Gene-specific primers were designed on the predicted canine
VMD2 sequence (http://genome.ucsc.edu/) using the Primer3 software (http://frodo.wi.mit.edu/primer3/primer3_code.html;
Supplementary Table S1). The complete cDNA sequence of canine
VMD2 was verified by constructing RPE/choroid 5′- and 3′-RACE libraries (BD Smart Race cDNA Amplification Kit; Clontech, Mountain View, CA) according to the manufacturer’s protocol and then were cloned into pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) using a cloning kit according to the manufacturer’s instructions (TOPO TA Cloning kit; Invitrogen).
Promoter regions of VMD2 were screened through the CONSITE database (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite) for dog, human, and mouse (http://genome.ucsc.edu/).
Gene alignments were assembled from experimental data or hypothetical loci for human (Homo sapiens, NM_004183; NP_004174), chimpanzee (Pan troglodytes, XM_522029; XP_5220291), rhesus monkey (Macaca mulatta, ENSG00000167995), crab-eating macaque (Macaca fascicularis, AY357925.1; AAQ56049), cow (Bos taurus, scaffold130366.2; XP_585778), pig (Sus scrofa, AY064707, BI343182; AAL40882), mouse (Mus musculus, NM_011913; NP_036043), rat (Rattus norvegicus, XM_574621; NP_001011940), chicken (Gallus gallus, XM_421055; XP_421055), opossum (Monodelphis domestica, scaffold14923.4), zebrafish (Danio rerio, ENSDART00000007569.2; XP_689098), xenopus (Xenopus tropicalis, CR760914; NP_988974), Drosophila (Drosophila melanogaster, NM_144346; NP_652603), and Caenorhabditis elegans (C. elegans, NM_061231; NP_493632.1). Proposed reading frames for the chicken and opossum were shortened at the 5′-end to start at the same position suggested for all other species. Hypothetical sequences posted for the chimpanzee and cow were modified at the 3′-end to splice into exon 11 at the same site as the orthologous human sequence; in the same manner, the predicted dog open-reading frame was corrected, matching the C-terminal protein sequence with that found in other nonrodent mammals. Finally, the published pig sequence did not have exons 1 to 3 or part of exon 4. The complete gene was reconstructed through an independent EST (BI343182) representing the beginning of the pig VMD2 (however, the complete pig VMD2 sequence is missing 25 bp in exon 4 that have not yet been sequenced).
Multiple nucleotide and protein sequence alignments were derived using CLUSTAL W, with a gap penalty of 10 and a gap extension penalty of 0.2. The nucleotide distance was based on a bootstrapped (
n = 1000) F84 model.
16 Protein distances were calculated using a bootstrapped (
n = 1000) Jones-Taylor-Thornton model, and the protein weight matrix was Blossom 30. An overall phylogenetic tree was inferred from the multiple sequence alignment using PHYLIP (version 3.5c) based on a neighbor-joining algorithm.
16