Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

ABSTRACT

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