Russell C B, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Bacteriol. 1989 May;171(5):2614-8. doi: 10.1128/jb.171.5.2614-2618.1989.
Transfer of an allele from a donor DNA to a recipient DNA molecule was selected by the loss of a dominant conditional lethal mutation previously incorporated ito the gene of interest in the recipient DNA. Both the Escherichia coli chromosome and plasmids carrying E. coli genes were used successfully as donor molecules. Recipient molecules for these exchanges were constructed in vitro by using the rpsL gene, which confers sensitivity to streptomycin, to replace segments of specific E. coli genes located either on multicopy plasmids or in the E. coli chromosome. Plasmids carrying such replacements were capable of acquiring chromosomal alleles of the gene(s) of interest, and strains carrying rpsL replacements in the chromosome were capable of acquiring plasmid-encoded alleles at the sight of the rpsL replacement. In both situations, these allele transfers resulted in loss of the rpsL gene from the recipient DNA molecule. The desired transfer events constituted a large percentage of these events, which gave rise to viable colonies when appropriate donor-recipient pairs were subjected to streptomycin selection. Thus, this is a useful approach for transferring alleles of interest from plasmids to the E. coli chromosome and vice versa.
通过先前整合到受体DNA中目标基因的显性条件致死突变的缺失，选择将一个等位基因从供体DNA转移到受体DNA分子上。携带大肠杆菌基因的大肠杆菌染色体和质粒均成功用作供体分子。通过使用赋予对链霉素敏感性的rpsL基因，在体外构建用于这些交换的受体分子，以取代位于多拷贝质粒或大肠杆菌染色体上的特定大肠杆菌基因片段。携带此类替换的质粒能够获得目标基因的染色体等位基因，而在染色体中携带rpsL替换的菌株能够在rpsL替换位点获得质粒编码的等位基因。在这两种情况下，这些等位基因转移导致受体DNA分子中rpsL基因的缺失。当合适的供体-受体对进行链霉素选择时，所需的转移事件在这些事件中占很大比例，从而产生了活菌落。因此，这是一种将目标等位基因从质粒转移到大肠杆菌染色体，反之亦然的有用方法。
