PHYSICAL CHARACTERIZATION OF KATG, ENCODING CATALASE HPI OF ESCHERICHIA-COLI
PHYSICAL CHARACTERIZATION OF KATG, ENCODING CATALASE HPI OF ESCHERICHIA-COLI
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DOI：
10.1016/0378-1119(87)90038-2
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发表时间：
1987-01-01
期刊：
影响因子：
3.5
通讯作者：
LOEWEN, PC
中科院分区：
文献类型：
--
作者：
TRIGGSRAINE, BL;LOEWEN, PC
使用转座子Tn 5插入，将编码来自大肠杆菌的双功能过氧化氢酶-过氧化物酶HPI的基因定位在Clarke和Carbon质粒pLC 36 -19的3.8-kb HindIII片段上。将该片段亚克隆到pAT 153的HindIII位点中以产生pBT 22。通过BAL 31消化一端将插入片段的大小减小至过氧化氢酶表达的表观最小大小约100 μ g。2.5通过互补和Maxicall菌株中的表达确定。进一步减小大小或从相反端消化使基因失活。通过将320-bp Bgl Ⅱ片段克隆到启动子克隆载体pKK 232 -8中，证实了pBT 22中插入片段0 kb末端启动子的位置和方向。来自野生型菌株和katG 17：：Tn 10突变体的基因组DNA的Southern印迹中的差异用HincII消化并用pBT 22探测，证实先前定位在katG中的转座子位于HPI的2.5-kb编码区中。
The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions. This fragment was subcloned into the HindIII site of pAT153 to create pBT22. The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx. 2.5 kb as determined by complementation and expression in maxicall strains. Further reduction in size or digestion from the opposite end inactivated the gene. The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8. Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI.