HOMOLOGOUS RECOMBINATION SYSTEMS OF PHAGES P1 & LAMBDA

长期目标是了解同源重组是如何 在已知重组酶过量的两种情况下增强， RecA介导的重组在E.大肠杆菌和重组 由噬菌体λ "红"系统催化 由于不受控制的遗传 重排可能在癌症和一些遗传疾病中起作用，而 "受控"同源重组是生物学上的一个重要来源。 多样性，重要的是要了解影响因素 复合频率 研究噬菌体PL（"ref"）的新功能的具体目的是， 刺激E.杆菌 是：（一）鉴定，纯化，和表征的参考 基因产物;（ii）确定ref功能的机制 促进重组;（三）调节ref的一般特征; (iv)ref在Pl生长和溶原性中作用。 对应 方法包括：（i）克隆过量生产的参考，放射化学 纯化，测试可能的活动，（ii）测试各种 用于ref效应的重组过程，动员已知的lacDelta （iii）鉴定推定的 阴性和阳性对照元件， 各种调控情况下，鉴定共同调控的P1功能 （iv）测试噬菌体生长、建立和/或维持 溶原性，当E.大肠杆菌同源重组和/或P1位点特异性 重组（cre/lox）是缺陷的。 Lambda Red研究的具体目标是：（i）确定 除了Lambda核酸外切酶和β蛋白（如果有的话）之外，Lambda PL还具有其他功能 除了λ复制功能外，还需要重组; (ii)体外系统的开发;（iii）功能的确定 （iv）Lambda ssb蛋白的表征; 体内核酸酶相互作用。 相应的方法包括： 使用我们的exo bet lacPO质粒，结合适当的 用于体内重组研究的Lambdabio置换试剂盒 遗传标记的缺失（几乎）所有PL功能， Lambdadv质粒;（ii）通过PCR测定体外Lamdadv重组; 电泳或更敏感的方法;（iii）测试 Lambdassb在E. coli ssb（ts）突变体;（iv）确定 γ-过表达质粒对E.大肠杆菌（rec+）紫外线敏感性和 重组能力

Long-term objectives are to understand how homologous recombination is enhanced in two situations where known recombination enzymes are in excess, or nearly so: RecA-mediated recombination in E. coli and recombination catalyzed by the phage Lambda "Red" system. Since uncontrolled genetic rearrangement may play a role in cancer and some genetic diseases, whereas "controlled" homologous recombination is an important source of biological diversity, it is important to understand the factors that affect recombination frequencies. Specific aims of the study of a novel function of phage PL ("ref") that stimulates certain RecABC-dependent homologous recombinations in E. coli are: (i) identification, purification, and characterization of the ref gene product; (ii) determination of the mechanism by which ref function promotes recombination; (iii) the general features of regulation of ref; (iv) the role of ref in Pl growth and lysogeny. Corresponding methodologies include: (i) cloning ref for overproduction, radiochemical purification, testing for likely activities, (ii) testing a variety of recombination processes for ref effects, mobilizing the known lacDelta substrates for ref onto Lambda phages; (iii) identification of putative negative and positive control elements by testing cloned Pl fragments in various regulatory situations, identification of P1 functions coregulated with ref; (iv) testing for phage growth, establishment and/or maintenance of lysogeny when E. coli homologous recombination and/or Pl site-specific recombination (cre/lox) is deficient. Specific aims of the Lambda Red studies are: (i) determination of which Lambda PL functions besides Lambda exonuclease and Beta protein (if any) are needed, in addition to Lambda replication functions, for recombination; (ii) development of an in vitro system; (iii) determination of the function of Lambda ssb protein; (iv) characterization of the Lambdagam-RecBC nuclease interaction in vivo. Corresponding methodologies include: (i) use of our exo bet lacPO plasmids, in conjunction with appropriate Lambdabio substitution phages for in vivo recombination studies with genetically marked phages lacking (nearly) all PL functions, and with Lambdadv plasmids; (ii) assay of in vitro Lamdadv recombination by electrophoretic, or more sensitive, methods; (iii) testing the effect of Lambdassb expression in E. coli ssb (ts) mutants; (iv) determining the effect of gam-overproducing plasmids on E. coli (rec+) UV-sensitivity and recombination proficiency.