A CLONED IMMUNOGLOBULIN CDNA FRAGMENT ENHANCES TRANSPOSITION OF IS-ELEMENTS INTO RECOMBINANT PLASMIDS
A CLONED IMMUNOGLOBULIN CDNA FRAGMENT ENHANCES TRANSPOSITION OF IS-ELEMENTS INTO RECOMBINANT PLASMIDS
复制标题
DOI：
10.1093/nar/10.15.4525
复制
发表时间：
1982-01-01
影响因子：
14.9
通讯作者：
ZAMIR, A
中科院分区：
文献类型：
--
作者：
AMSTER, O;SALOMON, D;ZAMIR, A
有证据表明，体外重组产生的新 DNA 序列排列可能引发 IS 元件的高频转座。将来自 MOPC 321 骨髓瘤的免疫球蛋白 K 轻链 cDNA 的 109 bp Bam HI 片段克隆到 pBR313 的 Bam HI 位点。克隆片段从 Gly 57 密码子延伸至 V-J 连接处。从新鲜转化的克隆中分离出 50 个质粒 DNA，其中有 6 个被鉴定出 IS1 或 IS5 插入。在传代培养几个生长周期后检测到额外的转座事件。 IS1 的三个独立插入发生在 TcRoperon 的启动子区域。所有 IS5 和剩余的 IS1 插入均位于克隆 DNA 序列上游的 TcR 区域。与IS1末端同源的序列，或对应于IS5靶位点处共有序列的序列分别存在于IS1或IS5的估计插入位点附近。与携带 pBR313 的细胞相比，携带携带克隆 DNA 的重组质粒的细菌的生长速度较携带 pBR313 的细胞降低，这表明由两种类型的质粒制成的融合基因产物对细胞生长具有抑制作用。 IS 插入减轻了这种抑制作用，从而提供了选择性优势，仅在仅以两个方向之一携带克隆 DNA 的质粒中发现。在其他类型的重组质粒中没有观察到 IS 元件的事实表明，仅选择压力不足以解释观察到的频繁 IS 插入，并且距 IS 插入位点一定距离的序列可能在转座频率的调节中至关重要。
Evidence is presented indicating that a novel DNA sequence arrangement generated byin vitrorecombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin K 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 TcRoperon. All IS5 and the remaining IS1 insertions were located in the TcRregion 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.