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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.

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 gamma-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.

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The increasing recognition of the importance of biodiversity and genetic variation has led to more frequent calls for their preservation. Moreover, novel molecular and advanced genomic tools can be effectively employed to incorporate genetic variability and genomic information into conservation planning and sustainable programs, aimed at species conservation and slowing the erosion of biological diversity. The particular geomorphological and hydrographic features of Gulf of Taranto (Northern Ionian Sea, Central Mediterranean Sea), supports the occurrence of different cetacean species among that the striped dolphin is the more frequent and abundant species. The aim of this study was to investigate the genetic variability of this species in the study area, deepening the possibility to identify a subpopulation in the basin.

The bottlenose dolphin (Tursiops truncatus) belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T. truncatus T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3′ end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.

Background 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. Results Our analyses revealed that although the organization of the goat TRB locus is broadly similar to that of the other artiodactyl species, with three in-tandem D-J-C clusters located at the 3′ end, a complex and extensive series of duplications have occurred in the V genes at the 5′ end, leading to a marked expansion in the number of the TRBV genes. This phenomenon appears to be a feature of the ruminant lineage since similar gene expansions have also occurred in sheep and cattle. Likewise, the general organization of the goat TRG genes is typical of ruminant species studied so far, with two paralogous TRG loci, TRG1 and TRG2, located in two distinct and distant positions on the same chromosome as result of a split in the ancestral locus. Each TRG locus consists of reiterated V-J-J-C cassettes, with the goat TRG2 containing an additional cassette relative to the corresponding sheep and cattle loci. Conclusions Taken together, these findings demonstrate that strong evolutionary pressures in the ruminant lineage have selected for the development of enlarged sets of TRB and TRG genes that contribute to a diverse T cell receptor repertoire. However, differences observed among the goat, sheep and cattle TRB and TRG genes indicate that distinct evolutionary histories, with independent expansions and/or contractions, have also affected each ruminant species.

These data are presented in support of structural and evolutionary analysis of the published article entitled "The occurrence of three D-J-C clusters within the dromedary TRB locus highlights a shared evolution in Tylopoda, Ruminantia and Suina" (Antonacci et al., 2017) [1]. Here we describe the genomic structure and the gene content of the T cell receptor beta chain (TRB) locus in Camelus dromedarius. As in the other species of mammals, the general genomic organization of the dromedary TRB locus consists of a pool of TRBV genes located upstream of in tandem TRBD-J-C clusters, followed by a TRBV gene with an inverted transcriptional orientation. A peculiarity of the dromedary TRB locus structure is the presence of three TRBD-J-C clusters, which is a common feature of sheep, cattle and pig sequences.

The emergent availability in public databases of more complete genome assemblies allows us to improve genomic data obtained by classical molecular cloning. The main goal of this study was to refine the genomic map of the dromedary TRG locus by integrating our previous genomic data with the analysis of recent genomic assemblies. We identified an additional TRGC cassette, defined as a V-J-C recombination unit, located at the 5’ of the locus and made up of five TRGV genes followed by three TRGJ genes and one TRGC gene. Hence, the complete dromedary TRG locus spans about 105 Kb and consists of three in tandem TRGC cassettes delimited by AMPH and STARD3NL genes at the 5′ and 3′ end, respectively. An expression assay carried out on peripheral blood showed the functional competency for the dromedary TRGC5 cassette and confirmed the presence of the somatic hypermutation mechanism able to enlarge the repertoire diversity of the dromedary γδ T cells.

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.

The present study identifies the genomic structure and the gene content of the T cell receptor beta (TRB) locus in the Oryctolagus cuniculus whole genome assembly. The rabbit locus spans less than 600 Kb and the general genomic organization is highly conserved with respect to other mammalian species. A pool of 74 TRB variable (TRBV) genes distributed in 24 subgroups are located upstream of two in tandem-aligned D-J-C gene clusters, each composed of one TRBD, six TRBJ genes, and one TRBC gene, followed by a single TRBV gene with an inverted transcriptional orientation. All TRB genes (functional, ORF, pseudogenes) of this paper have been approved by the IMGT/WHO-IUIS nomenclature committee. Additionally, five potentially functional protease serine (PRSS) trypsinogen or trypsinogen-like genes were identified: two in tandem PRSS-like genes, followed by two PRSS genes with unique traits, lie downstream of the TRBV1 gene and one PRSS gene is located about 400 Kb away downstream of the TRBV genes. Comparative and phylogenetic analyses revealed that multiple duplication events within a few subgroups have generated the germline repertoire of the rabbit TRBV genes, which is substantially larger than those described in humans, mice, and dogs, suggesting that a strong evolutionary pressure has selected the development of a species-specific TRBV repertoire. Hence, the genomic organization of the TRB locus in the genomes appears to be the result of a balance between the maintenance of a core-number of genes essential for the immunological performances and the requirement of newly arisen genes.

In mammals, T cells develop along two discrete pathways characterized by expression of either the αβ or the γδT cell receptors. Human, mouse, and dog display a low peripheral blood γδ T cell percentage, while sheep accounts for a high proportion of γδ T lymphocytes. In all these species, the genomic organization of the T cell receptor gamma (TRG) locus is well known. To gain further insight into the evolutionary significance of the γδ T cell lineage, the present study has defined the genomic organization of the TRG locus in rabbit (Oryctolagus cuniculus), another mammalian γδ high species, as deduced from the genome assembly. The rabbit TRG locus spans about 70 kb and consists of ten TRGV, two TRGJ genes, and one TRGC gene located 5' to 3' in the locus. When we compared the rabbit sequence with the human, mouse, sheep, and dog counterparts, a higher identity with human as well as sheep with respect to mouse and dog was evident, providing that in the different mammalian species, the TRG locus appears to have evolved independently without any correlation with the γδ condition. The complete sequence of the rabbit TRG locus described here provides also a resource for supporting functional studies especially in the context of the γδ T cell function.

Background The bottlenose dolphin (Tursiops truncatus) is a mammal that belongs to the Cetartiodactyla and have lived in marine ecosystems for nearly 60 millions years. Despite its popularity, our knowledge about its adaptive immunity and evolution is very limited. Furthermore, nothing is known about the genomics and evolution of dolphin antigen receptor immunity. Results Here we report a evolutionary and expression study of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) genes. We have identified in silico the TRG and TRA/TRD genes and analyzed the relevant mature transcripts in blood and in skin from four subjects. The dolphin TRG locus is the smallest and simplest of all mammalian loci as yet studied. It shows a genomic organization comprising two variable (V1 and V2), three joining (J1, J2 and J3) and a single constant (C), genes. Despite the fragmented nature of the genome assemblies, we deduced the TRA/TRD locus organization, with the recent TRDV1 subgroup genes duplications, as it is expected in artiodactyls. Expression analysis from blood of a subject allowed us to assign unambiguously eight TRAV genes to those annotated in the genomic sequence and to twelve new genes, belonging to five different subgroups. All transcripts were productive and no relevant biases towards TRAV-J rearrangements are observed. Blood and skin from four unrelated subjects expression data provide evidence for an unusual ratio of productive/unproductive transcripts which arise from the TRG V-J gene rearrangement and for a “public” gamma delta TR repertoire. The productive cDNA sequences, shared both in the same and in different individuals, include biases of the TRGV1 and TRGJ2 genes. The high frequency of TRGV1-J2/TRDV1- D1-J4 productive rearrangements in dolphins may represent an interesting oligo-clonal population comparable to that found in human with the TRGV9- JP/TRDV2-D-J T cells and in primates. Conclusions Although the features of the TRG and TRA/TRD loci organization reflect those of the so far examined artiodactyls, genomic results highlight in dolphin an unusually simple TRG locus. The cDNA analysis reveal productive TRA/TRD transcripts and unusual ratios of productive/unproductive TRG transcripts. Comparing multiple different individuals, evidence is found for a “public” gamma delta TCR repertoire thus suggesting that in dolphins as in human the gamma delta TCR repertoire is accompanied by selection for public gamma chain. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2841-9) contains supplementary material, which is available to authorized users.

In jawed vertebrates the V-(D)-J rearrangement is the main mechanism generating limitless variations of antigen-specific receptors, immunoglobulins (IGs), and T-cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T-cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V-J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.

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Background In mammals, T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T cell receptors. Human and mouse display a low peripheral blood γδ T cell percentage ("γδ low species") while sheep, bovine and pig accounts for a high proportion of γδ T lymphocytes ("γδ high species"). While the T cell receptor alpha (TRA) and delta (TRD) genes and the genomic organization of the TRA/TRD locus has been determined in human and mouse, this information is still poorly known in artiodactyl species, such as sheep. Results The analysis of the current Ovis aries whole genome assembly, Oar_v3.1, revealed that, as in the other mammalian species, the sheep TRD locus is nested within the TRA locus. In the most 5’ part the TRA/TRD locus contains TRAV genes which are intermingled with TRDV genes, then TRD genes which include seven TRDD, four TRDJ genes, one TRDC and a single TRDV gene with an inverted transcriptional orientation, and finally in the most 3’ part, the TRA locus is completed by 61 TRAJ genes and one TRAC gene. Comparative sequence and analysis and annotation led to the identification of 66 TRAV genes assigned to 34 TRAV subgroups and 25 TRDV genes belonging to the TRDV1 subgroup, while one gene was found for each TRDV2, TRDV3 and TRDV4 subgroups. Multiple duplication events within several TRAV subgroups have generated the sheep TRAV germline repertoire, which is substantially larger than the human one. A significant proportion of these TRAV gene duplications seems to have occurred simultaneously with the amplification of the TRDV1 subgroup genes. This dynamic of expansion has also generated novel multigene subgroups, which are species-specific. Ovis aries TRA and TRD genes identified in this study were assigned IMGT definitive or temporary names and were approved by the IMGT/WHO-IUIS nomenclature committee. The completeness of the genome assembly in the 3' part of the locus has allowed us to interpret rearranged CDR3 of cDNA from both TRA and TRD chain repertoires. The involvement of one up to four TRDD genes into a single transcript makes the potential sheep TRD chain much larger than any known TR chain repertoire. Conclusions The sheep genome, as the bovine genome, contains a large and diverse repertoire of TRA and TRD genes when compared to the “γδ T cell low” species genomes. The composition and length of the rearranged CDR3 in TRD V-delta domains influence the three-dimensional configuration of the antigen-combining site thus suggesting that in ruminants, γδ T cells play a more important and specific role in immune recognition. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1790-z) contains supplementary material, which is available to authorized users.

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.

A genomic region of 41,045 bp encompassing the 3'-end of the sheep T cell receptor beta chain was sequenced. Extensive molecular analysis has revealed that this region retains a unique structural feature for the presence of a third D-J-C cluster, never detected in any other mammalian species examined so far. A total of 3 TRBD, 18 TRBJ and 3 substantially identical TRBC genes were identified in about 28kb. At 13kb, downstream from the last TRBC gene, in an inverted transcriptional orientation, lies a TRBV gene. Sequence comparison and phylogenetic analyses have demonstrated that the extra D-J-C cluster originated from an unequal crossing over between the two ancestral TRBC genes. Interspersed repeats spanning 22.2% of the sequence, contribute to the wider size of the sheep TRB locus with respect to the other mammalian counterparts, without modifying the general genomic architecture. The nucleotide and predicted amino acid sequences from peripheral T cells cDNA clones indicated that the genes from cluster 3 are fully implicated in the beta chain recombination machinery. Closer inspections of the transcripts have also shown that inter-cluster rearrangements and splice variants, involving the additional cluster, increase the functional diversity of the sheep beta chain repertoire.

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.

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.

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.

In jawed vertebrates the V-(D)-J rearrangement is the main mechanism generating limitless variations of antigen-specific receptors, immunoglobulins (IGs), and T-cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T-cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V-J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.

Here is an updated report on the genomic organization of T cell receptor beta (TRB) locus in the domestic dog (Canis lupus familiaris) as inferred from comparative genomics and expression analysis. The most interesting results we found were a second TRBD–J–C cluster, which is absent from the reference genome sequence, and the annotation of two additional TRBV genes. In dogs, TRB locus consists of a library of 37 TRBV genes positioned at the 50 end of two in tandem aligned D–J–C gene clusters, each composed of a single TRBD, 6 TRBJ and one TRBC genes, followed by a single TRBV gene with an inverted transcriptional orientation. The TRB genes are distributed in less than 300 kb, making the canine locus, one of the smaller mammalian TRB locus studied so far. The small size may be ascribed to reduced gene duplication occurrences and a lower density of total interspersed repeats compared to humans and mice. Despite the low TRBV gene content, a large and diversified beta chain repertoire is displayed in the dog peripheral blood. A full usage of TRBV and TRBJ genes, including pseudogenes, and a high level of allelic polymorphism contribute to generate diversity. Finally, this study suggests that the overall TRB locus organization is evolutionarily conserved supporting the dog as a highly suited model system for immune development and diseases.

By a combination of rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR) we identified three T cell receptor delta variable (TRDV) subgroups and five joining (TRDJ) genes expressed in spleen, tonsils and blood of Camelus dromedarius. We provide evidence that the high diversity in sequence and length of the third complementarity determining region (CDR3) is a major component of the TR delta chain variability. Moreover, the identification of the corresponding germline genes allowed us to find out for the first time in a mammalian organism that productively rearranged TRDV genes undergo somatic mutation: the mutation rate of the analysed TRDV4 region is 0.013 per base pair in spleen and 0.009 in blood. The point mutations are scattered throughout the length of the variable domain from framework region FR1 to FR4. This random distribution of the amino acid changes, instead of its CDR clustering observed in immunoglobulins (IG), indicates that somatic mutation in dromedary, while contributing to the development of the TRDV repertoire, is not under antigen selection. © 2011 Elsevier Ltd.

Two genomic clones for the TCRA/TCRD locus have been isolated and characterized in sheep. The first clone corresponds to a new sheep Valpha element, the other to the entire Cdelta gene. Chromosomal mapping by fluorescence in situ hybridization (FISH) of Valpha and Cdelta clones localized the TCRA/TCRD locus on sheep chromosome region 7q14--q22. This is the first physical assignment for genomic clones on sheep chromosome 7 by FISH. Moreover, the present data put the ovine 7q14--q22 and the human 14q11.2 regions in the same syntenic group.

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.

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We have isolated a new interspersed sequence present in a high copy number in the ovine genome. This patchwork sequence, named 3.79 AS1, is part of a larger element encompassing similarities to constant region of reverse transcriptase and to art2 shared with the Bovine Dimer Driven Family (BDDF). The 3.79 AS1 sequence includes homologies to amplification promoting sequences (APS), to a potential origin of bidirectional DNA replication (OBR), to the Alu core sequence motif GGAGGC required for RNA polymerase III promoter function and to the ATGGCTGCCAT sequence that has been shown to be able to induce amplification-dependent transformation in murine cells. Fluorescent in situ hybridization experiments using probes derived from both ends of the 3.79 AS1 sequence showed a widespread signal over all sheep chromosomes, except the Y chromosome. We propose that the structural features of the 3.79 AS1 patchwork sequence, that is likely to be a subfamily of Bov B LINE that invaded the Artiodactyl genome prior to the separation of the Bovidae species, facilitated its massive amplification and dispersion in the ovine genome.

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.

A series of genomic clones derived from a sheep library were used to determine the germline configuration and the exon-intron organization of TRGC2, TRGC3, and TRGC4 genes. Based on the outcomes of molecular analysis, we compared and aligned the genomic sequences with the known complete cDNA sequences of sheep and deduced the exon-intron organization of TRGC genes in this ruminant animal, EX1, corresponding to the disulfide-linked constant domain, and EX3, corresponding to the transmembrane and cytoplasmatic domains, are similar in length in all genes. Conversely, the hinge-encoding EX2A, EX2B, and EX2C exons differ in number and length between genes, and EX2A contains the TTKPP motif irrespective of whether it occurs in single or triplicate form. The molecular data also indicate that at least one additional gene is present in sheep. Phylogenetic analysis grouped the ruminant TRGC genes in two clusters that could have emerged from two ancestral forms that underwent a series of duplications giving rise to the new sequences that were selected and then fixed in the ruminant lineages. A correlation between the cluster distribution in the phylogenetic tree of TRGC genes and their expression during fetal development is discussed.

cDNA sequences obtained from polymerase chain reaction products of reverse-transcribed RNA from sheep thymus showed the presence of a large number of members of the TRDV1 gene family. Some are TRDV1 genes also found in peripheral blood lymphocytes, while four genes had not been described so far. The cDNA sequences also showed extensive junctional diversity and a preferential usage of the three TRDJ elements. We characterized the genomic organization of the sheep TRDJ locus and detected a correlation between the nonrandom usage of TRDJ elements during development and their chromosomal order.

Sertoli cell-only seminiferous tubules of sterile XX, Sxr /-male mice served as an excellent source of pure Sertoli cells. When H-2-compatible female mice were immunized 3 times with these Sertoli cells, resulting antibodies recognized two antigens on the plasma membrane of testicular Sertoli cells. They were male-specific, but ubiquitously expressed H-Y antigen and the cell lineage-specific antigen which Sertoli cells shared with ovarian follicular cells. Doubly primed (2 or 3 times in vivo, and once in vitro) cytotoxic T cells from these females lysed target Sertoli cells in both H-2-restricted and nonrestricted manners. While H-2-restricted killings were attributable to H-Y antigen, further work is needed to identify the Sertoli follicular cell lineage-specific antigen as the cause of H-2-nonrestricted killings.

beta 2m(-), HLA (-) Daudi human male Burkitt lymphoma cells excreted a group of protein subunits that shared three distinctive characteristics; their conspicuously longer half-lives compared to more hydrophilic Daudi excreted proteins, their tendency to form progressively larger polymers by means of interchain disulfide bridges, and the extreme hydrophobicity of these polymers. The plasma membrane of extragonadal somatic cells absorbed 1.2 to 2.8% of these hydrophobic proteins. The unoccupied H-Y receptor sites residing on the plasma membrane of bovine fetal ovarian cells, on the other hand, selectively absorbed polymers of 18,000 mol. wt. subunits, and this antigen-receptor interaction, if allowed to continue for five days, induced the formation of tunica albuginea and seminiferous tubules in bovine XX embryonic indifferent gonads. In this manner, human H-Y antigen excreted by Daudi cells has functionally been identified as a series of polymers derived from 18,000 mol. wt. subunits. While, the H-Y antigenic determinants were retained even by the largest polymeric form that became irreversibly water insoluble, the receptor binding activity was shown only by 36.8% of the available polymeric forms of 18,000 mol. wt. subunits, at the most. Nevertheless, once bound to the receptor site, these polymers were rapidly reduced to the monomeric form on the plasma membrane of bovine fetal ovarian cells. Accordingly, the 18,000 mol. wt. monomer might actually represent the functional form of H-Y antigen.

In accordance with the anchorage site hypothesis, Daudi beta 2-microglobulin (-) HLA (-) human male Burkitt lymphoma cells are incapable of stably maintaining H-Y antigen on their plasma membrane; instead, they excrete it into the culture medium. The proposed testis-organizing function of Daudi H-Y antigen in solution, previously demonstrated in bovine ovarian embryonic cells, has been tested for the first time in human undifferentiated gonads. Cultured in the presence of Daudi excreted H-Y antigen, gonads obtained from early human embryos of 46, XX chromosomal constitution underwent precocious and complete testicular differentiation.

Publisher Summary This chapter discusses the sex-determining mechanism of mammals. The uniqueness of mammals lies in the clear partitioning of their sex-determining mechanism into two components: the primary or gonadal determination and the secondary or extragonadal sex determination. By this partitioning, the first component becomes independent and beyond the reach of sex steroids. The embryonic indifferent gonads of mammals have the inherent tendency to develop toward the ovary. The precise meaning of this general statement is likely to be the bisexual expression of the ovary-organizing plasma membrane component and its specific membrane-bound receptor. Testicular organogenesis that normally, but not always, depends upon the presence of the Y-chromosome is the responsibility of the two plasma membrane components; the male-specific but ubiquitously expressed H-Y antigen and its specific receptors expressed only by gonadal cells but of both sexes. Testis-organizing H-Y antigen is not an integral component of the plasma membrane. Instead, it utilizes β2 -microglobulin-MHC antigen dimers as its plasma membrane anchorage sites. This enables H-Y antigen to play a hormone like role during testicular organogenesis. Thus, the primary sex of mammals is determined not so much by the presence or the absence of the Y chromosome but rather by the expression or nonexpression of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S−F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors—the characteristic of fetal Leydig cells.

Among experimentally produced BALB/C3H aggregation (Blastocyst fusion) chimaeras of the mouse, one fertile XX (BALB)/XY (C3H) male was identified who maintained 50% or more female cells in many parts of his body. Results of H-Y antibody absorption tests revealed an XY to XX transfer of testis-organizing H-Y antigen among testicular Sertoli and Leydig cells, but not among spleen and epidermal cells.

The H-Y antigen status was determined in nine transsexual patients. Our results indicate that sexual behaviour is independent of H-Y antigen constitution, in fact all male-to-female transsexual patients typed as H-Y antigen positive, and the female-to-male transsexual patients were H-Y antigen negative.

This paper reports the isolation and the sequence determination of rat phosphoglycerate kinase (PGK) cDNA clones. This cDNA, derived from an X-linked PGK gene transcript, contains a reading frame of 1254 nt and 5′ and 3′ non coding regions of 40 and 380 nt respectively. Analysis of the nucleotide sequence at the three codon position shows a biased codon usage with a prevalence of the triplet G non G N. Comparison of the inferred rat amino acid sequence with that of other organisms makes possible the calculation of the unit evolutionary period (UEP) for this enzyme, placing it at around 40 million years (My). Thus PGK is one of the oldest housekeeping enzymes.

In this paper we report a detailed comparative and evolutionary analysis of the sequences of constant T-cell receptor (Tcr) C gamma delta genes of artiodactyls compared to the homologous sequences of rodents and primates. Because of the frequency and physiological distribution of gamma delta T-cells in different animals, rodents and humans are defined as "gamma delta low" species and ruminants as "gamma delta high" species. Such a characteristic seems to be due to an adaptive role of gamma delta T-cell function. By analyzing the ruminant gene phylogeny of Tcr C gamma we were able to estimate the distance between cattle and sheep at 18 million years ago, a time that is in agreement with other nonmolecular estimates. For Tcr C gamma delta genes a peculiar phylogenetic relationship was found, with human and mouse clustering together and leaving artiodactyls apart. By using appropriate outgroups, the same phylogenetic pattern was obtained with other T-cell related sequences: namely, Tcr C alpha chain, CD3 gamma and delta invariant subunits. Interleukin-2. Interleukin-2 receptor alpha chain and Interleukin-1 beta with the exception of Tcr C beta chain and Interleukin-1 alpha. In contrast, the analysis of all other T-cell nonrelated genes, available in primary databases reveals a different tree, where primates and artiodactyls are sister taxa and rodents are apart in accordance with the current view of mammalian phylogeny. These data are relevant to important evolutionary issues. They show how misleading a phylogeny based on a single or on a few homologous genes may be. In addition they demonstrate that genes with correlated functions may evolve in a lineage specific manner probably in relation to environmental conditions.

A suspension of free testicular cells were obtained by mild trypsin treatment from newborn BALB/c testes, and their plasma membrane H-Y antigen sites were blocked (lysostripped) by an excess of H-Y antibody of proven specificity and potency (45 min in ice). Upon 16 h of the Moscona-type rotation culture, these treated testicular cells yielded primarily spherical aggregates, more than half of which demonstrated a strong resemblance to ovarian follicles. The resemblance was particularly striking between the smallest testicular folliculoids and primordial ovarian follicles that abound in the newborn female gonad. Under the same condition, control serum-treated testicular cells primarily yielded cylindrical tubular structures that can be very long. Over a critical range, concentrations of H-Y antibody apparently influenced the frequency of testicular folliculoid formation. The above directly supports the proposed testis-organizing function of H-Y antigen and is certainly compatible with the genetic situation encountered in the wood lemming (Myopus schisticolor), that in the functional absence of H-Y antigen, XY gonadal cells readily organize an ovary.