Amster O, Salomon D, Zamir A
Nucleic Acids Res. 1982 Aug 11;10(15):4525-42. doi: 10.1093/nar/10.15.4525.
Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.
有证据表明，体外重组产生的一种新的DNA序列排列可能引发IS元件的高频转座。将来自MOPC 321骨髓瘤免疫球蛋白κ轻链cDNA的109 bp Bam HI片段克隆到pBR313的Bam HI位点。克隆片段从甘氨酸57密码子延伸至V-J连接点。从新转化的克隆中分离出的50个质粒DNA中有6个鉴定出IS1或IS5的插入。在经过几个生长周期的传代培养后检测到了更多的转座事件。IS1在TcR操纵子的启动子区域发生了三次独立插入。所有的IS5和其余的IS1插入都位于克隆DNA序列上游的TcR区域。分别在IS1或IS5估计的插入位点附近存在与IS1末端同源的序列，或与IS5靶位点的共有序列相对应的序列。携带以任一方向携带克隆DNA的重组质粒的细菌相对于携带pBR313的细胞生长速率降低，这表明由两种类型质粒产生的融合基因产物对细胞生长具有抑制作用。发现仅在以两种方向之一携带克隆DNA的质粒中存在可减轻这种抑制作用从而提供选择优势的IS插入。在另一种重组质粒中未观察到IS元件这一事实表明，仅选择性压力不足以解释观察到的频繁IS插入，并且与IS插入位点有一定距离的序列可能在转座频率的调节中起关键作用。
