The Genomic Organisation of the TRA/TRD Locus Validates the Peculiar Characteristics of Dromedary δ-Chain Expression

The role of gamma/delta T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., “ gamma/delta low species”) with a restricted diversity of gamma/delta T cell receptors (TR). Conversely, artiodactyl species (i.e., “ gamma/delta high species”) account for a high proportion of gamma/delta T cells with large gamma and delta chain repertoires. The genomic organisation of the gamma TR (TRG) and delta (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for gamma and delta chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR alpha (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary delta chains. Furthermore, the comparison between genomic and expressed sequences reveals that D genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary delta chain antigen binding-site

Comparative Analysis of the TRB Locus in the Camelus Genus

T cells can be separated into two major subsets based on the heterodimer that forms their T cell receptors. αβ T cells have receptors consisting of α and β chains, while γδ T cells are composed of γ and δ chains. αβ T cells play an essential role within the adaptive immune responses against pathogens. The recent genomic characterization of the Camelus dromedarius T cell receptor β (TRB) locus has allowed us to infer the structure of this locus from the draft genome sequences of its wild and domestic Bactrian congeners, Camelus ferus and Camelus bactrianus. The general structural organization of the wild and domestic Bactrian TRB locus is similar to that of the dromedary, with a pool of TRBV genes positioned at the 5′ end of D-J-C clusters, followed by a single TRBV gene located at the 3′ end with an inverted transcriptional orientation. Despite the fragmented nature of the assemblies, comparative genomics reveals the existence of a perfect co-linearity between the three Old World camel TRB genomic sequences, which enables the transfer of information from one sequence to another and the filling of gaps in the genomic sequences. A virtual camelid TRB locus is hypothesized with the presence of 33 TRBV genes distributed in 26 subgroups. Likewise, in the artiodactyl species, three in-tandem D-J-C clusters, each composed of one TRBD gene, six or seven TRBJ genes, and one TRBC gene, are placed at the 3′ end of the locus. As reported in the ruminant species, a group of four functional TRY genes at the 5′ end and only one gene at the 3′ end, complete the camelid TRB locus. Although the gene content is similar, differences are observed in the TRBV functional repertoire, and genes that are functional in one species are pseudogenes in the other species. Hence, variations in the functional repertoire between dromedary, wild and domestic Bactrian camels, rather than differences in the gene content, may represent the molecular basis explaining the disparity in the TRB repertoire between the Camelus species. Finally, our data contribute to the knowledge about the evolutionary history of Old World camelids

The expansion of the TRB and TRG genes in domestic goats (Capra hircus) is characteristic of the ruminant species

Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects their successful adaptation to diverse environmental conditions. Despite the relevance of this species, the genetic research on the goat traits is limited compared to other domestic species. Thanks to the latest goat reference genomic sequence (ARS1), which is considered to be one of the most continuous assemblies in livestock, we deduced the genomic structure of the T cell receptor beta (TRB) and gamma (TRG) loci in this ruminant species

The Organization of the Pig T-Cell Receptor γ (TRG) Locus Provides Insights into the Evolutionary Patterns of the TRG Genes across Cetartiodactyla

The domestic pig (Sus scrofa) is a species representative of the Suina, one of the four suborders within Cetartiodactyla. In this paper, we reported our analysis of the pig TRG locus in comparison with the loci of species representative of the Ruminantia, Tylopoda, and Cetacea suborders. The pig TRG genomic structure reiterates the peculiarity of the organization of Cetartiodactyla loci in TRGC “cassettes”, each containing the basic V-J-J-C unit. Eighteen genes arranged in four TRGC cassettes, form the pig TRG locus. All the functional TRG genes were expressed, and the TRGV genes preferentially rearrange with the TRGJ genes within their own cassette, which correlates the diversity of the γ-chain repertoire with the number of cassettes. Among them, the TRGC5, located at the 5′ end of the locus, is the only cassette that retains a marked homology with the corresponding TRGC cassettes of all the analyzed species. The preservation of the TRGC5 cassette for such a long evolutionary time presumes a highly specialized function of its genes, which could be essential for the survival of species. Therefore, the maintenance of this cassette in pigs confirms that it is the most evolutionarily ancient within Cetartiodactyla, and it has undergone a process of duplication to give rise to the other TRGC cassettes in the different artiodactyl species in a lineage-specific manner

Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains

In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total of 331 mutations from a single animal, and comparing the properties of the mutation profiles of dromedary TRG and TRD variable domains. The transition preference and the significant mutation frequency in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lineages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rearrangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by the fact that our structural models, computed by adopting a comparative procedure, are consistent with the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural stability of the \u3b3\u3b4 subunits

An overall presentation of the T cell receptor (TR) genomics in Camelidae: future perspectives through Illumina sequencing in Camelus dromedarius

Here we review the structural characteristics and the current status of the detailed genomic organization of α/ β and γ/δ T cell receptors (TRs) loci in the Camelus genus. We discuss on the organization of loci and chains from γ/δ TR, during the synthesis of which somatic hypermutations occur, a process which is known for an extremely limited number of species for TR. Thanks to identification of the reference (AMPH and STARD3NL) genes in Camelus dromedarius and Camelus ferus, we report the updating of the gamma (TRG) locus discussing evolutionary aspects with respect to human and sheep loci upon the identification of the TRGC5 cassette. We also review the comparative genomics of the T cell receptor beta (TRB) locus starting from the Camelus dromedarius and moving forward through the draft genome sequences of its wild and domestic Bactrian congeners, Camelus ferus and Camelus bactrianus. Therefore, with the aim of investigating the genomic variability of the T cell receptors loci in Camelus dromedarius considered as useful and promising model for therapeutic applications and for phylogenetic studies in the adaptive immune responses, we intend to take advantage of the large-scale project of Illumina genome sequencing. The 2019 Illumina® Agricultural Greater Good Initiative Award is expected to generate hundreds of whole-genome sequences of Camelus dromedarius samples collected from a wide intercontinental geographic range, thus paving the way toward an improved understanding of the genetic architecture of T cell receptors loci and their interindividual diversity

Brief research report: 3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how gamma delta T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary gamma delta T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted gamma delta T cell receptor complex

Cytochrome b marker reveals an independent lineage of Stenella coeruleoalba in the Gulf of Taranto

Heterogeneity in geomorphological and hydrographical conditions throughout the Mediterranean Sea could be the driving factors behind the significant differences between putative sub-populations, although the existence of a large panmictic population of striped dolphin Stenella coeruleoalba (Meyen 1833) in this marine region could not be excluded. However, understanding the ecological implications of such genetic differentiation is difficult, as inferences about gene flow are usually made on evolutionary time scales and not along the ecological time frame over which most management and conservation practices are applied. In fact, as stated by the IUCN Red List, in the case of species assessed as vulnerable, the degree of genetic exchange between populations within a biogeographic region and its ecological implications represent a fascinating challenge that should be very deeply explored. This is even more significant in the Gulf of Taranto (Northern Ionian Sea, Central-eastern Mediterranean Sea), where the geomorphological and hydrographic characteristics support the hypothesis of a separated striped dolphin population genetically diverging from its original Mediterranean counterpart. To assess this hypothesis, a genetic analysis was carried out on DNA fragments of the mitochondrial cyt b gene to explore the evolutionary origin of S. coeruleoalba in the investigated area and its genetic diversity in comparison with available sequences from other Mediterranean and Atlantic populations. Results were discussed indicating ecological implications and suggesting conservation objectives. Moreover, a delphinid systematic was also suggested