大肠杆菌中TOL基因xylB和xylE的分子克隆
Molecular cloning of TOL genes xylB and xylE in Escherichia coli.
作者信息
Inouye S, Nakazawa A, Nakazawa T
出版信息
J Bacteriol. 1981 Mar;145(3):1137-43. doi: 10.1128/jb.145.3.1137-1143.1981.
Abstract
The xylB and xylE genes in the TOL plasmid of Pseudomonas putida mt-2, which code for benzyl alcohol dehydrogenase and catechol 2,3-oxygenase, respectively, were cloned onto plasmid pBR322 in Escherichia coli for detailed mapping. The xylB gene was mapped in a 2.9-kilobase region within the BamHI BC fragment of pTN2, an in vivo RP4-TOL recombinant, whereas the xylE gene was mapped in a 1.8-kilobase region within the BamHI BD fragment. The directions of transcription of these genes were deduced from the expression of the cloned genes which had been ligated in orientations opposite pBR322 at its BamHI site within the tetracycline resistance gene. The xylB and xylE genes are inducible by a specific inducer of the TOL pathway genes in the RP4-TOL recombinant, whereas they are not inducible in the pBR322-TOL hybrids. The regulatory regions involved in expression of the xylB and xylE genes do not appear to be located in the vicinity of the structural genes. Catechol 2,3-oxygenase formed in E. coli carrying an xylE-containing plasmid is similar, or identical, to that formed in P. putida carrying the TOL plasmid.
摘要
恶臭假单胞菌mt - 2的TOL质粒中的xylB和xylE基因，分别编码苄醇脱氢酶和儿茶酚2,3 - 加氧酶，被克隆到大肠杆菌的质粒pBR322上用于详细定位。xylB基因定位在体内RP4 - TOL重组体pTN2的BamHI BC片段内的一个2.9千碱基区域，而xylE基因定位在BamHI BD片段内的一个1.8千碱基区域。这些基因的转录方向是从在四环素抗性基因内其BamHI位点处与pBR322反向连接的克隆基因的表达推导出来的。xylB和xylE基因在RP4 - TOL重组体中可被TOL途径基因的特异性诱导剂诱导，而在pBR322 - TOL杂种中则不能被诱导。参与xylB和xylE基因表达的调控区域似乎不在结构基因附近。携带含xylE质粒的大肠杆菌中形成的儿茶酚2,3 - 加氧酶与携带TOL质粒的恶臭假单胞菌中形成的儿茶酚2,3 - 加氧酶相似或相同。
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