Elsevier

Gene

Volume 397, Issues 1–2, 1 August 2007, Pages 12-25
Gene

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

https://doi.org/10.1016/j.gene.2007.03.011Get rights and content

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 (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 (L1–L4) 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.

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 eukaryotic cells, mitochondrial ATP production and cellular ATP consumption are tightly linked to ANT, the kinetics of which have been proposed to play a major regulatory role in mitochondrial oxidative phosphorylation. Also, ANT is recognized as a central component of the mitochondrial permeability transition pore associated with apoptosis (Halestrap and Brennerb, 2003).

The ANTs described are relatively conserved in sequence and structure among species (Santamaria et al., 2004). Many species of eukaryote studied to date possess more than one ANT isoform, but the number of isoforms and their expression and/or function can vary among species (Santamaria et al., 2004). For instance, in humans, multiple isoforms (i.e., ANT-1, ANT-2, ANT-3 and ANT-4 = AAC4) have been isolated and characterized (Battini et al., 1987, Neckelmann et al., 1987, Houldsworth and Attardi, 1988, Cozens et al., 1989, Dolce et al., 2005), with each isoform displaying a relatively tissue-specific expression profile (Neckelmann et al., 1987, Houldsworth and Attardi, 1988, Li etal., 1989, Dolce et al., 2005). Also, the free-living nematode, Caenorhabditis elegans (order Rhabditida), possesses at least three distinct ANT isoforms (encoded by the genes tag-61, tag-316 and K01H12.2; see http://www.wormbase.org). Utilizing double-stranded RNA interference (RNAi; Fire et al., 1998), it has been demonstrated that tag-61 can be silenced in C. elegans, leading to phenotypes including larval arrest (Lva) (Simmer et al., 2003), embryonic lethality (Let) (Gönczy, 2000, Sonnichsen, 2005), general pace of development abnormal early embryo (Emb) (Sonnichsen et al., 2005) and maternal sterility (Ste) (Rual et al., 2004). Knock-down of each of the other two genes (tag-316 and K01H12.2) has been associated with the same RNAi phenotypes (Kamath, 2003, Simmer, 2003, Rual, 2004, Sonnichsen, 2005). In spite of this information, little is known about the precise functional roles of C. elegans ANTs, reflecting the current designations (i.e., tag = temporarily assigned genes, or a code). Nonetheless, achieving knock-down in C. elegans has demonstrated that these genes or gene products are central to the development and/or survival of this free-living nematode, which stimulated our interest in exploring homologues in Trichostrongylus vitrinus (order Strongylida; an economically important parasitic nematode of livestock), particularly now that RNAi has been shown to achieve knock-down for some genes in some parasitic nematodes, including members of the family Trichostrongylidae (e.g., Issa et al., 2005, Kotze and Bagnall, 2006; reviewed by Geldhof et al., 2007).

In spite of the functional significance of ANTs in C. elegans (e.g., Gönczy, 2000, Sonnichsen, 2005), there is no published information on them for any parasitic nematode. Clearly, the isolation and characterization of ANTs and their genes from a parasitic nematode would provide a starting point for a better understanding of their roles in developmental and/or survival processes, and would enable comparative analyses between C. elegans and the parasitic nematodes. Recently, in a genomic study (Nisbet and Gasser, 2004), we isolated and characterized an expressed sequence tag (EST), designated TVf01_D06 (181 bp), from an adult female-enriched cDNA library for T. vitrinus. The amino acid sequence inferred from this EST had the highest similarity/identity (E-value: 3e−23) to the protein encoded by the tag-61 gene (or T27E9.1; see http://www.wormbase.org/) from C. elegans (unpublished findings). Therefore, the aims of this study were to isolate and characterize full-length complementary and genomic DNAs corresponding to EST TVf01_D06 and to carry out comparative analyses with related molecules encoded by other organisms (at both the protein and genomic levels), to explore transcription in different developmental stages of T. vitrinus and to attempt gene knock-down in C. elegans using the T. vitrinus homologue.

Section snippets

Propagation of the parasite

Merino lambs (males; 8–12 weeks of age), maintained under helminth-free conditions, were infected intraruminally with 30,000 infective third-stage larvae (L3) of T. vitrinus. The patency of the infection (∼ 24 days) was established by the detection of strongylid eggs in the faeces using the McMaster flotation method (MAFF, 1977). First- and second-stage larvae (L1 and L2) and L3 were collected after 1, 3 and 7 days of incubation of faeces at 28 °C, respectively, and purified by repeated

Characterization of the cDNA of Tv-ant-1 from T. vitrinus and comparison with homologues at the amino acid sequence level

The cDNA representing Tv-ant-1 isolated by RACE was 1324 bp in size, including an open reading frame (ORF) of 894 bp, a 5′-UTR of 45 bp, and a 3′-UTR of 385 bp. In the 3′-UTR, a polyadenylation signal (AATAAA) located 13 nucleotides 5′ to the poly-A tail was predicted. The uninterrupted ORF encoded a predicted protein (Tv-ANT-1) of 297 amino acids, which contained a consensus sequence (RRRMMM) representing ADP/ATP carriers but which is absent from other mitochondrial carriers (Pebay-Peyroula et

Discussion

In the present study, full-length cDNA and gene sequences (designated Tv-ant-1) of an ANT (Tv-ANT-1) from the parasitic nematode T. vitrinus were isolated and characterized. Although ADP/ATP translocases have been studied in a range of vertebrates, including human, mouse, cattle, and invertebrates, such as Drosophila melanogaster (vinegar fly), Saccharomyces cerevisiae (yeast) and C. elegans (see Fiore, 1998, Santamaria et al., 2004), this is the first detailed report of a translocase gene from

Acknowledgements

We thank Youssef G. Abs EL-Osta for producing parasite material. Funding support to RBG from the Australian Research Council (LP0346983 and LP0667795), Genetic Technologies Limited and Meat and Livestock Australia is gratefully acknowledged.

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