Determination of aspartic protease gene dosage in the Onchocerca volvulusgenome

Document Type: Short paper

Author

Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

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. gibsonigenome. Nylon
membrane was loaded with serial dilutions ofgenomic 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 blotanalysis 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.

Keywords