Herzig et al. BMC Genomics 2010, 11:100 http://www.biomedcentral.com/1471-2164/11/100 Sequence analyses Nucleotide sequences were aligned and analyzed using BioEdit version 7.0.5.3 [43]. Exon-intron structure sche- matics were based on alignments of cDNA and genomic DNA sequence using SIM4 [44] and visualized with LalnView http://pbil.univ-lyon1.fr/software/lalnview. html. GLEAN numbers of the annotated bovine gene sequences used in sequence analyses are reported in Table 1. Published T cell receptor delta sequences, derived from cloned RT-PCR products, were submitted to GenBank (http://www.ncbi.nlm.nih.gov/Genbank/ index.html; see Table 5 for accession numbers) and assigned to TRDV genes identified here. CDR3 sequence data described in Table 6, from bovine TRD rearranged genes, are available upon request. Rearranged CDR3 lengths were determined from positions 105 to 117, that is, between the 2nd-CYS 104 and J-PHE 118 of the FGXG motif, according to the IMGT unique numbering for V-DOMAIN [29]. The accession numbers of addi- tional sequences used in analyses are as follows: human (Homo sapiens): TRDV1 (M22198), TRDV2 (X15207), TRDV3 (M23326), TRDJ1 (M20289, AE000661), TRDJ2 (L36386, AE000661), TRDJ3 (M21508, AE000661), TRDJ4 (AJ249814, AE000661), TRDD1 (M23325), TRDD2 (M22153), TRDD3 (M22152); mouse (Mus mus- culus): TRDV1 (M94080), TRDV2-1 (AE008686), TRDV2-2 (AE008686), TRDV4 (AE008686), TRDV5 (AE008686), TRDJ1 (AF019412), TRDJ2 (X64903, NT_039614), TRDD1 (X64900), TRDD2 (X64901); pig (Sus scrofa): TRDJ1 (D49560, AB053451), TRDJ2 (D49561, AB053451), TRDJ3 (D49562, AB053451), TRDJ4 (D49563, AB053451); cattle (Bos taurus): TRAV (BC148926), TRGJ1-1; sheep (Ovis aries): TRDV1S1 (Z12970, AJ786827), TRDV1S2 (Z12981), TRDV1S3 (Z12988), TRDV1S4 (Z12989), TRDV1S5 (Z12990, AJ786828), TRDV1S7 (Z12992), TRDV1S8 (Z12993, AJ786830), TRDV1S9 (Z12994, AJ786831), TRDV1S10 (Z12971, AJ786832), TRDV1S11 (Z12972), TRDV1S12 (Z12973), TRDV1S13 (Z12974, AJ786833), TRDV1S14 (Z12975), TRDV1S15 (Z12976), TRDV1S16 (AJ786834), TRDV1S17 (Z12978), TRDV1S18 (Z12979, AJ786835), TRDV1S19 (Z12980), TRDV1S20 (Z12982), TRDV1S21 (Z12983), TRDV1S22 (Z12984), TRDV1S24 (Z12986), TRDV1S25 (Z12987), TRDV1S29 (AJ786836), TRDV1S30 (AJ786837), TRDV1S31 (AJ786838), TRDV1S32 (AJ786839), TRDV1S33 (AJ786840), TRDV1S34 (AJ786841), TRDV1S35 (AJ809502), TRDV1S36 (AJ809503), TRDV1S37 (AJ809504), TRDV1S38 (AJ809505), TRDV1S39 (AJ809506), TRDV1S40 (AJ809507), TRDV2 (Z12995), TRDV3 (Z12996), TRDV4 (Z12997, AJ810117), TRDJ1 (AJ277510), TRDJ2 (AJ277511), TRDJ3 (AJ277512). Multiple sequence alignments were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index. Page 17 of 19 html; [28]) and the default parameters and were visua- lized using JalView [30]. IMGT unique numbering was determined for the V-DOMAIN [29] and TRDV second- ary structures were evaluated and displayed as IMGT Collier de Perles [33] as determined using IMGT/V- QUEST [32]. Sequence logos were generated using WebLogo3 [45]. Phylogenetic analyses Phylogenetic analyses were performed using nucleotide sequences, unless otherwise indicated, of functional (and ORF) TRDV and TRDJ genes. TRDV sequences were truncated to include the V-REGION (IMGT amino acid positions 1 through 108) and comprised those from bovine (identified here in Btau_3.1), human, mouse and ovine (see accession numbers above). TRDJ sequences were truncated to include sequence between the RS (5’ end) and splice site (3’ end) and comprised those from bovine (identified here in Btau_3.1), human, mouse, ovine and swine (see accession numbers above). Phylogenies were constructed in MEGA4 [46] using the neighbor-joining method and the p-distance model for nucleotide sequences. Phylogenies were tested using an interior branch test or bootstrap analysis, as indi- cated, with 1000 replications. cDNA analysis Bovine TRDV cDNA was obtained as described in pre- vious studies [16,17]. Briefly, blood was collected via jugular venipuncture into heparin and peripheral blood mononuclear cells were isolated via density gradient centrifugation over Ficoll-Paque™ PLUS (GE Healthcare Bio-Sciences, Piscataway, NJ) according to the manufac- turer’s protocol. Animal use complied with federal guidelines and had IACUC approval. Pelleted cells were resuspended in TRIzol (Invitrogen, Carlsbad, CA) and RNA was isolated according to the manufacturer’s pro- tocol. Polymerase chain reaction (PCR) was performed following reverse transcription (RT) and using primers designed to specifically amplify TRDV transcripts as previously described [16,17]. In addition, amplification of TRD rearranged CDR3 was performed using a com- mon reverse primer called TRDC-rev (5’-CTC CTT CAC CAA ACA AGC GAC G-3’) and the following for- ward primers: TRDV1S1S2-for (5’-GCA GAT AAA TCC ATC AGC CTC ACC-3’), TRDV1S3-for (5’-CAC AGA ACT CCA TCA GCC TCA CC-3’), TRDV1S4-for (5’-TCA CGT AAA GCC ATC AGC CTC ATT-3’), TRDV2-for (5’-GGA AAA AAA ATC ATC AGC CTC ACC-3’), TRDV3-for (5’-TAC AAA CCC AAC CAA ATG CTG AAA-3’), TRDV4-for (5’-AGC ATG AGC CAA AAA ACC TTC CAC-3’). PCR products were ana- lyzed on TAE agarose gels and cloned into the pCR2.1 vector (Invitrogen) according to the manufacturer’s