Herzig et al. BMC Genomics 2010, 11:100 http://www.biomedcentral.com/1471-2164/11/100 for by diversity of the genes that rearrange to form the CDR3 (i.e. TRDD and TRDJ genes). Moreover, because many bovine TRDV1 amino acid sequences are very similar, structurally it might be expected that their ligand binding regionsincluding CDR3 would also need to be as diverse as that in humans and mice. Humans have three TRDD and four TRDJ genes while mice have two TRDD and two TRDJ genes. With the exception of the human TRDJ genes, these numbers all fall short of the five TRDD and three TRDJ genes identified in cattle. We demonstrated here that in cattle the TRD CDR3 contained combinations that include between one and five TRDD genes and would range in length from nine to 37 amino acids. With the exception of TRDD4, the germline bovine TRDD genes predominantly encode glycine and the hydrophobic residues valine and trypto- phan, with the neutral residues threonine and tyrosine being encoded to a lesser extent (see Additional File 1). This is reminiscent of findings for the equine VH CDR3 loops in which a higher proportion of glycine and lower proportion of cysteine content was identified when sequences were compared with those from humans, sheep and pigs [40]. As was suggested for equine VH CDR3, this indicates that bovine TRDV CDR3 loops have increased flexibility and are therefore better suited to recognize a large number of antigenic conformations. Considering this, it is unlikely that CDR3 diversity in mice and humans compensates for their less diverse TRDV repertoire and, in fact, it seems that cattle have more scope for antigen binding on all levels. If the highly diverse CDR3 is indeed the main struc- tural component involved in antigen recognition, then the TRDV gene products might be most important for interactions with co-receptors or antigen presenting cells, whatever their nature may be. While the interac- tions between gδ T cell receptor and the antigens that they bind are still largely unknown, it has been demon- strated that for ab T cell receptor the CDR1 loops (which are germline encoded) are involved with binding peptide as well as MHC. The CDR2 loops (also germline encoded) bind only MHC. The TRDV CDR1 in cattle exhibited diverse lengths and were generallyfound to be five to ten amino acids long as for mouse and human TRDV, TRBV and TRAV CDR1. Thisdiverse bovine TRDV CDR1 repertoire may contribute to antigen bind- ing in contrast to what has been reported for murine gδ T cell recognition of T22 as described above. It was found that bovine TRDV1 CDR2 were either only three amino acids long or absent completely (e.g., TRDV1f, TRDV1ae, TRDV1ar and TRDV1o) in contrast to TRBV CDR2 lengths of five to seven amino acids in mammals. It is yet to be determined whether the TRDV1 genes lacking CDR2 are functional, however, if they are found to be so this lackof, or abbreviation of, the CDR2 Page 16 of 19 inbovine TRDV1 genes is logical since gδ T cells are not MHC-restricted. Conclusions Based on annotations of the bovine genome we identi- fied the TRD genes including 56 TRDV genes, five TRDD genes, three TRDJ genes and the single TRDC gene and described their organization within the TRD locus. Furthermore, we report that the TRDV1 subgroup contains 52 genes, indicating that this subgroup under- went expansion as has been found for sheep and swine. Also, because this large gene subgroup has been main- tained in the bovine genome this indicates a unique role for these genes in gδ T cell biology. Methods Genome Annotation In conjunction with the Bovine Genome Sequencing Consortium http://genomes.arc.georgetown.edu/ bovine/, manual annotation of the T cell receptor delta genes was performed using the Apollo Genome Annotation and Curation Tool, version 1.6.5 [41] and the bovine genome assembly Btau_3.1 [42]. First, a BLAST search of bovine T cell receptor delta cDNA sequences against the Bovine Official Gene Set (GLEAN) was performed in order to identify predicted gene models. These were then analyzed using the Apollo software and the following actions were per- formed where applicable: (i) models were checked for correct exon-intron structure, (ii) initiation and termi- nation codons were identified where applicable and in other cases 5’ and 3’ ends were set based on RS and splice sites, (iii) exons were either added or deleted if it was determined that the coding region in the pre- dicted model was incorrect, (iv) predicted gene models were split when a single model encompassed more than one gene or merged when two models coded for a single gene and (v) RS were identified for TRDV, TRDD and TRDJ genes. Predicted gene models identi- fied from the BLAST search were considered pseudo- genes, and were not included in subsequent analyses, when premature stop codons or frameshifts occurred in areas where the sequence integrity was deemed ade- quate. Furthermore, predicted gene models were con- sidered Open Reading Frame (ORF), as defined by IMGT [31] if the coding region had an open reading frame but the sequence structure differed significantly from known TRDV sequences; ORF gene models were included in subsequent analyses. Following annotation, predicted gene model identity and classification were verified using BLAST searches and IMGT/V-QUEST [32]; only gene models that were unambiguously iden- tified as TRDV genes, and not TRAV/DV genes, based on those results were included in subsequent analyses.