Herzig et al. BMC Genomics 2010, 11:100 http://www.biomedcentral.com/1471-2164/11/100 T cell receptor alpha (TRA) locus. For both humans and mice these are located on chromosome 14 and encom- pass over 1 megabase (Mb) for the combined TRA/TRD locus [13]. The TRD locus embedded within the TRA locus spans approximately 60 kb in humans and 275 kb in mice. The loci comprise TRDV genes (two in humans and five in mice), followed by TRDD genes (three in humans and two in mice), TRDJ genes (four in humans and two in mice), a single TRDC gene and an additional TRDV gene that is located 3’ of the TRDC gene in an inverted transcriptional orientation [10,14]. In addition, five and ten functional TRAV/DV genes (that rearrange to either TRDD or TRAJ) have been identified for humans and mice, respectively, and these are found upstream of the embedded TRD locus [10,14]. Limited evidence derived from analysis of cDNA clones from cattle, sheep and swine, as well as limited germline information for sheep, suggests that the gen- eral organization of the bovine TRA/TRD locus does not differ greatly from that of humans and mice [17,20]. It also suggests that a much larger repertoire of TRDV genes exists for cattle, as well as for the other “gδ T cell high” species sheep and swine [15,17,20,21]. Indeed, recent mapping of the bovine TRA/TRD locus identified over 100 TRDV and over 300 TRAV or TRAV/DV genes in cattle [22]. For cattle, four genes belonging to three different small subgroups (TRDV2, TRDV3 and TRDV4) have been identified that are orthologous to those same subgroups in sheep [17,23]. In contrast, while the bovine TRDV1 genes have been found to be related to the single TRDV1 gene that occurs in humans, expansion of the TRDV1 subgroup accounts for the larger number of TRDV genes in the “gδ T cell high” species [15,17,20-22]. The swine and sheep TRDV1 subgroups have been esti- mated to contain at least 31 and 40 genes, respectively [15,21] while the bovine TRDV1 subgroup has been reported to contain at least 37 genes [20]. However, cur- rent knowledge about ruminant and swine TRD genes is predominantly based on cDNA evidence rather than on genomic DNA. Because of this, it has been unclear whether the large number of cDNA TRDV1 sequences is the result of multiple genes or polymorphisms among animals; thus, there has been no clear way to classify and name these transcripts. By annotating the bovine genome Btau_3.1 assembly, here we demonstrated the presence of 56 TRDV genes, 52 of which belong to the TRDV1 sub- group. We also proposed TRDV1 sets to classify the bovine and ovine TRDV1 genes based on their phyloge- netic grouping. The existence of multiple D and J genes also contri- butes to diversity of possible T cell receptor delta chain amino acid sequences since a particular TRDV gene is expected to be able to recombine with any D and J gene. The region of the protein derived from the V-D-J Page 2 of 19 gene rearrangement is known as the complementarity determining region 3 (CDR3), while the CDR1 and CDR2 loops are germline-encoded by the TRDV gene. The V-D-J gene rearrangement involves recognition of the recombination signal (RS) sequences for subsequent DNA cleavage by the enzymes RAG1 and RAG2 [24]. T cell receptor delta chain sequence diversity is augmented by junctional flexibility and the addition of N and P nucleotides. RS flank the V, D and J genes and are com- posed of highly conserved heptamer and nonamer sequences separated by either a 12 base pair (bp) spacer (located 5’ of TRDD and TRDJ genes) or a 23 bp spacer (located 3’ of TRDV and TRDD genes). Since the con- served heptamer and nonamer sequences and spacer lengths have been found to be important for efficient recombination [25] they were also evaluated here. Finally, recent evidence demonstrated that diversity of T cell receptor delta chain sequences is amplified by the occurrence of multiple TRDD genes within a single CDR3 [20,26]. Thus we also evaluated TRDD gene usage by analyzing cDNA sequences and report those findings here. Results Gene structure and genomic organization of bovine TRD genes Here we set out to annotate and classify the bovine TRD genes. Prior to the initiation of the studies described here many cDNA sequences representing TRDV1 gene transcripts had been reported [16-20,27] but the actual number of functional bovine TRDV1 genes and their sequences was unknown. As a result it was not possible to differentiate between cDNA sequences derived from distinct genes and those that represented polymorphisms of the same gene among animals. Furthermore, the existence of TRDV2, TRDV3 and TRDV4 genes had been demonstrated [17], along with the TRDJ and TRDD genes [20], but their numbers and placement within the bovine genome had not been definitively described. The exon-intron structures of TRDV genes (Figure 1A) and of the single TRDC gene (Figure 1B) were determined. A TRDV gene encompasses a coding region of approximately 560 bp and comprises two exons. The first exon, L-PART1, is 49, 55 and 37 bp in length for TRDV1/TRDV2, TRDV3 and TRDV4, respectively. The second exon, V-EXON, is between 271 and 335 bp in length for TRDV1, and is 299, 293 and 309 bp long for TRDV2, TRDV3 and TRDV4, respectively. The TRDC gene encompasses a coding region of 1282 bp compris- ing three exons. RS sequences were identified, as expected, adjacent to the 3’ end of each V-EXON (Fig- ure 1A) while the termination codon was at the 3’ end of TRDC exon 3.