Kulakauskas S, Wikström P M, Berg D E
Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110.
J Bacteriol. 1991 Apr;173(8):2633-8. doi: 10.1128/jb.173.8.2633-2638.1991.
An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.
开发了一种将克隆的大肠杆菌DNA中的突变从质粒载体转移到细菌染色体的有效方法。携带因插入抗性基因而发生突变的质粒的细胞，用含有同源克隆DNA的λ噬菌体感染，所得裂解物用于转导。通过其氨苄青霉素敏感表型将染色体转导子（重组体）与质粒转导子区分开来，或者通过使用recBC sbcB大肠杆菌菌株作为受体来避免质粒转导子。在非溶原菌中获得的染色体转导子通常是单倍体，这是由于对λ载体的选择，而在溶原菌中获得时则是部分二倍体。从转导细菌中获得了携带抗性标记并能高频转导它的噬菌体纯株。本文所述的基于λ噬菌体的将突变等位基因转移到细菌染色体中的方法，对于基因功能和基因组结构的各种分析应该是有用的。
