The deduced structure of the T cell receptor gamma locus in Canis lupus familiaris.

Analyzing the recent high-quality genome sequence of the domestic dog (Canis lupus familiaris), we deduced for the first time in a mammalian species belonging to Carnivora order, the genomic structure and the putative origin of the TRG locus. New variable (TRGV), joining (TRGJ) and constant (TRGC) genes for a total of 40 are organized into eight cassettes aligned in tandem in the same transcriptional orientation, each containing the basic recombinational unit V-J-J-C, except for a J-J-C cassette, that lacks the V gene and occupies the 3' end of the locus. Amphiphysin (AMPH) and related to steroidogenic acute regulatory protein D3-N-terminal like (STARD3NL) genes flank, respectively, the 5' and 3' ends of the canine TRG locus that spans about 460kb. Moreover LINE1 elements, evenly distributed along the entire sequence, significantly (20.59%) contribute to the architecture of the dog TRG locus. Eight of the 16 TRGV genes are functional and belong to 4 different subgroups. Canine TRGJ genes are two for each cassette and only seven out of 16 are functional. The germline configuration and the exon-intron organization of the 8 TRGC genes was determined, six of them resulting functional. The dot plot similarity genomic comparison of human, mouse and dog TRG loci highlighted the occurrence of reiterated duplications of the cassettes during the dog TRG locus evolution. On the other hand the low ratio of functional genes to the total number of canine TRG genes (21/40), suggest that there is no correlation between the extensive duplications of the cassettes and a need for new functional genes. Furthermore the comparison revealed that the TRGC6, C7 and C8 genes are highly related across species suggesting these existed before the primate-rodent-canidae lineages diverged.

Genomic organization of the sheep TRG1@ locus and comparative analyses of Bovidae and human variable genes.

gammadelta T cells commonly account for 0.5%-5% of human (gammadelta low species) circulating T cells, whereas they are very common in chickens, and they may account for >70% of peripheral cells in ruminants (gammadelta high species). We have previously reported the ovine TRG2@ locus structure, the first complete physical map of any ruminant animal TCR locus. Here we determined the TRG1@ locus organization in sheep, reported all variable (V) gamma gene segments in their germline configuration and included human and cattle sequences in a three species comparison. The TRG1@ locus spans about 140 kb and consists of three clusters named TRG5, TRG3, and TRG1 according to the constant (C) genes. The predicted tertiary structure of cattle and sheep V proteins showed a remarkably high degree of conservation between the experimentally determined human Vgamma9 and the proteins belonging to TRG5 Vgamma subgroup. However systematic comparison of primary and tertiary structure highligthed that in Bovidae the overall conformation of the gammadelta TCR, is more similar to the Fab fragment of an antibody than any TCR heterodimer. Phylogenetic analysis showed that the evolution of cattle and sheep V genes is related to the rearrangement process of V segments with the relevant C, and consequentely to the appartenence of the V genes to a given cluster. The TRG cluster evolution in cattle and sheep pointed out the existence of a TRG5 ancient cluster and the occurrence of duplications of its minimal structural scheme of one V, two joining (J), and one C.

Comparative analyses of sheep and human TRG joining regions: evolution of J genes in Bovidae is driven by sequence conservation in their promoters for germline transcription.

The availability of genomic clones representative of the T cell receptor gamma (TRG1@ and TRG2@) ovine loci enabled us to compare the germline genomic organization and nucleotide diversity of joining (J) segments and reconstruct their evolutionary history by phylogenetic analysis of cattle, sheep and human expressed sequences. Expression profiling (RT-PCR data) in fetus and adult indicated that only the ovine J genes in which two or more of the key sequence features, such as recombination signal sequences (RSS), 3' splice sites, and core sequences, are missing or severely altered fail to be transcribed. Comparative genomic examination of the two human with the six sheep germline transcription promoters located at 5' of the relevant constant (C)-distal J segments showed a strong conservation of the redundant STAT consensus motifs, indicating that TRG1@ and TRG2@ loci are under the influence of IL-7 and STAT signalling. These findings support the phylogenetic analysis of human and Bovidae (cattle and sheep) that revealed a different grouping pattern of C-distal compared to C-proximal J segments. Likewise, the phylogenetic behaviour of either C-distal and C-proximal J segments is in accordance with the Bovidae TRG clusters evolution. Comparison of sheep and human structures of recombination signal sequences (RSS) has highlighted a greater conservation in sheep 12 RSS rather than 23 RSS thus suggesting that the initial recruitment of recombination activating genes (RAG) products requires at least one relatively high-affinity RSS per recombination event.

Artiodactyl emergence is accompanied by the birth of an extensive pool of diverse germline TRDV1 genes.

Molecular cloning of cDNA from gamma/delta T cells has shown that in sheep, the variable domain of the delta chain is chiefly determined by the expression of the TRDV1 subgroup, apparently composed of a large number of genes. There are three other TRDV subgroups, but these include only one gene each. To evaluate the extent and the complexity of the genomic TRDV repertoire, we screened a sheep liver genomic library from a single individual of the Altamurana breed and sheep fibroblast genomic DNA from a single individual of the Gentile di Puglia breed. We identified a total of 22 TRDV1 genes and the TRDV4 gene. A sequence comparison between germline and the rearranged genes indicates that, in sheep, the TRDV repertoire is generated by the VDJ rearrangement of at least 40 distinct TRDV1 genes. All germline TRDV1 genes present a high degree of similarity in their coding as well as in 5' and 3' flanking regions. However, a systematic analysis of the translation products reveals that these genes present a broadly different and specific repertoire in the complementarity-determining regions or recognition loops, allowing us to organize the TRDV genes into sets. We assume that selection processes operating at the level of ligand recognition have shaped the sheep TRDV germline repertoire. A phylogenetic study based on a sequence analysis of the TRDV genes from different mammalian species shows that the diversification level of these genes is higher in artiodactyl species compared to humans and mice.

Evolution of TRG clusters in cattle and sheep genomes as drawn from the structural analysis of the ovine TRG2@ locus.

The availability of genomic clones representative of the T-cell receptor constant gamma (TRGC) ovine genes enabled us to demonstrate, by fluorescent in situ hybridization (FISH) on cattle and sheep metaphases, the presence of two T-cell receptor gamma (TRG1@ and TRG2@) paralogous loci separated by at least five chromosomal bands on chromosome 4. Only TRG1@ is included within a region of homology with human TRG locus on chromosome 7, thus TRG2@ locus appears to be peculiar to ruminants. The structure of the entire TRG2@ locus, the first complete physical map of any ruminant animal TCR gamma locus, is reported here. The TRG2@ spans about 90 kb and consists of three clusters that we named TRG6, TRG2, and TRG4, according to the constant genes name. Phylogenetic analysis has highlighted the correlation between the grouping pattern of cattle and sheep variable gamma (TRGV) genes and the relevant TRGC; variable (V), joining (J), and constant (C) rearrange to be found together in mature transcripts. The simultaneous results on the TRG2@ locus molecular organization in sheep and on the phylogenetic analysis of cattle and sheep V expressed sequences indicate that at least six TRG clusters distributed in the two loci are present in these ruminant animals. The inferred evolution of TRG clusters in cattle and sheep genomes is consistent with a scenario where a minimal ancient cluster, containing the basic structural scheme of one V, one J, and one C gene, has undergone a process of duplication and intrachromosomal transposition.