CHARACTERIZATION OF THE MANGANESE SUPEROXIDE-DISMUTASE CDNA AND GENE FROM THE HUMAN PARASITE ONCHOCERCA-VOLVULUS

The manganese-containing superoxide dismutase (MnSOD) is a major compo nent of the cellular defence mechanisms against the toxic effects of t he superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onc hocerca volvulus, sequences encoding the MnSOD were isolated and exami ned in this study. Degenerate primers were designed based upon conserv ed regions of MnSOD sequences from other organisms, and were used in P CR on reverse-transcribed O. volvulus total RNA and genomic DNA to ide ntify 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 s equence of the longest clone determined. The complete 5'-end of the Ov MnSOD cDNA was obtained using the rapid amplification of cDNA ends (RA CE) procedure with O. volvulus total RNA and was found to possess a sp liced leader sequence at the 5'-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required f or enzyme activity and metal binding. The 24 N-terminal amino acids en coded by the OvMnSOD cDNA comprise a putative mitochondrial transit pe ptide. The OvMnSOD gene was also isolated from an O. volvulus adult wo rm lambda fix II genomic library, a restriction map was constructed an d, the; nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctio ns. Potential regulatory elements were identified in the 5' genomic fl anking sequence. Southern-blot analysis with total worm genomic DNA in dicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.