Qadri M I, Flores C C, Davis A J, Lichtenstein C P
Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, England.
J Mol Biol. 1989 May 5;207(1):85-98. doi: 10.1016/0022-2836(89)90442-7.
The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.
大的（14 kb；kb = 10³个碱基）细菌转座子Tn7（编码对甲氧苄啶和链霉素/壮观霉素的抗性）具有不同寻常的特性。与其他元件一样，Tn7转座效率低且靶位点特异性低，但Tn7还会以独特的方向高频转座到大肠杆菌染色体中一个称为attTn7的首选“附着”位点，同样也会转座到含有attTn7的质粒中。我们开发了一种基于新型噬菌体M13的检测系统，以测量Tn7转座到在克隆的1 kb染色体DNA片段上含有attTn7的M13mp噬菌体载体的频率。从Tn7供体菌株收获的噬菌体用于感染受体细菌，并选择对甲氧苄啶具有抗性的细菌。转座频率表示为每噬菌斑形成单位中对甲氧苄啶抗性菌落的数量，发现转座到M13mp::attTn7的频率约为10⁻⁴，相比之下，转座到插入了约1 kb其他“通用型”DNA的M13mp重组体的频率为10⁻¹⁰。通过对M13mp::attTn7的缺失分析，我们表明attTn位于一个64个碱基对的区域内；与实际插入位点相邻且编码glmS基因羧基末端的序列是必需的。该检测还为Tn7右端赋予的转座免疫提供了证据。
