Herzig et al. BMC Genomics 2010, 11:100 http://www.biomedcentral.com/1471-2164/11/100 CDR, as indicated. TRDV1 genes reported here are tem- porarily designated TRDV1a - TRDV1bp until the entire chromosomal sequence has been assembled. TRDV1 genes encode CDR1 that range in length from five to ten amino acids (except for TRDV1o which encodes an 18 amino acid long CDR1) while TRDV2, TRDV3 and TRDV4 genes all encode CDR1 that are seven amino acids in length. In contrast, most CDR2 of TRDV1, TRDV2 and TRDV3 genes were three amino acids in length, except for four of the TRDV1 genes as noted below, while that of TRDV4 was five amino acids long. In all but two cases (TRDV1bc and TRDV1w), TRDV1 and TRDV2 genes encode a QXS motif in the CDR2. Interestingly, TRDV1f, TRDV1ae, TRDV1ar and TRDV1o (see Figure 3A) all contained a deletion which spans the last FR2 amino acid to the fifth FR3 amino acid, resulting in a lack of CDR2 in those genes. An identical deletion had also been found in the sheep TRDV1S29 gene [15]. Also, the RS of TRDV1bg and TRDV1bj were found to occur farther downstream than those of the other TRDV1 genes identified here, result- ing in a putative transcript that extends 15 (for TRDV1bg) or 12 (for TRDV1bj) amino acids beyond the conserved 2nd-CYS 104, as opposed to extending only three or four amino acids which is the case for the other TRDV1 genes. Because of the unusual nature of those six genes, until their expression by gδ T cells is validated they will be considered ORF instead of func- tional as defined by IMGT [31]. 2D structure graphical representations of TRDV1, TRDV2, TRDV3 and TRDV4 genes (Figure 3B) were generated using IMGT/V- QUEST [32] and the IMGT/Collier-de-Perles tool pro- gram [33]. The CDR1 and CDR2 loops are indicated in red and yellow, respectively. The amino acids 1st-CYS 23, CONSERVED-TRP 41, hydrophobic amino acid (Leu) 89 and 2nd-CYS 104, which are conserved in all four TRDV subgroups, are shown with letters in red. As for sheep, genes in the bovine TRDV1 family share many characteristics including a 20 amino acid long highly conserved leader region and the YFC motif found in the 3’ end of FR3 (see Figure 3A). However, bovine TRDV1 genes described here were further classified into eleven sets by constructing a phylogenetic tree using the neighbor-joining method allowing us to compare them to equivalent human, mouse and ovine sequences (Figure 4). The grouping of ovine TRDV1 genes by this method corresponded with what had been previously described [15]. The tree shows grouping of bovine TRDV1 gene sequences into eleven sets, as indicated, and the percentage interior test values (based on 1000 replicates) are shown for each set. Classification was further supported based on CDR characteristics used to classify ovine TRDV1 genes [15] (Table 3). Previously described characteristics used to classify TRDV1 genes Page 8 of 19 included CDR1 length (five, seven, nine or ten amino acids), the chemical characteristics of the amino acid at position 57 (within CDR2) and the presence or absence of Trp 107 in CDR3. The number of TRDV1 genes eval- uated here that occur within each set is also shown (Table 4) with set 1 being the largest and set 4 being the smallest. While some sets can share the same fea- tures reported in Table 3 (i.e. sets 5 and 8, 4 and 6) the classification of TRDV1 genes into those separate sets is supported based on the phylogenetic analysis (Figure 4) and additional sequence features (data not shown). When bovine TRDV1 RS (total of 52 sequences) and ovine TRDV1 RS (total of 12 sequences) were evaluated, a high level of sequence conservation was observed both among and within species, as depicted in the sequence logos shown in Figure 5A. When the RS of bovine TRDV4 orthologs were compared (including those for human TRDV3, mouse TRDV5 and ovine TRDV4) a similarly high level of sequence conservation was observed among species (Figure 5B). In contrast, com- parison of the remaining TRDV RS (those for bovine TRDV2 and TRDV3, ovine TRDV2 and TRDV3, mouse TRDV1, TRDV2 and TRDV4, human TRDV1 and TRDV2) revealed conservation in only the heptamer and nonamer sequences (Figure 5C). This is consistent with the lack of relatedness of these genes as determined based on sequence analysis (refer to Figure 4). Overall, sequence conservation was observed within the RS hep- tamer and nonamer of all TRDV genes, regardless of species (compare Figure 5A, Figure 5B and Figure 5C). TRDD and TRDJ gene analysis Genomic locations, sequences and RS of bovine TRDD and TRDJ genes were also determined (Table 1 and Additional File 1) and confirmed what had been reported previously [20]. Evidence for five TRDD genes was found and deduced amino acid sequences for all three open reading frames are shown in Additional File 1. Additional putative TRDD genes were identified based on potential RS (data not shown); however, subse- quent cDNA sequence analyses provided little or no evi- dence that those putative TRDD genes were valid and thus they are not presented. Three TRDJ genes were found and their nucleotide and deduced amino acid sequences are also reported in Additional File 1. Align- ments comparing bovine, ovine, swine, human and mouse TRDJ gene nucleotide and deduced amino acid sequences are shown (Figure 6A and Figure 6B). A phy- logenetic tree (Figure 6C), constructed using the neigh- bor-joining method to evaluate the relatedness of the above sequences, reflected the alignment. Bovine, human and murine 5’D-RS and 3’D-RS of TRDD genes and J-RS of TRDJ genes were evaluated for sequence conservation (data not shown). Despite a low number of