Characterization of the manganese superoxide dismutase cDNA and gene from the human parasite Onchocerca volvulus.

Abstract

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.