Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family.

BACKGROUND: Several novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach. RESULTS: We have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX3C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX6C motif in Ig1 and a CX7C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX3C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs. CONCLUSIONS: This report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.

Atlantic salmon eggs favour sperm in competition that have similar major histocompatibility alleles.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp.

Peptide-binding motif prediction by using phage display library for SasaUBA*0301, a resistance haplotype of MHC class I molecule from Atlantic Salmon (Salmo salar).

The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7 mer and 12 mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, beta 2m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of beta 2-microglobulin is joined together with a flexible (GGGGS)3 linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to beta 2m/SasaUBA*0301 were isolated after four rounds of panning from the 7 mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12 mer peptides was also screened against beta 2m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.

Natural selection acts on Atlantic salmon major histocompatibility (MH) variability in the wild.

Pathogen-driven balancing selection is thought to maintain polymorphism in major histocompatibility (MH) genes. However, there have been few empirical demonstrations of selection acting on MH loci in natural populations. To determine whether natural selection on MH genes has fitness consequences for wild Atlantic salmon in natural conditions, we compared observed genotype frequencies of Atlantic salmon (Salmo salar) surviving in a river six months after their introduction as eggs with frequencies expected from parental crosses. We found significant differences between expected and observed genotype frequencies at the MH class II alpha locus, but not at a MH class I-linked microsatellite or at seven non-MH-linked microsatellite loci. We therefore conclude that selection at the MH class II alpha locus was a result of disease-mediated natural selection, rather than any demographic event. We also show that survival was associated with additive allelic effects at the MH class II alpha locus. Our results have implications for both the conservation of wild salmon stocks and the management of disease in hatchery fish. We conclude that natural or hatchery populations have the best chance of dealing with episodic and variable disease challenges if MH genetic variation is preserved both within and among populations.

Biocomplexity in a highly migratory pelagic marine fish, Atlantic herring.

The existence of biologically differentiated populations has been credited with a major role in conferring sustainability and in buffering overall productivity of anadromous fish population complexes where evidence for spatial structure is uncontroversial. Here, we describe evidence of correlated genetic and life history (spawning season linked to spawning location) differentiation in an abundant and highly migratory pelagic fish, Atlantic herring, Clupea harengus, in the North Sea (NS) and adjacent areas. The existence of genetically and phenotypically diverse stocks in this region despite intense seasonal mixing strongly implicates natal homing in this species. Based on information from genetic markers and otolith morphology, we estimate the proportional contribution by NS, Skagerrak (SKG) and Kattegat and western Baltic (WBS) fish to mixed aggregations targeted by the NS fishery. We use these estimates to identify spatial and temporal differences in life history (migratory behaviour) and habitat use among genetically differentiated migratory populations that mix seasonally. Our study suggests the existence of more complex patterns of intraspecific diversity than was previously recognized. Sustainability may be compromised if such complex patterns are reduced through generalized management (e.g. area closures) that overlooks population differences in spatial use throughout the life cycle.

Major histocompatibility lineages and immune gene function in teleost fishes: the road not taken.

It has become increasingly clear over the course of the past decade that the immune system genes of teleosts and tetrapods are plainly derived from common ancestral genes. The last 5 years, however, have also made it abundantly clear that in the teleost genome some of these genes are organized in a manner very different from that seen in mammals. These differences are probably the result of differences in life history traits, such as fecundancy, within each group of species when faced with an evolutionary fork in the road shortly after their divergence from each other. One group, the tetrapods, including mammals, chose a highly organized linked major histocompatibility complex, while in teleosts the major histocompatibility genes remained unlinked. In this review we will discuss the structural and functional implications of this different organization, particularly for major histocompatibility genes, but drawing on the current knowledge of some other genes for further support for the hypothesis that each group took a different road, one more traveled and one less taken.

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.

The molecular evolution of the interleukin-1 family of cytokines; IL-18 in teleost fish.

The cytokine network is an important homeostatic system with potent activities in immune surveillance, growth, developmental and repair processes. Although interleukin-1beta (IL-1beta) is considered a pivotal pro-inflammatory cytokine, merely focussing on its inflammatory role would be too narrow an approach. Elucidation of the human, the mouse and the Fugu rubripes (pufferfish) genome now enables a more comprehensive overview of this cytokine family and its receptors in several vertebrate classes. Phylogenetic analyses of the IL-1 family members, comprising over 80 sequences of various fish, amphibian, avian and mammalian species, reveal that for only a few mammalian IL-1 family members unambiguous orthologues have been found in fish, indicating a recent origin of some of the mammalian IL-1 family members. Interestingly, the Fugu genome did reveal teleost orthologues for IL-18 and its putative receptor complex. All teleost IL-1beta sequences cluster separately from IL-1beta sequences of other species. In contrast, a number of IL-1 receptor family members have well conserved fish orthologues. This supports the concept of an ancestral role of this family, possibly in the brain.

Varying disease-mediated selection at different life-history stages of Atlantic salmon in fresh water.

Laboratory studies on associations between disease resistance and susceptibility and major histocompatibility (MH) genes in Atlantic salmon Salmo salar have shown the importance of immunogenetics in understanding the capacity of populations to fight specific diseases. However, the occurrence and virulence of pathogens may vary spatially and temporally in the wild, making it more complicated to predict the overall effect that MH genes exert on fitness of natural populations and over several life-history stages. Here we show that MH variability is a significant determinant of salmon survival in fresh water, by comparing observed and expected genotype frequencies at MH and control microsatellite loci at parr and migrant stages in the wild. We found that additive allelic effects at immunogenetic loci were more likely to determine survival than dominance deviation, and that selection on certain MH alleles varied with life stage, possibly owing to varying pathogen prevalence and/or virulence over time. Our results highlight the importance of preserving genetic diversity (particularly at MH loci) in wild populations, so that they have the best chance of adapting to new and increased disease challenges as a result of projected climate warming and increasing aquaculture.

Rainbow trout (Oncorhynchus mykiss) possess multiple novel immunoglobulin-like transcripts containing either an ITAM or ITIMs.

The recognition of pathogens by the innate immune system relies on a wide range of inhibitory and activating receptors. Some of these non-rearranging receptors belong to the immunoglobulin superfamily (IgSF) and in teleost fish the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) have been reported. Here we describe the identification and characterisation of three new NILTs from rainbow trout (Oncorhynchus mykiss), with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain. The cytoplasmic regions contain either immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or an immunoreceptor tyrosine-based activating motif (ITAM) for downstream signalling. Alignment of the various NILT Ig domains revealed a high similarity, especially between Ig domains from NILTs found in this study. Furthermore, a phylogenetic tree showed that NILTs are more closely related to the triggering receptor expressed on myeloid (TREM) cells and NKp44 than to NITRs. The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues. Expression of NILTs was also shown to be an early event in development, with both eyed eggs and embryos expressing all four genes. The results obtained in this study and future experiments will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in rainbow trout.

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.).

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity.

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.).

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon.

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.

Head kidney-derived macrophages of common carp (Cyprinus carpio L.) show plasticity and functional polarization upon differential stimulation.

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005.

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species.

The 16 African 'large' barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44.

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.

The major histocompatibility class I locus in Atlantic salmon (Salmo salar L.): polymorphism, linkage analysis and protein modelling.

A cDNA library screening using the conserved exon 4 of Atlantic salmon Mhc class I as probe provided the basis for a study on Mhc class I polymorphism in a breeding population. Twelve different alleles were identified in the 82 dams and sires studied. No individual expressed more than two alleles, which corresponded to the diploid segregation patterns of the polymorphic marker residing within the 3'-untranslated tail. Close linkage between the Sasa-UBA and Sasa-TAP2B loci strengthens the claim that Sasa-UBA is the major Mhc class I locus in Atlantic salmon. We found no evidence for a second expressed classical or non-classical Mhc class I locus in Atlantic salmon. A phylogenetic analysis of salmonid Mhc class I sequences showed domains conserved between rainbow trout, brown trout and Atlantic salmon. Evidence for shuffling of the alpha(1) domain was identified and lineages of the remaining alpha(2) through the cytoplasmic tail gene segment can be defined. The coding sequence of one allele was found associated with two different markers, suggesting recombination within the 3'-tail dinucleotide repeat itself. Protein modelling of several Sasa-UBA alleles shows distinct differences in their peptide binding domains and enables a further understanding of the functionality of the high polymorphism.