Genomic characterization of Tv-ant-1,a Caenorhabditis elegans tag-61 homologue from the parasitic nematode Trichostrongylus vitrinus ☆ Min Hu a , Bronwyn E. Campbell a , Mark Pellegrino a , Alex Loukas b , Ian Beveridge a , Shoba Ranganathan c,d , Robin B. Gasser a, ⁎ a Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b Helminth Biology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006, Australia c Department of Chemistry and Biomolecular Sciences & Biotechnology Research Institute, Macquarie University, Sydney, New South Wales 2109, Australia d Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore Received 14 February 2007; received in revised form 19 March 2007; accepted 24 March 2007 Received by A. Bernardi Available online 30 March 2007 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 α-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 (5V -UTR) upstream of Tv-ant-1 identified some promoter components, including GATA transcription factor, CAATand 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Adenine nucleotide translocator; ADP/ATP translocase; Caenorhabditis elegans; Genomic structure; Promoter elements; Transcription; Trichostrongylus vitrinus 1. Introduction The adenine nucleotide translocator (ANT) family of proteins, also called ADP/ATP translocases or ADP/ATP carrier proteins, belongs to the mitochondrial carrier family (MCF) (Walker and Runswick, 1993; Dahout-Gonzalez et al., 2006). These molecules represent the most abundant proteins in the inner membrane of the mitochondrion (Adrian et al., 1986) and transport ADP into and ATP out of the mitochondrion. In Gene 397 (2007) 12 – 25 www.elsevier.com/locate/gene Abbreviations: ANT, adenine nucleotide translocator; dsRNA, double- stranded RNA; EST, expressed sequence tag; L 1 , first-stage larva; L 2 , second- stage larva; L 3 , third-stage larva; L 4 , fourth-stage larva; RACE, rapid amplification of cDNA ends; RNAi, double-stranded RNA interference; RT- PCR, reverse transcription PCR. ☆ Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under accession nos. AM696280–AM696282. ⁎ Corresponding author. Tel.: +61 3 97312000; fax: +61 3 97312366. E-mail address: robinbg@unimelb.edu.au (R.B. Gasser). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.03.011