA和环状细菌染色体的重组产生了一种菌株，其中LacZ被CAT取代。与起始菌株不同，这些重组子既对抗生素具有抗药性，又不能利用糖乳糖；它们很容易选择和计数。通过实验来探索各种重组促进基因在Red介导的基因替换中的作用。将特别研究细菌基因recf、reco、recr、recA和recQ，以及lambda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物种结构特征的依赖性。有待解决的问题包括该系统是否对某些序列比对其他序列更有效地响应，以及带有不可消化的硫代键的各种人工DNA是否在Red介导的重组中活跃。