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首页> 外文期刊>Journal of bacteriology >hisT is part of a multigene operon in Escherichia coli K-12.
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hisT is part of a multigene operon in Escherichia coli K-12.

机译：hisT是大肠杆菌K-12中多基因操纵子的一部分。

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The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.
机译：克隆了大肠杆菌K-12 hisT基因，并在多拷贝质粒上分析了其组织和表达。在已知含有purF基因的Clarke-Carbon质粒上分离出编码tRNA伪尿苷合酶I（PSUI）的hisT基因。通过在hisT突变菌株中恢复PSUI酶活性的产生和抑制琥珀突变的能力，暗示了该质粒上hisT基因的存在。将含有hisT基因的2.3碱基对的HindIII-ClaI限制性片段亚克隆到质粒pBR322中，并将所得的质粒（命名为psi 300）用限制性酶作图。具有不同种类的hisT突变的互补分析和tRNA结构分析证实质粒psi 300包含hisT结构基因。酶分析显示，质粒psi 300大约在30分钟内产生了PSUI活性。是野生型水平的20倍。包含来自插入到lac启动子下游的psi 300质粒的限制性片段的亚克隆确定了hisT基因从HindIII位点朝向ClaI位点定向。构建含有插入或缺失突变的质粒psi 300的其他亚克隆和衍生物，并测定其在小细胞中的PSUI活性的产生和蛋白质的产生。这些实验表明：（i）近端的1.3碱基碱基的HindIII-BssHII限制性片段含有hisT基因的启动子，并编码不是PSUI的45,000道尔顿的多肽; （ii）1.0碱基对的BssHII-ClaI远端限制性片段编码31,000道尔顿的PSUI多肽; （iii）与PSUI相比，合成了45,000道尔顿的多肽大约过量了八倍; （iv）两个多肽的合成是偶联的，表明这两个基因是操纵子的一部分。 mini-Mu d1（lac Km）噬菌体插入质粒psi 300证实了hisT基因是操纵子的下游基因。

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