Nakanishi S, Tazumi A, Aihara N, Sekizuka T, Amano K, Moore J E, Millar B C, Matsuda M
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Fuchinobe, Sagamihara, Japan.
Br J Biomed Sci. 2010;67(3):133-9. doi: 10.1080/09674845.2010.11730310.
Two sets of PCR primers are constructed to clone the cytochrome P450 structural gene, including putative promoter and terminator structures, and its adjacent genetic loci in Campylobacter lari isolates. The putative open reading frames (ORFs) of the P450 genes from 11 C. lari isolates (n=5 for urease-negative (UN) C. lari; n=6 urease-positive thermophilic campylobacters [UPTC]) examined consisted of 1365 or 1371 bases (455 or 457 amino acid residues), differing from those of the other thermophilic campylobacters (1359 [453] for C. jejuni and C. upsaliensis; 1368 [456] for C. coli). Each of the putative ORFs from the 11 isolates examined was also shown to carry start and stop codons and ribosome binding sites. Two putative promoter structures, consisting of sequences at the -35- and -10-like regions were also identified upstream of the ORFs. A single copy of the P450 gene in the genome was identified with UN C. lari JCM2530(T) and UPTC CF89-12, based on Southern blot hybridisation analysis. In addition, when reverse transcription polymerase chain reaction (RT-PCR) analyses were carried out, the transcription of the P450 structural gene in C. lari organisms in vivo was confirmed. The transcription initiation site for the gene was also determined. High nucleotide sequence similarities (95.2-98.8%) of the full-length P450 structural gene were shown with each of the 12 C. lari isolates. The UN C. lari and UPTC organisms showed similar findings with the neighbour-joining method, based on the sequence information of the P450 structural gene.
构建了两组聚合酶链式反应（PCR）引物，用于克隆空肠弯曲菌分离株中的细胞色素P450结构基因（包括推定的启动子和终止子结构）及其相邻的基因位点。对11株空肠弯曲菌分离株（脲酶阴性（UN）空肠弯曲菌5株；脲酶阳性嗜热弯曲菌（UPTC）6株）的P450基因推定开放阅读框（ORF）进行检测，其由1365或1371个碱基（455或457个氨基酸残基）组成，与其他嗜热弯曲菌不同（空肠弯曲菌和乌普萨拉弯曲菌为1359个碱基（453个氨基酸）；结肠弯曲菌为1368个碱基（456个氨基酸））。所检测的11株分离株的每个推定ORF也显示带有起始和终止密码子以及核糖体结合位点。在ORF上游还鉴定出两个推定的启动子结构，由类似-35和-10区域的序列组成。基于Southern印迹杂交分析，在UN空肠弯曲菌JCM2530(T)和UPTC CF89-12的基因组中鉴定出P450基因的单拷贝。此外，当进行逆转录聚合酶链反应（RT-PCR）分析时，证实了空肠弯曲菌体内P450结构基因的转录。还确定了该基因的转录起始位点。12株空肠弯曲菌分离株的全长P450结构基因均显示出较高的核苷酸序列相似性（95.2-98.8%）。基于P450结构基因的序列信息，采用邻接法，UN空肠弯曲菌和UPTC菌株显示出相似的结果。
