Hu Min, Campbell Bronwyn E, Pellegrino Mark, Loukas Alex, Beveridge Ian, Ranganathan Shoba, Gasser Robin B
Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
Gene. 2007 Aug 1;397(1-2):12-25. doi: 10.1016/j.gene.2007.03.011. Epub 2007 Mar 30.
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.
从玻璃细颈线虫（圆线目）中分离出了一个编码腺嘌呤核苷酸转运体（ANT或ADP/ATP转运酶）（Tv-ANT-1）的全长cDNA。玻璃细颈线虫是一种对小型反刍动物具有重要经济意义的寄生线虫。894个核苷酸的不间断开放阅读框（ORF）编码了一个预测的297个氨基酸的蛋白质，包含特征基序[RRRMMM]和PX(D,E)XX(K,R)。与自由生活线虫秀丽隐杆线虫、牛和人类的选定序列进行比较表明Tv-ANT-1相对保守。在共有序列RRRMMM及其附近以及预测与α-螺旋相关并穿越细胞膜的六个疏水区域中，序列同一性最高。使用邻接法和最大简约法对选定氨基酸序列数据进行系统发育分析，结果显示Tv-ANT-1与从秀丽隐杆线虫tag-61基因推断出的分子（Ce-ANT-3）关系最为密切。全长Tv-ant-1基因的基因组组织与tag-61相似。对Tv-ant-1上游区域（5'-UTR）的分析确定了一些启动子成分，包括GATA转录因子、CAAT和E-box元件。通过逆转录聚合酶链反应（RT-PCR）进行的转录分析表明，Tv-ant-1在玻璃细颈线虫的所有发育阶段均有转录，包括第一至第四期幼虫（L(1)-L(4)）以及雌雄成虫。通过用来自Tv-ant-1 cDNA的双链RNA（dsRNA）喂养秀丽隐杆线虫进行RNA干扰（使用秀丽隐杆线虫的同源基因作为阳性对照），未发现基因沉默现象。尽管Tv-ant-1和tag-61基因在23 - 30 bp的序列片段中核苷酸同一性为100%，但这些同一性似乎不足以使用寄生虫同源物/直系同源物Tv-ant-1在秀丽隐杆线虫中实现有效的沉默。对玻璃细颈线虫ANT的这一初步了解为探索其在毛圆科线虫发育和/或生存过程中的作用奠定了基础。
