短小芽孢杆菌质粒pCJ3的衍生质粒pAl32的酶谱及抗性基因定位

Restriction Map and Tc^r Gene Localization of the Truncated Plasmid pAL32 Derived from the Plasmid pCJ3 in Bacillus pumilus

从短小芽孢杆菌四环素抗性质粒pCJ3截短的衍生质粒pAL32,经用6种限制酶双酶解试验,根据琼脂糖凝胶电泳带估算各片段的分子量,作出了pAL32的6种限制酶图谱。利用已除去复制功能的金黄色葡萄球菌质粒pUB110与pAL32构建的Km^rTc^r的双抗性重组质粒pSC33为载体,在EcoRV位点克隆的λDNA片段的重组子均为Km^rTc^s。用pUB110与pAL32在PvuⅡ位点连接后的重组质粒也为Km^rTc^s。根据这些重组子四环素抗性的失活,推知pAL32上EcoRV与PvuⅡ切点均位于其四环素抗性基因上。

The truncated Tc plasmid pAL32 derived from the plasmid pCJ3 wascleaved by reciprocal double digestions with 6 restriction endonucleases. Themolecular weights of the fragments were estimated by agarose gel electrophoresis.A simple restriction map of pAL32 was thus constructed. Recombinants were obtain-ed by cloning EcoR Ⅴ fragments of λDNA using Km^r Tc^r plasmid pSC33 as vectorwhich was constructed from the plasmid pAL32 and the removed origin plasmidpUB110. All of them are resistant to Km and sensistive to Tc. A Km^rTc^n recom-binant plasmid was also obtained by Pvu Ⅱ digest from the plasmid pAL32 andpUB110. Based on the inactivation of the tetracycline resistance of these recombi-nants, it is suggested that EcoRⅤ and Pvu Ⅱ sites are located on Tc^r gene of thePlasmid pAL32.