We are interested in cloning acetylcholine receptor (AChR) subunit genes from the human parasitic nematode Onchocerca volvulus and expressing the genes in the laboratory nematode C. elegans to facilitate study of potential therapeutic drug targets. Onchocerca causes riverblindness in tropical regions of Africa and Central and South America. We have screened an Onchocerca volvulus cDNA library at reduced stringency with four different C. elegans hybridization probes corresponding to highly conserved region of the AChR subunit genes
unc-29,
unc-38,
lev-1, and a new AChR alpha subunit located near
unc-74. Hybridization probes were made by PCR using nonradioactively labeled digoxigenin dUTP and PCR primers that flanked highly conserved subunit sequences. Two strongly positive Onchocerca clones were identified and plaque-purified in screening 470,000 clones (47 plates, 94 filters) from the cDNA library. The two clones are different isolates of an Onchocerca homolog to the C. elegans
unc-29 nonalpha AChR subunit gene. The longer Onchocerca cDNA contains the sequence expected for the amino terminal two-thirds of the AChR subunit, including the signal sequence. Over this region, the Onchocerca clone is 88% identical to C. elegans
unc-29 at the amino acid level and about 75% identical at the nucleic acid level. The sequences of the first three (TM1-3) of the four highly conserved AChR transmembrane domains are virtually identical between Onchocerca and C. elegans. These results indicate that the function of the
unc-29 nonalpha subunit of the levamisole AChR has been highly conserved in evolution during the separation of free-living from parasitic nematodes. As only the 3' end of our clone is missing, reconstruction of a full-length Onchocerca clone should be possible, allowing tests of subunit functionality by substitution of the Onchocerca subunit for the C. elegans
unc-29 subunit in transgenic nematodes and in frog oocyte expressions assays.