大肠杆菌中过氧化氢酶HPII表达所需的katE和katF的克隆及物理特性分析

Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli.

作者信息

Mulvey M R, Sorby P A, Triggs-Raine B L, Loewen P C

机构信息

Department of Microbiology, University of Manitoba, Winnipeg, Canada.

出版信息

Gene. 1988 Dec 20;73(2):337-45. doi: 10.1016/0378-1119(88)90498-2.

DOI:10.1016/0378-1119(88)90498-2

PMID:2977357

Abstract

Two genes, katE and katF, affecting the synthesis of catalase HPII in Escherichia coli, have been cloned. The multistep cloning protocol involved: screening for the tet gene in a transposon interrupting the genes, selecting DNA adjacent to the transposon, and using it to probe a library of wild-type DNA to select clones from which katE and katF were subcloned into pAT153. The clones were physically characterized and the presence of the genes confirmed by complementation of their respective mutations. The location of the transposon insertions in the two genes was determined by Southern blotting of genomic digests to further confirm the identity of the cloned genes. A 93-kDa protein, the same size as the subunit of HPII, was encoded by the katE plasmid, indicating that katE was the structural gene for HPII. A 44-kDa protein was encoded by the katF plasmid.

摘要

已克隆出影响大肠杆菌中过氧化氢酶HPII合成的两个基因katE和katF。多步克隆方案包括：在中断这些基因的转座子中筛选tet基因，选择与转座子相邻的DNA，并使用其探测野生型DNA文库以选择克隆，然后将katE和katF亚克隆到pAT153中。对这些克隆进行物理表征，并通过各自突变的互补作用确认基因的存在。通过基因组消化产物的Southern印迹法确定转座子在这两个基因中的插入位置，以进一步确认克隆基因的身份。katE质粒编码一种93 kDa的蛋白质，其大小与HPII的亚基相同，表明katE是HPII的结构基因。katF质粒编码一种44 kDa的蛋白质。