Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.
Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.
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Tn1000 介导的产气克雷伯菌组氨酸利用 (hut) 基因簇的插入诱变：hut 的遗传分析和 Tn1000 的异常靶标特异性。
DOI：
10.1128/jb.172.10.5991-5998.1990
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发表时间：
1990
影响因子：
3.2
通讯作者：
Bender,RA
中科院分区：
文献类型：
--
作者：
Schwacha,A;Cohen,JA;Gehring,KB;Bender,RA
将产气克雷伯氏菌的组氨酸利用（hut）基因以两个方向克隆到质粒pBR 325的HindIII位点中，并用Tn 1000对所得两个质粒pCB 120和pCB 121进行诱变。Tn 1000在pCB 121中的插入位点在整个质粒中均匀分布，而在pCB 120中的插入位点则不是。在一个小的，约1000的Tn 1000插入在“正”或γ-δ方向有很大的过量。3.5-质粒的内切酶区域。对pCB 120和pCB 121中Tn 1000插入的遗传分析表明，hutUH基因形成从hutU转录的操纵子，而hutC基因（编码hut特异性阻遏物）独立地从其自身的启动子转录。hutIG簇似乎不形成操纵子。奇怪的是，在hutI中的插入在针对hutG 504的互补测试中给出了两种不同的表型，这表明hutI包含两个功能不同的结构域，或者在hut簇内可能存在另一个未定义的位点。Tn 1000插入的集合允许在hut簇内分配基因边界，并且从携带hut基因中的插入的质粒表达的多肽的小细胞分析显示，hutI、hutG、hutU和hutH基因分别编码43、33、57和54千道尔顿的多肽。
The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of Tn1000 into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of Tn1000 insertions in the "plus" or gamma delta orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the Tn1000 insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter. The hutIG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of Tn1000 insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.