Raczyńska-Pawelec A, Pawelec D, Rozynek E, Jagusztyn-Krynicka E K
Institute of Microbiology, University of Warsaw, Poland.
Acta Microbiol Pol. 1995;44(3-4):227-41.
asd gene of the human enteric pathogen, Campylobacter jejuni Dz72/92, has been isolated from a genomic library constructed in the expression plasmid vector pUC8-2 in Escherichia coli. The gene has been fished out by complementation of the asd gene deletion present in the genome of E. coli chi 6097. The smallest recombinant plasmid (pUWM3) able to confer Asd+ phenotype contains a 1.8 kb insert cloned into HindIII site located within the multi-cloning site of the pUC8-2 vector. The origin of the insert has been confirmed by hybridization. Several pieces of evidence indicate that the expression of the cloned house-keeping gene is driven from its own promoter, which can be recognised by E. coli RNA polymerase. The asd gene promoter has been located on 300 kb HindIII-DraI fragment of pUWM3. Recombinant plasmid pUWM3 specifies a new 38 kDa protein which we believe is the asd gene product. Overproduction of the 38 kDa protein due to the transcription originating from the vector lacZ gene promoter is toxic for the cells.
从空肠弯曲菌Dz72/92（一种人类肠道病原体）中分离出的asd基因，是从构建于大肠杆菌表达质粒载体pUC8 - 2中的基因组文库中获得的。该基因是通过对大肠杆菌chi 6097基因组中存在的asd基因缺失进行互补筛选出来的。能够赋予Asd⁺表型的最小重组质粒（pUWM3）包含一个1.8 kb的插入片段，该片段克隆到位于pUC8 - 2载体多克隆位点内的HindIII位点。通过杂交证实了插入片段的来源。几条证据表明，克隆的管家基因的表达由其自身启动子驱动，该启动子可被大肠杆菌RNA聚合酶识别。asd基因启动子位于pUWM3的300 kb HindIII - DraI片段上。重组质粒pUWM3编码一种新的38 kDa蛋白，我们认为它就是asd基因的产物。由于源自载体lacZ基因启动子的转录导致38 kDa蛋白过量产生，对细胞有毒性。
