Genomic characterization of Tv-ant-1, a Caenorhabditis elegans tag-61 homologue from the parasitic nematode Trichostrongylus vitrinus

Abstract

A full-length cDNA (Tv-ant-1) encoding an adenine nucleotide translocator (ANT or ADP/ATP translocase) (Tv-ANT-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 894 nucleotides encoded a predicted protein of 297 amino acids, containing characteristic motifs [RRRMMM] and PX(D,E)XX(K,R). Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, cattle and human showed that Tv-ANT-1 is relatively conserved. Sequence identity was the greatest in and near the consensus sequence RRRMMM, and in the six hydrophobic regions predicted to be associated with alpha-helices and to traverse the cell membrane. Phylogenetic analyses of selected amino acid sequence data, using the neighbor-joining and maximum parsimony methods, revealed Tv-ANT-1 to be most closely related to the molecule (Ce-ANT-3) inferred from the tag-61 gene of C. elegans. Comparison of the genomic organization of the full-length Tv-ant-1 gene was similar to that of tag-61. Analysis of the region (5'-UTR) upstream of Tv-ant-1 identified some promoter components, including GATA transcription factor, CAAT and E-box elements. Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-ant-1 was transcribed in all developmental stages of T. vitrinus, including the first- to fourth- stage larvae (L(1)-L(4)) as well as female and male adults. RNA interference, conducted by feeding C. elegans with double-stranded RNA (dsRNA) from Tv-ant-1 cDNA (using the homologous gene from C. elegans as a positive control), revealed no gene silencing. In spite of nucleotide identities of 100% in 23-30 bp stretches of sequence between the genes Tv-ant-1 and tag-61, these identities seem to be insufficient to achieve effective silencing in C. elegans using the parasite homologue/orthologue Tv-ant-1. This first insight into an ANT of T. vitrinus provides a foundation for exploring its role in developmental and/or survival processes of trichostrongylid nematodes.