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. 2003 Aug;109(4):515–526. doi: 10.1046/j.1365-2567.2003.01695.x

Genomic structure around joining segments and constant regions of swine T-cell receptor α/δ (TRA/TRD) locus

Hirohide Uenishi *, Hideki Hiraiwa *,†, Ryuji Yamamoto *,‡, Hiroshi Yasue *, Yohtaroh Takagaki ‡, Takashi Shiina §, Eri Kikkawa §, Hidetoshi Inoko §, Takashi Awata *
PMCID: PMC1783003  PMID: 12871218

Abstract

A complete genomic region of 131·2 kb including the swine T-cell receptor α/δ constant region (TRAC/TRDC) and joining segments (TRAJ/TRDJ) was sequenced. The structure of this region was strikingly conserved in comparison to that of human or mouse. All of the 61 TRAJ segments detected in the human genomic sequence were detected in the swine sequence and the sequence of the protein binding site of T early alpha, the sequence of the α enhancer element and the conserved sequence block between TRAJ3 and TRAJ4 are highly conserved. Insertion of the repetitive sequences that interspersed after the differentiation of the species in mammals such as short interspersed nucleotide elements is markedly suppressed in comparison to other genomic regions, while the composition of the mammalian-wide interspersed sequences is relatively conserved in human and swine. This observation indicates the existence of a highly selective pressure to conserve this genomic region around TRAJ throughout the evolution of mammals.

Introduction

Vertebrates have T and B cells that play essential roles in immune responses in an antigen-specific manner. T cells are divided into two major groups, αβ and γδ T cells, by their heterodimeric T-cell antigenic receptors (TCR). TCR α and γ glycoprotein chains consist of variable (V), joining (J) and constant (C) regions, while β and δ chains consist of V, diversity (D), J and C regions, encoded by genes at discrete locations on the chromosome. These genes are combined through a rearrangement process before the TCR chains are appropriately expressed.1

It has been revealed by analyses in human and mouse that the TRD gene, encoding the TCR δ chain, lies within the TRA locus, encoding the TCR α chain. The TRD locus was excised from the genome when the TRA locus was rearranged. Complete sequences of the TRA/TRD locus of human and mouse have been already revealed and their comparison indicated striking conservation in this region:2 61 TCR Jα (TRAJ) segments have been found between the TCR Cδ gene segment (TRDC) and the TCR Cα gene segment (TRAC). Furthermore, many conserved sequences, including enhancers, promoters and a sequence that enhances the activity of enhancers, were found and their functions were intensively investigated. The organization of this region was examined and a partial sequence proximal to TRAC revealed in chicken.3 In puffer fish, the TRA locus is unique in structure, i.e. variable segments of TRA (TRAV) are located downstream of the TRAC and TRA enhancer (Eα).4,5 However, the complete sequence and entire structure of this region in mammals except human and mouse have not been clarified. The sequencing of this region in other mammals is required for comparison to elucidate cis-acting regulatory elements for the expression and rearrangement of the TRA/TRD gene.

Pigs are considered a promising candidate for donors in xenotransplantation, and for incubators of regenerated human organs to be supplied for transplantation. A comprehensive understanding of the swine immune system is indispensable for overcoming the rejection of a transplant, and the problems that inevitably occur. It is plausible that T cells have an essential role in acquired immunity that is closely involved with acute vascular rejection in transplantation by their direct cytotoxicity or the regulatory function of other lymphocytes.6 Furthermore, analysis of the resistance of swine to various infections that affect livestock productivity such as viral respiratory diseases requires elucidation of the repertoire of TCR-recognizing antigens derived from the pathogen.

Here we report the swine genomic sequence including the entire region between TRDC and TRAC, and also four TCR Jδ (TRDJ) segments located in the region upstream of TRDC. The results of this study revealed the potential of swine to produce a sufficiently divergent repertoire of TCR α and δ chains to recognize antigens, and will facilitate elucidation of the evolutional formation of this region and lineage difference of the αβ/γδ T cell between primates, rodents and artiodactyls.

Materials and methods

Genomic DNA cloning and template preparation

BAC clones, provided by the DNA Bank of the National Institute of Agrobiological Sciences (Tsukuba, Ibaraki, Japan), containing both TRDC and TRAC were isolated from a swine genomic BAC library constructed with kidney cells derived from an LWD pig, the offspring of a LW sow and a boar of the Duroc breed. The LW pig was the offspring of a sow of the Landrace breed and a boar of the Large White breed.7 The procedure used for the polymerase chain reaction (PCR) screening of the library was previously described.8 The BAC clones obtained were mapped to the portion of swine chromosome 7 that corresponds to the portion of human chromosome 14 where human TRA/TRD is located.8 BAC DNA was purified twice by ultracentrifugation with a caesium gradient and cleaved to 2–4 kb by sonication with a Branson 430 sonifier (Branson Ultrasonics, Danbury, CT). The sonicated DNA fragments were blunt-ended using Klenow fragments (Toyobo, Osaka, Japan) and ligated into the EcoRV site of the pUC19 vector and subjected to shotgun sequencing. DNA of the plasmids of the shotgun library was extracted and purified using the NA and FB filter system (Millipore, Bedford, MA) with a Biomek2000 (Beckman Coulter, Fullerton, CA).

DNA sequencing, sequence assembly and editing

DNA sequencing by the dideoxynucleotide chain termination method was performed as previously described9 with a BigDye Terminator ready reaction kit, using an ABI PRISM377 DNA sequencer and an ABI373XL DNA sequencer (Applied Biosystems, Foster City, CA). Nucleotide sequencing of a BAC clone (1025C2) was performed by the shotgun method.10 Assembly of the shotgun sequences was performed with ATGC sequence assembly software (Genetyx, Tokyo, Japan) and gaps between the sequence islands were filled by obtaining the PCR fragments with the primers constructed in the end of the islands. Each base position was sequenced at least twice, with an average of seven times. The same result in terms of an assembled nucleotide sequence was obtained with Phred basecalling and Phrap assembling software.11,12 The integrity of the assembled sequence was also confirmed by the comparison of fragment size after digestion with combinations of the restriction enzymes NotI, EcoRV, SalI, PvuI, SacII, SmaI and XhoI with the information obtained from the assembled sequence.

Data analysis

The assembled DNA sequence was analysed for similarity with blast13 in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Dot-plot analysis was performed with Genetyx genetic analysis software (Genetyx, Tokyo, Japan). The homology analysis of each joining segment between swine, humans and mice, and the sequence alignment were performed with ClustalX 1.81.14 The determination of positions of repetitive sequences was performed with RepeatMasker ver. 07/26/2001 (http://ftp.genome.washington.edu/RM/RepeatMasker.html) and repetitive sequence database Repbase 7.2.15

Results

Using PCR primers designed with known cDNA sequences, BAC clones containing the germline DNA sequence of TRD and TRA were obtained. From the swine BAC library7 clones 860C5 and 1025C2 were isolated. To confirm the integrity of the sequence of the isolated genomic clones containing TRAC and/or TRDC, their restriction fragments were compared. No deletions or chimerisms were observed. We chose clone 1025C2 as a template for sequencing analysis. Its entire genomic structure revealed in this study is schematically summarized in Fig. 1.

Figure 1.

Figure 1

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Genomic structure of the swine TRDJ-TRDC-TRAJ-TRAC region. The exon–intron structure of TRAC, TRDC and TRDV3, and distribution of the TRAJ and TRDJ segments are indicated. Pseudo TRAJ segments assumed by including termination codon(s) are indicated by shaded arrows. The newly detected pseudo TRAJ segment (TRAJ-P) is indicated as ‘JP’. Putative locations of Eα, Eδ and TEA promotor are also indicated. The revealed sequence contained the 3′ part of the DAD1 gene in the reverse orientation of TRD-TRA transcription. Locations of PRE-1, which is a swine-specific SINE, MIR, and LINE are indicated in this figure. All LINE were partial sequences and less than 400 bp in length except one (more than 773 bp, indicated by an open arrow). Restriction sites and GC-contents are also shown. Overall sequence similarity to the human sequence was calculated with ClustalX. The human (NT_037845) and swine (this study) sequences were aligned, and variation in the ratio of the swine nucleotides matched to those of human is indicated.

Coding regions of constant genes

Since the sequence presented in this study is the first complete sequence of germline DNA covering the TRAC and TRDC locus in swine, the TCR Cδ (TRAC) and TCR Cα (TRAC) genes were identified by alignment with the swine cDNA sequences of the TCR α or δ chain gene16,17 and the genomic sequence of the TRA/TRD locus, and by analogy with the organization of the locus in humans and mice. Each of these swine TRAC and TRDC genes consists of three translated exons and a fourth exon that does not have a translated region, similar to human and mouse. The first, second and third exons of swine TRAC are 270, 45 and 108 bp in length, respectively. The fourth exon is assumed to be 582 bp based on the mRNA sequence revealed by the preceding analysis.16 These four exons of TRAC are separated by introns that are 1964, 896 and 677 bp in length, respectively. The four exons of TRDC are assumed to be 279, 66, 114 and 939 bp, respectively. The introns of TRDC are 568, 658 and 1159 bp in length, respectively.

A variable gene segment included between TRDC and TRAJ

Although almost all of the TRDV segments are located on the 5′ side of the diversity (D) segments of TRD, one was identified between TRDC and the most 5′-TRAJ in humans and mice. In mice, the Trdv5 segment is located 2506 bp downstream from the fourth TRDC exon and 5790 bp upstream from the most 5′-TRAJ segment. In humans, correspondingly, the TRDV3 segment is located 2486 bp downstream from the fourth TRDC exon and 5687 bp upstream from the most 5′-TRAJ segment. In swine, similar to humans and mice, one TRDV segment that has high homology with murine TRDV5 and human TRDV3 exists between TRDC and TRAJ (designated TRDV3, Fig. 1). It is located 2425 bp downstream from the fourth TRDC exon and 6946 bp upstream from the most 5′-TRAJ segment. This TRDV segment inverted in the germline was formerly identified in TRD mRNA sequences.17

Joining gene segments and whole structure of TRDJ-TRDC-TRAJ-TRAC locus

Complete sequencing of the region between TRDC and TRAC in mouse revealed that it contains more than 50 TRAJ segments.18,19 Later, the same region of the human genomic sequence was determined and a comparison of these sequences clarified that this region contains at least 61 TRAJ segments.20 The swine genomic sequence between TRDC and TRDA was now clarified and a comparative analysis among humans, mice and swine revealed that one more TRAJ pseudogene segment exists between TRAJ47 and TRAJ48 according to the nomenclature in the reference20 (Fig. 2a). It has three stop codons within the segments between the rearrangement signal and the putative splicing site, and the most C-terminal amino acid that is well conserved in the other TRAJ segments as proline is changed to asparagine. Otherwise, TRAJ19, 51 and 55 are assumed to be pseudogenes because of the existence of stop codons. All mRNAs of swine TRA that have been cloned and sequenced hitherto had joining segments that are included in the TRAJ segments presented in this study16 (Yamamoto et al., unpublished data).

Figure 2.

Figure 2

Figure 2

Figure 2

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Joining gene segments detected around the germline sequence of the TRDC-TRAC region. (a) Deduced sequences of amino acids of swine TRAJ and TRDJ segments. A pseudo TRAJ segment (TRAJ-P) was newly detected between TRAJ47 and TRAJ48. Conserved amino acids are indicated by shading. Stop codons are indicated by asterisks. (b) A phylogenetic tree of TRAJ segments of swine, human and mouse without roots. The phylogenetic tree was drawn by the Neighbor-Joining method. TRAJ segments of different species at the same location have higher similarity to each other than those of the same species (indicated by open circles). The horizontal bar in the lower left of the figure indicates the substitution rate (one substitution per 10 sites). (c) Phylogenetic tree of TRDJ segments. Conditions and descriptions were the same as for the tree indicated in (b). (d) Alignment of TRDJ segments among swine, human and mouse. Identical nucleotides among three species are indicated by asterisks.

Figure 2(b) shows a phylogenetic tree of TRAJ segments of swine, humans and mice. This figure indicates that TRAJ segments of different species at the same location have higher similarity to each other than those of the same species.

Previous studies identified four TRDJ segments in humans,21–23 two in mice,24 three in sheep25 and three in bovine.26 In the germline genomic sequence we identified all four swine TRDJ segments isolated in the previous study.17 The order of the TRDJ segments was Jδ3, Jδ2, Jδ4 and Jδ1 from adjacent to the TRDC region (designated TRDJ3, 2, 4 and 1, respectively, in this paper). All of the sequences except TRDJ1 identified in the genomic sequence that we report here were identical to those in the previous report.17 Phylogenetic analysis suggests that the TRDJ segment between TRDJ1 and TRDJ2, corresponding to human and swine TRDJ2 and TRDJ4, was deleted after the differentiation of rodents (Fig. 2c). Figure 2(b, c) indicates that most of the J segments of swine are more similar to human than mouse. However, there are some exceptions, for example, the human TRDJ1 is more similar to that of mouse than swine (Fig. 2c). Some of the swine TRAJ segments, such as TRAJ26 and 33, are more similar to those of mouse than those of human (Fig. 2b). Alignment of TRDJ sequences among swine, humans and mice indicates that lengths of the swine TRDJ segments were varied in comparison with those of human (Fig. 2d). This may reflect the unique selection in swine or artiodactyls because of greater usage of γδT lymphocytes in the periphery than in primates and rodents.

DNA rearrangement signals

All TRAJ and TRDJ segments are flanked by standard recombination signal sequences (RSS) with nonamer (GGTTTTTGT) and heptamer (CACTGTG) sequences more than 50% similar to those of human and mouse. All RSS except that of TRDJ2 have a 12-bp spacer sequence between the nonamer and heptamer. The spacer sequence in TRDJ2 is 11 bp in length. The percent conservation of bases in each part of the RSS is shown in Fig. 3. The first four bases, AAAG, of the spacer sequences of the RSS of TRAJ are highly conserved in swine, similar to human and mouse.

Figure 3.

Figure 3

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Usage of bases in the recombination signal sequences (RSS) of swine. ‘Position’ indicates the location of the base in the sequence from the 5′ side. Bold letters indicate that the percentage of the usage of the particular base is more than 50%.

Regulatory elements

The rearrangement and transcription of TCR is controlled by various kinds of regulatory elements interspersed among the locus of the TCR. Studies in human and mouse revealed the existence of putative regulatory elements around the TRA/TRD constant region, such as the TRD enhancer (Eδ)27–29 T early α (TEA)30,31 conserved sequence block (CSB)32 and TRA enhancer (Eα).33–35 Analysis of the regulatory factors around the TRA/TRD locus may provide a clue as to the lineage difference of T cells between ‘high-γδ’ animals, such as artiodactyls or chicken, and other animals such as humans and mice.36,37

In swine, a putative Eδ, based on the similarity of sequences with human Eδ, is found 1·2kb 3′ to the TRDC gene segment. Swine putative Eα is located 5·0kb 3′ to the TRAC gene, also similar to humans and mice. Comparison of the swine Eα and Eδ sequences with those of humans or mice shows strong conservation of the sequences of putative binding sites for regulatory proteins, such as CREB and Ets in Eα20 and AML-1a and c-Myb in Eδ38,39 (Fig. 4a, b).

Figure 4.

Figure 4

Figure 4

Figure 4

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Sequence alignment of regulatory elements (Eα (a), Eδ (b), TEA (c) and CSB (d)) within the TRA/TRD locus among swine, human and mouse. Putative binding sites for transcriptional elements are indicated in open boxes. Putative binding sites of transcriptional factors, enclosed by open boxes, were cited from the literature.20, 52, 53 Consensus sequences for transcriptional elements are indicated above the sites.

TEA is known as a cis-regulatory element for activating germline transcription of the TRAJ locus in thymocyte ontogeny, and the germline transcript may indicate that the structure around TRAJ is opened for recombinase. de Chasseval et al. revealed that there were many putative binding sites for T-cell-specific transcription factors in the TEA of human and mouse.30 Figure 4(c) shows that these sites previously proposed according to a comparison of the sequences of humans and mice are also highly conserved in the swine sequence.

CSB is a highly conserved sequence between humans and mice, located between TRAJ3 and TRAJ4.19,20 CSB itself does not have enhancer activity, however, it was demonstrated to interact with various nuclear factors and up-regulate the activity of Eα.40,41 A swine putative CSB sequence was also detected between TRAJ3 and TRAJ4. It is 244 bp downstream of TRAJ4 and extends into the signal sequence of TRAJ3. Nucleotide similarities of the swine putative CSB sequence against those of humans and mice are both 91% (Fig. 4d).

Comparison of entire genomic structure and repetitive sequences reveals conservation of this region through evolution

A comparison of the genomic structure of the TRDJ-TRDC-TRAJ-TRAC region between swine and humans by dot-plot analysis demonstrated that no significant insertion/deletion had occurred in this region throughout evolution, except certain areas such as the sequence around TRAJ31 and TRAJ32 (Fig. 5). Figure 5 implies that the sequence around TRAJ31 and TRAJ32 in swine was slightly longer than that in humans, which may reflect the insertion of repetitive sequences such as long interspersed nucleotide elements (LINE) and short interspersed nucleotide elements (SINE).

Figure 5.

Figure 5

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Dot-plot analysis of the TRDJ-TRDC-TRAJ-TRAC region of human and swine. Two sequences were divided into pieces, and each piece contained 1020 bp. The pieces derived from swine and human sequences were compared, and dots were plotted when the compared pieces shared more than one set of five identical bases.

The overall sequence similarity of the TRDJ-TRDC-TRAJ-TRAC as investigated by alignment demonstrated the degree of similarity in the entire genomic region (Fig. 1). The region around the downstream portion of TRDJ segments (TRDJ2 and TRDJ3) and the region covering TRAJ segments except the centre had significant homology between humans and swine. The upstream region of TRDJ segments, especially between TRDJ1 and TRDJ4, showed significantly lower similarity than the downstream region. The region around TEA had higher similarity between humans and swine than the sequences adjacent to TEA, which did not show significant homology. The region around TRAJ31 and TRAJ32 demonstrated lower similarity than the neighbouring sequences also in the alignment analysis.

Repetitive sequences both specific to the species and widely distributed among species are scattered ubiquitously in the genome of vertebrates.42 These repetitive sequences occupy almost half of the whole genome. We analysed the distribution of these repetitive sequences in the TRAC/TRDC locus of swine and compared it with that of humans and mice (Table 1). As shown in Fig. 1, the distribution of species-specific SINE is not identical among these species. SINE including PRE-1, a representative SINE sequence of swine, occupy 7·5% of the sequence between TRAC and TRDC in swine. The percentage of total interspersed repeats in this region in swine is 15·1%, while that in humans and mice is 13·2% and 7·2%, respectively. On the other hand, percentages of SINE and total interspersed repeats in 13 kb of the swine lysozyme genomic sequence are 17·0% and 40·8%, respectively. In swine, other regions like SLA contain more than 15% SINE and 40% repetitive sequences. Therefore, unlike in other genomic regions, the insertion of SINE seems to be highly suppressed in the TRAD/TRDC locus throughout swine, human and mouse. The composition of mammalian interspersed repeats (MIR) in the TRDC-TRAC region of swine, human and mouse was also investigated. The rate of occupation of mammalian interspersed repeats (MIR)43 in this region of swine is 3·6%, and not significantly different from that in other species (human: 3·1%, mouse: 1·9%). Although, in mouse, most MIR are undetectable probably because of the propagation of microsatellite sequences, MIR are well conserved between humans and swine.

Table 1.

Analysis of repetitive sequences in the germline sequences of the TRDJ-TRDC-TRAJ-TRAC region in swine, human and mouse. Results for the genomic sequence of SLA (DDBJ/EMBL/GenBank Accession: AJ131112) are also indicated for comparison.

Swine TRDC-TRAC germline Swine MHC (SLA) class I Mouse TRDC-TRAC germline Human TRDC-TRAC germline
Number of elements Length occupied Percentage of sequence Number of elements Length occupied Percentage of sequence Number of elements Length occupied Percentage of sequence Number of elements Length occupied Percentage of sequence
SINE 39 6625 bp 7·51% 157 33 930 bp 21·91% 27 3317 bp 4·08% 38 7613 bp 8·54%
″PRE-1 11 2782 bp 3·15% 136 30 872 bp 19·93%
″B1s 8 919 bp 1·13%
″ALUs 14 4118 bp 4·62%
″MIRs 22 3208 bp 3·64% 9 1048 bp 0·68% 13 1563 bp 1·92% 24 3495 bp 3·92%
LINE 23 4767 bp 5·40% 53 22 971 bp 14·83% 8 1710 bp 2·11% 13 3117 bp 3·50%
″LINE1 7 1407 bp 1·59% 43 19 930 bp 12·87% 2 621 bp 0·76% 2 392 bp 0·44%
″LINE2 8 1860 bp 2·11% 9 2781 bp 1·80% 2 326 bp 0·40% 6 1692 bp 1·90%
″L3/CR1 8 1500 bp 1·70% 1 260 bp 0·17% 4 763 bp 0·94% 5 1033 bp 1·16%
LTR elements 0 0 bp 0·00% 23 8687 bp 5·61% 0 0 bp 0·00% 0 0 bp 0·00%
DNA elements 5 955 bp 1·08% 17 5433 bp 3·51% 5 820 bp 1·01% 5 1076 bp 1·21%
Unclassified 9 980 bp 1·11% 41 3261 bp 2·11% 0 0 bp 0·00% 0 0 bp 0·00%
Total interspersed repeats 13 327 bp 15·11% 74 282 bp 47·97% 5847 bp 7·20% 11 806 bp 13·24%
Simple repeats 11 454 bp 0·51% 24 811 bp 0·52% 33 1974 bp 2·43% 6 255 bp 0·29%
Low complexity 3 84 bp 0·10% 21 762 bp 0·49% 9 281 bp 0·35% 8 239 bp 0·27%
Total length 88 226 bp 154 867 bp 81 214 bp 89 170 bp
GC level 45·24% 48·17% 45·26% 43·71%
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Discussion

The TRA gene has been hitherto identified in lower vertebrates such as sharks,44 rainbow trout45 and puffer fish.4,5 Sequencing analysis of the CDR3 region of TRA cDNA revealed that rainbow trout has more than 32 TRAJ segments.45 On the other hand, genomic DNA and cDNA sequences of the TRA locus of Tetraodon nigroviridis showed that there are more than 12 TRAJ and two TRDJ segments in pufferfish. Analysis of the genomic structure of puffer fish revealed that the TRA locus has different features from that of mammals, for example, TRAV/TRDV segments are located downstream of TRAC and Eα, and TRV upstream of TRAC/TRDC has not been identified so far.5 The genome of Takifugu rubripes is about 365 Mb46 and markedly smaller than that of other bony fish, for example, the genome of rainbow trout is about 2400 Mb.47 Therefore, it cannot be ruled out that puffer fish has a unique genomic structure in this region distinct from other fish. However, these findings suggest that the structure between TRDC and TRAC that includes more than 60TRAJ segments was constructed after the phylogenesis of bony fish. A recent review article proposed the hypothesis that the TRA/TRD locus was constructed by duplication of the immunoglobulin/TCR ancestral gene, inversion of the V-D-J-C region for TRD, and successive duplication of V and J segments.48 Our result that this genomic region was conserved to a strikingly large extent among mammals suggests that the inferred duplication occurred in the process of evolution of vertebrates lower than mammals. The observation that the composition of MIR, propagated before the mammalian radiation43 is not clearly different among mammals also supports this hypothesis. Structural comparison of the TRA/TRD locus between swine, humans and mice did not indicate a recently duplicated region. Further analysis of the genomic structure of the TRA and TRD locus in amphibians and reptiles will help to elucidate the phylogenetic evolution of this genomic region.

Comparison of the entire structure of the TRDJ-TRDC-TRAJ-TRAC region between human and swine also revealed some positions that had lower similarity than the neighbouring areas in this genomic region. There may be a difference between humans and swine in the recruitment of the factors that regulate rearrangement processes because of these regions, which may affect the lineage of αβ/γδ T lymphocytes.

The identification of repetitive sequences between TRDC and TRAC indicated that the insertion of SINE or LINE sequences in this region has been highly suppressed as reported by others48 and this study. The insertion of repetitive sequences tends to destroy the structure of the TRAJ coding region, and, as a consequence, may impair the ability to produce a sufficient diversity of TRA chains corresponding to various antigens. Therefore, the integration of repetitive sequence had a tendency to be avoided because it might be a cause of reduced resistance to infection through the evolutionary process.

The TRB and TRD genes have D segments and more than two locations where an insertion of N-nucleotides, addition of P-nucleotides, or nucleotide deletion can occur, and, as a result, are assured a highly diversified repertoire instead of a relatively small number of joining segments in comparison to the TRA gene, which does not have D segments and has only one location for insertions/deletions of nucleotides through the recombination process. To ensure a highly diversified repertoire of TCR α chains, an adequately large number of TRAJ segments may be indispensable. Hence, the number and sequences of TRAJ segments may be highly conserved as a consequence of necessity.

Recent study has revealed that swine are phylogenetically more distant from humans than are mice.49,50 However, many nucleotide sequences submitted in databases indicate swine sequences have much higher similarity with humans than mouse sequences. The present study also showed higher conservation between humans and swine, than between humans and mice, or swine and mice, in simple nucleotide homology as well as in genomic structure such as the content of repetitive sequences in this region. This may reflect the genomic instability in mice, such as a higher mutation rate and fluctuation of simple sequence repeats and other repetitive sequences.

Artiodactyls, such as swine and cattle, and chicken are known as ‘high-γδ’ animals that have more than 40%γδ T cells in their peripheral blood.36,37,51 The difference in lineage and function of the αβ/γδ T cells between these ‘high-γδ’ animals and human or mouse is of interest for hygiene and the application of regenerative medicine to livestock. Comparison of the sequences of the TRA/TRD locus between other ‘high-γδ’ animals, which remain to be determined, and that of swine, humans or mice will provide a clue as to the lineage bias of αβ/γδ T cells in these animals.

Acknowledgments

We are grateful to Ai Hoshikawa, Kazuko Tsuboi and Noriko Sugai-Hayashi for technical assistance. We also thank Takeshi Hayashi, Masahide Nishibori and Asako Ando for helpful discussions. This work was supported by Animal Genome Research Program of Japanese Racing Association, Animal Genome Research Project of Ministry of Agriculture, Forestry and Fisheries of Japan, and Organized Research Combination System of Ministry of Education, Culture, Sport, Science and Technology of Japan.

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