Triggs-Raine B L, Loewen P C
Gene. 1987;52(2-3):121-8. doi: 10.1016/0378-1119(87)90038-2.
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 maxicell 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.
利用转座子Tn5插入，将来自大肠杆菌的双功能过氧化氢酶-过氧化物酶HPI的编码基因定位在克拉克和卡本质粒pLC36 - 19的一个3.8 kb HindIII片段上。该片段被亚克隆到pAT153的HindIII位点，构建成pBT22。通过BAL 31从一端消化，将插入片段的大小减小到过氧化氢酶表达的明显最小尺寸，约为2.5 kb，这是通过在大细胞菌株中的互补和表达确定的。进一步减小尺寸或从另一端消化会使基因失活。通过将一个320 bp的BglII片段克隆到启动子克隆载体pKK232 - 8中，证实了pBT22中插入片段0 kb端启动子的位置和方向。用HincII消化野生型菌株和katG17::Tn10突变体的基因组DNA，并用地高辛标记的pBT22进行Southern杂交，结果表明，先前定位在katG中的转座子位于HPI的2.5 kb编码区域内。
