Evolution by recombination and transspecies polymorphism in the MHC class I gene of Xenopus laevis

The patterns of major histocompatibility complex (MHC) evolution involve duplications, deletions, and independent divergence of loci during episodes punctuated by natural selection. Major differences in MHC evolution among taxa have previously been attributed to variation in linkage patterns of class I and class II MHC genes. Here we characterize patterns of evolution in the MHC class Ia gene of Xenopus laevis in terms of polymorphism, recombination, and extent of transspecies polymorphism. We also compare these patterns to see if a correlation exists with linkage or separation of the MHC class I and class II regions as seen in amphibians and teleost fishes. In X. laevis, we find high levels of polymorphism. Also, genetic exchange is relatively frequent and occurs in intron II, reshuffling allelic forms of exons 2 and 3. Evolutionary relationships among class I alleles show an intermingling of alleles from divergent Xenopus species rather than a species-specific clustering. Results indicate that the patterns of evolution are similar to those found in salmonid fishes and are different from the mode of evolution seen in primates. Similar patterns of class Ia evolution in salmonid fishes and X. laevis suggest that nonlinkage of class I and class II regions alone is insufficient to explain some patterns of MHC evolution in salmonids.     

Two ancient allelic lineages at the single classical class I locus in the Xenopus MHC

Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and alpha2 peptide-binding domains, and there are highly diverse alleles (defined by the alpha1 peptide-binding domain) in each lineage. Surprisingly, the alpha3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki     

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.     

MHC evolution in three salmonid species: a comparison between class II alpha and beta genes

The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II α and class II β loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II α and β genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIα gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.     

Classical MHC class I genes composed of highly divergent sequence lineages share a single locus in rainbow trout (Oncorhynchus mykiss).

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the alpha3 and transmembrane domains is unprecedented.     

The MHC class I linkage group is a major determinant in the in vivo rejection of allogeneic erythrocytes in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Despite accumulating sequence data, information on the function of major histocompatibility complex (MHC) genes in fish is scarce. In contrast to the genome organization in higher vertebrates, the polymorphic MHC class I and II genes are not linked in the teleost genome. A previous study found an MHC class II linkage group to be a major determinant in the rejection of allogeneic scales by a teleost species (Cardwell et al. 2001). The present study investigated whether the teleost MHC class I linkage group can be involved in allograft rejection. Erythrocytes were chosen as grafts since they express MHC class I, but do not express class II. Rainbow trout erythrocytes expressing different MHC class I alleles were differentially stained, mixed and injected into recipients that were of the same sibling group as the donors. The MHC class I linkage group was the major determinant for in vivo graft rejection.     

Patterns of Historical Balancing Selection on the Salmonid Major Histocompatibility Complex Class II β Gene

Allelic variation in the major histocompatibility class (MHC) IIB gene of salmonids is analyzed for patterns indicative of natural selection acting at the molecular level. Sequence data for the second exon of this MHC gene were generated for 11 species in three salmonid genera: Oncorhynchus, Salmo, and Salvelinus. Phylogenetic analysis of nucleotide sequences revealed: (1) monophyletic grouping of alleles from each genus, (2) transspecies evolution of alleles within Salmo and Salvelinus, and (3) differential patterns of transspecies evolution within the genus Oncorhynchus. Within Oncorhynchus, five of seven species had alleles that were species-specific or nearly so, while the remaining two, O. mykiss and O. clarkii, retained ancestral polymorphisms. The different patterns in Oncorhynchus and the other two genera could be due to historical demographic effects or functional differences in MHC molecules in the three genera, but the two hypotheses could not be distinguished with the current dataset. An analysis of recombination/gene conversion identified numerous recombinant alleles, which is consistent with what has been found in other vertebrate taxa. However, these gene conversion events could not account for the species-specific allelic lineages observed in five of the Oncorhynchus species. Analyses of the relative rates of nonsynonymous and synonymous substitutions revealed the signature of selection on the class IIB gene in all 11 of the salmonid species for both the ABS and the non-ABS codons. Codon-based analyses of selection identified seven codons that have experienced selection in the majority of the species. More than half of these sites were mammalian ABS codons, but several were not, suggesting subtle functional differences in the mammalian and teleost fish MHC molecules.     

Identification of novel major histocompatibility complex class I sequences in a marsupial,the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The major histocompatibility complex (MHC) is an essential part of the vertebrate immune response. MHC genes may be classified as classical, non-classical or non-functional pseudogenes. We have investigated the diversity of class I MHC genes in the brushtail possum, a marsupial native to Australia and an introduced pest in New Zealand. The MHC of marsupials is poorly characterised compared to eutherian mammal species. Comparisons between marsupials and eutherians may enhance understanding of the evolution and functions of this important genetic region. We found a high level of diversity in possum class I MHC genes. Twenty novel sequences were identified using polymerase chain reaction (PCR) primers designed from existing marsupial class I MHC genes. Eleven of these sequences shared a high level of homology with the only previously identified possum MHC class I gene TrvuUB and appear to be alleles at a single locus. Another seven sequences are also similar to TrvuUB but have frame-shift mutations or stop codons early in their sequence, suggesting they are non-functional alleles of a pseudogene locus. The remaining sequences are highly divergent from other possum sequences and clusters with American marsupials in phylogenetic analysis, indicating they may have changed little since the separation of Australian and American marsupials.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.     

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.     

Comprehensive analysis of medaka major histocompatibility complex (MHC) class II genes: Implications for evolution in teleosts

The major histocompatibility complex (MHC) class II molecules play central roles in adaptive immunity by regulating immune response via the activation of CD4 T cells. The full complement of the MHC class II genes has been elucidated only in mammalian species to date. To understand the evolution of these genes, we performed their first comprehensive analysis in nonmammalian species using a teleost, medaka (Oryzias latipes). Based on a database search, cDNA cloning, and genomic PCR, medaka was shown to possess five pairs of expressed class II genes, comprising one IIA and one IIB gene. Each pair was located on a different chromosome and was not linked to the class I genes. Only one pair showed a high degree of polymorphism and was considered to be classical class II genes, whereas the other four pairs were nonclassical. Phylogenetic analysis of all medaka class II genes and most reported teleost class II genes revealed that the IIA and IIB genes formed separate clades, each containing three well-corresponding lineages. One lineage contained three medaka genes and all known classical class II genes of Ostariophysi and Euteleostei and was presumed to be an original lineage of the teleost MHC class II genes. The other two lineages contained one nonclassical medaka gene each and some Euteleostei genes. These results indicate that multiple lineages of the teleost MHC class II genes have been conserved for hundreds of millions of years and that the tightly linked IIA and IIB genes have undergone concerted evolution.     

Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis)

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.Electronic Supplementary Material Supplementary material is available for this article at     

Genetic variation of the major histocompatibility complex (MHC class II β gene) in the threatened Gila trout, <Emphasis Type="Italic">Oncorhynchus gilae gilae</Emphasis>

Gila trout (Oncorhynchus gilae gilae) was federally protected in 1973 because of severe declines in abundance and geographic range size. At present, four relict genetic lineages of the species remain in mountain streams of New Mexico and Arizona, USA. Management actions aimed at species recovery, including hatchery production and restocking of formerly occupied streams, have been guided by information from non-functional genetic markers. In this study, we investigated genetic variation at exon 2 of the major histocompatibility complex (MHC) class II β gene that is involved in pathogen resistance and thus presumably under natural selection. Phylogenetic analysis revealed trans-species polymorphism and a significantly high ratio of non-synonymous to synonymous amino acid changes consistent with the action of historical balancing selection that maintained diversity at this locus in the past. However, Gila trout exhibited low allelic diversity (five alleles from 142 individuals assayed) compared to some other salmonid fishes, and populations that originated exclusively from hatcheries possessed three or fewer MHC alleles. Comparative analysis of genetic variation at MHC and six presumably neutrally evolving microsatellite loci revealed that genetic drift cannot be rejected as a primary force governing evolution of MHC in contemporary populations of Gila trout. Maintenance of diversity at MHC will require careful implementation of hatchery breeding protocols and continued protection of wild populations to prevent loss of allelic diversity due to drift.     

Major histocompatibility complex and kin discrimination in Atlantic salmon and brook trout

Many species of salmonids can discriminate kin from unrelated conspecifics using olfactory cues. In this study, we determined the role of the major histocompatibility complex (MHC) in kin discrimination by juvenile Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis). Genetic variation at the highly polymorphic exon coding for peptide-binding region of an MHC class II gene was studied using polymerase chain reaction and denaturing gradient gel electrophoresis. Experiments compared discrimination ability based on MHC haplotypes both within and among kin and non-kin groups. Juveniles chose kin sharing both alleles over kin sharing no alleles. Juveniles also preferred non-kin sharing both alleles to non-kin sharing no alleles. These data suggest that the MHC class II gene influence kin discrimination in juvenile Atlantic salmon and brook trout. The influence of additional genes was also apparent in trials where juveniles were able to recognize kin sharing no alleles over non-kin sharing no alleles. However, the inability of juveniles to discriminate between kin sharing no alleles and non-kin sharing either one or both alleles indicates that MHC is as potent as the rest of the genome in producing distinguishable odours.     

A divergent non-classical class I gene conserved in salmonids

 Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α₃ domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene. Received: 22 September 1998 / Revised: 24 November 1998     

Retained orthologous relationships of the MHC Class I genes during euteleost evolution

Major histocompatibility complex (MHC) class I molecules play a pivotal role in immune defense system, presenting the antigen peptides to cytotoxic CD8+ T lymphocytes. Most vertebrates possess multiple MHC class I loci, but the analysis of their evolutionary relationships between distantly related species has difficulties because genetic events such as gene duplication, deletion, recombination, and/or conversion have occurred frequently in these genes. Human MHC class I genes have been conserved only within the primates for up to 46-66 My. Here, we performed comprehensive analysis of the MHC class I genes of the medaka fish, Oryzias latipes, and found that they could be classified into four groups of ancient origin. In phylogenetic analysis using these genes and the classical and nonclassical class I genes of other teleost fishes, three extracellular domains of the class I genes showed quite different evolutionary histories. The α1 domains generated four deeply diverged lineages corresponding to four medaka class I groups with high bootstrap values. These lineages were shared with salmonid and/or other acanthopterygian class I genes, unveiling the orthologous relationships between the classical MHC class I genes of medaka and salmonids, which diverged approximately 260 Ma. This suggested that the lineages must have diverged in the early days of the euteleost evolution and have been maintained for a long time in their genome. In contrast, the α3 domains clustered by species or fish groups, regardless of classical or nonclassical gene types, suggesting that this domain was homogenized in each species during prolonged evolution, possibly retaining the potential for CD8 binding even in the nonclassical genes. On the other hand, the α2 domains formed no apparent clusters with the α1 lineages or with species, suggesting that they were diversified partly by interlocus gene conversion, and that the α1 and α2 domains evolved separately. Such evolutionary mode is characteristic to the teleost MHC class I genes and might have contributed to the long-term conservation of the α1 domain.     

Unique haplotypes of co-segregating major histocompatibility class II <Emphasis Type="Italic">A</Emphasis> and class II <Emphasis Type="Italic">B</Emphasis> alleles in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) give rise to diverse class II genotypes

Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3' untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.