Henkle-Dührsen K, Tawe W, Warnecke C, Walter R D
Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Hamburg, Germany.
Biochem J. 1995 Jun 1;308 ( Pt 2)(Pt 2):441-6. doi: 10.1042/bj3080441.
The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5'-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5'-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5' genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.
含锰超氧化物歧化酶（MnSOD）是细胞防御超氧自由基毒性作用机制的主要组成部分。在关于抗氧化酶及其在人体寄生线虫旋盘尾丝虫中的保护作用的研究框架内，本研究分离并检测了编码MnSOD的序列。基于其他生物体MnSOD序列的保守区域设计了简并引物，并将其用于对逆转录的旋盘尾丝虫总RNA和基因组DNA进行PCR，以鉴定编码旋盘尾丝虫MnSOD（OvMnSOD）的部分cDNA和基因组DNA片段。基因组DNA PCR产物用于筛选旋盘尾丝虫成虫λ unizap II cDNA文库，并确定最长克隆的核苷酸序列。使用cDNA末端快速扩增（RACE）程序，以旋盘尾丝虫总RNA获得了OvMnSOD cDNA的完整5'端，发现其在5'末端具有剪接前导序列。推导的一级序列编码一个25 kDa的蛋白质，该蛋白质具有酶活性和金属结合所需的保守残基。OvMnSOD cDNA编码的24个N端氨基酸包含一个推定的线粒体转运肽。还从旋盘尾丝虫成虫λ fix II基因组文库中分离出OvMnSOD基因，构建了限制性图谱并确定了核苷酸序列。发现OvMnSOD基因具有五个外显子和四个内含子，具有一致的剪接位点连接。在5'基因组侧翼序列中鉴定出潜在的调控元件。用全虫基因组DNA进行的Southern杂交分析表明该基因是单拷贝基因，其限制性图谱与分离出的基因一致。
