Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Aspartic proteases are a relatively small group of enzymes which express in various nematodes including Onchocerca volvulus. An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic protease of O. volvulus, was made by slot blot analysis in a closely related species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that [32]P-labeled cDNA insert. To prepare the initial probe, OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridization of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the [32]P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the same probe revealed a single band except when predicted internal restriction sites were affected. It was confirmed that Onchocerca contains a single copy of the gene corresponding to this cDNA insert per haploid genome

Molecular cloning of adenylate kinase from the human filarial parasite Onchocerca volvulus

Adenylate kinases [ADK] are ubiquitous enzymes that contribute to the homeostasis of adenine nucleotides in living cells. In this study, the cloning of a cDNA encoding an adenylate kinase from the filaria Onchocerca volvulus has been described. Using PCR technique, a 281 bp cDNA fragment encoding part of an adenylate kinase was isolated from an O. volvulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone through the searching the GenBank expressed sequence tag database. The full-length cDNA encodes 236 amino acid residues with a predicted molecular mass of 26.177 kDa. The deduced amino acid sequence exhibited 80% identity to the homologous adenylate kinase identified from Caenorhabditis elegans. Domain analysis of the resulting protein sequence was found to contain "adenylate kinase signature" motif which is highly conserved in all known ADKs. Multiple alignments showed that the N-terminal is well conserved, whereas the C-terminal is the most variable region

Hydrolytic activity of an onchocercoma

The onchocercoma or nodule produced by the nematode Onchocerca volvulus (Filarioidea) in the skin of patients suffering from onchocerciasis has not been examined by histochemical techniques. In this work we have used histochemical techniques to evaluate 5 hydrolytic enzymes, namely phosphatases, esterases and beta-glucuronidase. The results show increased enzymatic activity at the sites of major metabolic activity and within reactive cells including macrophages (mc) and giant cells (gc) of the onchocercoma