HOMOLOGOUS RECOMBINATION SYSTEMS OF PHAGES P1 & LAMBDA

长期目标是了解同源重组是如何 在已知重组酶过多的两种情况下增强， 或接近成功：重组蛋白A在大肠杆菌中的重组和重组 由噬菌体Lambda“Red”系统催化。因为不受控制的遗传 重排可能在癌症和一些遗传性疾病中起作用，而 “受控”同源重组是生物的重要来源 多样性，重要的是要了解影响 复合频率。 研究噬菌体PL(REF)的新功能的具体目的 在大肠杆菌中刺激某些依赖于RecABC的同源重组 是：(I)REF的鉴定、纯化和特性 基因产物；(2)REF作用机制的确定 促进重组；(3)REF监管的一般特征； (4)REF在Pl生长和溶源中的作用。对应 方法包括：(I)克隆REF，用于生产过剩、放射化学 提纯，测试可能的活动，(Ii)测试各种 REF效应的重组过程，动员已知的lacDelta Ref作用于Lambda噬菌体的底物；(Iii)推定的 通过检测克隆的Pl片段检测阴性和阳性对照元件 各种监管情况，识别共同监管的P1功能 (4)测试噬菌体的生长、建立和/或维持 当大肠杆菌同源重组和/或Pl位点特异性时的溶原性 重组(cre/lox)不足。 兰姆达红色研究的具体目标是：(一)确定 除了Lambda核酸外切酶和Beta蛋白(如果有)外，Lambda PL还具有其他功能 除了Lambda复制功能外，还需要用于重组； (Ii)体外系统的开发；(Iii)功能的测定 (Iv)Lambdagam-RecBC的特性 体内的核酸酶相互作用。相应的方法包括：(1) 使用我们的外源lacPO质粒，与适当的 Lambdabio替代噬菌体用于体内重组研究 缺乏(几乎)所有PL功能的遗传标记噬菌体，并具有 Lambdadv质粒；(Ii)体外重组Lamdadv的检测 电泳法或更灵敏的方法；(Iii)测试 Lambdassb在大肠杆菌SSB(Ts)突变体中的表达； GAM高产表达载体对大肠杆菌(REC+)紫外线敏感性和细胞毒性的影响 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.