A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny

In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups. Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of approximately 450 kb and to encompass at least 19 loci. These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit beta (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP). This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region. In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci. It probably represents a different haplotype of part of the class I region. The previously identified UAA gene shares an identical 5' part with UEA, but the two genes differ in their 3' parts. One of them is probably the result of an unequal crossing over. The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.     

Evidence that the separation of Mhc class II from class I loci in the zebrafish,Danio rerio,occurred by translocation

In the zebrafish, Danio rerio, and other teleosts, the class I and class II loci of the major histocompatibility complex ( Mhc) reside on different chromosomes. To shed light on the events that might have generated this difference from tetrapods, in which these two types of loci are clustered in a single chromosomal region, the organization of the class II loci in linkage group 8 of the zebrafish was determined by the characterization of contigs of PAC clones. Three contigs were defined: DAB, DCB, and DBB. The 350-kb-long DAB contig contained only four genes: DDB, DAB, SLC7A4, and DAA. The 150-kb-long DCB contig contained the DCB, DCA, and fz10 genes at an undetermined distance from the DAB contig. And the 120-kb-long DBB contig comprised the DBB gene presumably in another linkage group. The low gene density of the linkage group 8 contigs, contrasting with the high gene density of the zebrafish class I region, and the close association with genes [ SLC7A4 coding for an amino acid transporter, and fz10 (frizzled 10) coding for a receptor of the WNT glycoprotein] that are not linked with the tetrapod Mhc, is interpreted to mean that the separation of the class II from class I loci in teleosts occurred by translocation rather than by genomic or chromosomal duplication.     

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes)

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.     

Comparative genomic analysis of the major histocompatibility complex class I region in the teleost genus <Emphasis Type="Italic">Oryzias</Emphasis>

The major histocompatibility complex (MHC) class I region of teleosts harbors a tight cluster of the class IA genes and several other genes directly involved in class I antigen presentation. Moreover, the dichotomous haplotypic lineages (termed d- and N- lineages) of the proteasome subunit beta genes, PSMB8 and PSMB10, are present in this region of the medaka, Oryzias latipes. To understand the evolution of the Oryzias MHC class I region at the nucleotide sequence level, we analyzed bacterial artificial chromosome clones covering the MHC class I region containing the d- lineage of Oryzias luzonensis and the d- and N- lineages of Oryzias dancena. Comparison among these three elucidated sequences and the published sequences of the d- and N- lineages of O. latipes indicated that the order and orientation of the encoded genes were completely conserved among these five genomic regions, except for the class IA genes, which showed species-specific variation in copy number. The PSMB8 and PSMB10 genes showed trans-species dimorphism. The remaining regions flanking the PSMB10, PSMB8, and class IA genes showed high degrees of sequence conservation at interspecies as well as intraspecies levels. Thus, the three independent evolutionary patterns under apparently distinctive selective pressures are recognized in the Oryzias MHC class I region. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spatial arrangement of pig MHC class I sequences

Bacterial artificial chromosome (BAC) clones were assigned within the pig major histocompatibility complex (Mhc) by polymerase chain reaction-screening and Southern blot hybridization using sequence-tagged site (STS) markers and BAC end-rescued sequences. In all, 35 BAC clones were discovered containing 12 anchor genes of the SLA class I region and two genes of the SLA class III region. Twenty of these 35 clones comprised two distinct class I gene clusters, each spanning about 100 kilobases. One cluster enclosed three class I related genes (SLA-6 to -8) and two genes (MIC-1 and MIC-2) more distantly related to class I. The other cluster enclosed typical class I genes, of which three (SLA-1, -2, and -3) were transcribed by fibroblasts homozygous for the H01 haplotype which we used to construct a pig BAC library. Ordered clones are certainly helpful in isolating agronomically, biologically, and medically important genes. They would also be useful for inducing genetic modifications in pig cell lines.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

Comparative genomic analysis of two avian (quail and chicken) MHC regions

We mapped two different quail Mhc haplotypes and sequenced one of them (haplotype A) for comparative genomic analysis with a previously sequenced haplotype of the chicken Mhc. The quail haplotype A spans 180 kb of genomic sequence, encoding a total of 41 genes compared with only 19 genes within the 92-kb chicken Mhc. Except for two gene families (B30 and tRNA), both species have the same basic set of gene family members that were previously described in the chicken "minimal essential" Mhc. The two Mhc regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated genes with 7 class I, 10 class IIB, 4 NK, 6 lectin, and 8 B-G genes. Comparisons between the quail and chicken Mhc class I and class II gene sequences by phylogenetic analysis showed that they were more closely related within species than between species, suggesting that the quail Mhc genes were duplicated after the separation of these two species from their common ancestor. The proteins encoded by the NK and class I genes are known to interact as ligands and receptors, but unlike in the quail and the chicken, the genes encoding these proteins in mammals are found on different chromosomes. The finding of NK-like genes in the quail Mhc strongly suggests an evolutionary connection between the NK C-type lectin-like superfamily and the Mhc, providing support for future studies on the NK, lectin, class I, and class II interaction in birds.     

Identification and characterization of a beta proteasome subunit cluster in the Japanese pufferfish (Fugu rubripes)

The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.     

Pinpointing a selective sweep to the chimpanzee MHC class I region by comparative genomics

Chimpanzees experienced a reduction of the allelic repertoire at the major histocompatibility complex (MHC) class I A and B loci, which may have been caused by a retrovirus belonging to the simian immunodeficiency virus (SIV) family. Extended MHC haplotypes were defined in a pedigreed chimpanzee colony. Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees to investigate the genomic extent of the repertoire reduction. Multilocus demographic analyses underscored that chimpanzees indeed experienced a selective sweep that mainly targeted the chromosomal segment carrying the Mhc class I region. Probably due to genetic linkage, the sweep also affected other polymorphic loci, mapping in the close vicinity of the Mhc class I region genes. Nevertheless, although the allelic repertoire at particular Mhc class I and II loci appears to be limited, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep. However, recombination did not have sufficient time to erase the signal of the selective sweep.     

Highly divergent dimorphic alleles of the proteasome subunit beta type-8 (PSMB8) gene of the bichir Polypterus senegalus: implication for evolution of the PSMB8 gene of jawed vertebrates

The proteasome subunit beta type-8 (PSMB8) gene encodes a catalytic subunit of the immunoproteasome, which is involved in the generation of peptides presented by MHC class I molecules. To date, highly diverged dichotomous alleles of PSMB8 have been reported in Oryzias species (actinopterygian teleosts) and Xenopus species (sarcopterygian amphibians). These dimorphic alleles share a similar substitution (A/V(31)F/Y) at the 31st position of the mature protein, which is most probably involved in formation of the S1 pocket. This substitution likely confers different cleavage specificities on the dimorphic PSMB8s. In addition, two paralogous PSMB8 genes possessing the A and F residues at the 31st position have been reported in sharks. Phylogenetic analysis indicated that the two types of PSMB8 of Oryzias, Xenopus, and sharks arose by independent evolutionary events. Here, we identified another pair of dimorphic alleles of PSMB8, which have the A and F residues at the 31st position of the mature protein, from bichir, Polypterus senegalus, a basal actinopterygian. The sequences of the mature proteins-encoding region of the dimorphic alleles of bichir PSMB8, the A and F types, showed only 72.7% and 77.5% identities at the nucleotide and the deduced amino acid levels, respectively. Their intronic sequences show almost no similarity, indicating that the dimorphic alleles of bichir PSMB8 have a very ancient origin. However, phylogenetic analysis showed that the dimorphisms of PSMB8 of bichir, Xenopus, and Oryzias arose by independent evolutionary events, suggesting the presence of a strong selective pressure for possessing the dimorphism.     

Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense

Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.     

Organization of Mhc class II A and B genes in the tilapiine fish Oreochromis

Perch-like fishes of the family Cichlidae are models for the study of speciation. An important tool in these studies is the major histocompatibility complex (Mhc) and its organization. The present study takes the first step toward the elucidation of the Mhc class II gene organization in the tilapiine fish Oreochromis niloticus (Orni). Using class II A- and class II B-specific probes, Mhc-bearing clones were identified and isolated from a bacterial artificial chromosome (BAC) library. The analysis of these clones by a combination of molecular, genetic-mapping, and phylogenetic methods led to the identification of nine class II A and 15 class II B loci. Genes at these loci constitute two families, which we designate as class IIa and class IIb families. Each of the families contains A and B loci. Some genes in both families are expressed and functional. The two families differ in their chromosomal location (they are unlinked) and their mode of evolution. The class IIa family genes are conserved across different teleost taxonomical orders, whereas the class IIb family genes are apparently products of multiple, more recent, rounds of gene duplications. The rounds established at least five monophyletic groups of genes. The founding unit of each monophyletic group might have been a pair of class II A and B loci.     

Good copy, bad copy: choosing animal models for HLA-linked diseases

Recent large-scale sequencing and comparative analyses of the major histocompatibility complex (Mhc) provide a novel view of this long-studied region. The main insight is that even though Mhcs are defined by the presence of the Mhc class I and II genes, the regions encoding class I/II histocompatibility antigens are the least conserved among the species; hence the difficulty of modeling the human class I/II-linked diseases. Fortunately, the majority of the genes in the Mhc, the non-class I/II genes, are conserved among the investigated mammals. The full set of Mhc genes in their evolutionary context presents new possibilities to study Mhc-linked diseases by allowing systematic evaluation of the various experimental animals and approaches.     

Ancient allelism at the cytosolic chaperonin-alpha-encoding gene of the zebrafish

The T-complex protein 1, TCP1, gene codes for the CCT-alpha subunit of the group II chaperonins. The gene was first described in the house mouse, in which it is closely linked to the T locus at a distance of approximately 11 cM from the Mhc. In the zebrafish, Danio rerio, in which the T homolog is linked to the class I Mhc loci, the TCP1 locus segregates independently of both the T and the Mhc loci. Despite its conservation between species, the zebrafish TCP1 locus is highly polymorphic. In a sample of 15 individuals and the screening of a cDNA library, 12 different alleles were found, and some of the allelic pairs were found to differ by up to nine nucleotides in a 275-bp-long stretch of sequence. The substitutions occur in both translated and untranslated regions, but in the former they occur predominantly at synonymous codon sites. Phylogenetically, the alleles fall into two groups distinguished also by the presence or absence of a 10-bp insertion/deletion in the 3' untranslated region. The two groups may have diverged as long as 3.5 mya, and the polymorphic differences may have accumulated by genetic drift in geographically isolated populations.     

Natural selection of the major histocompatibility complex (Mhc) in Hawaiian honeycreepers (Drepanidinae)

The native Hawaiian honeycreepers represent a classic example of adaptive radiation and speciation, but currently face one the highest extinction rates in the world. Although multiple factors have likely influenced the fate of Hawaiian birds, the relatively recent introduction of avian malaria is thought to be a major factor limiting honeycreeper distribution and abundance. We have initiated genetic analyses of class II beta chain Mhc genes in four species of honeycreepers using methods that eliminate the possibility of sequencing mosaic variants formed by cloning heteroduplexed polymerase chain reaction products. Phylogenetic analyses group the honeycreeper Mhc sequences into two distinct clusters. Variation within one cluster is high, with dN > dS and levels of diversity similar to other studies of Mhc (B system) genes in birds. The second cluster is nearly invariant and includes sequences from honeycreepers (Fringillidae), a sparrow (Emberizidae) and a blackbird (Emberizidae). This highly conserved cluster appears reminiscent of the independently segregating Rfp-Y system of genes defined in chickens. The notion that balancing selection operates at the Mhc in the honeycreepers is supported by transpecies polymorphism and strikingly high dN/dS ratios at codons putatively involved in peptide interaction. Mitochondrial DNA control region sequences were invariant in the i'iwi, but were highly variable in the 'amakihi. By contrast, levels of variability of class II beta chain Mhc sequence codons that are hypothesized to be directly involved in peptide interactions appear comparable between i'iwi and 'amakihi. In the i'iwi, natural selection may have maintained variation within the Mhc, even in the face of what appears to a genetic bottleneck.     

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.     

QUOD ERAT FACIENDUM: sequence analysis of the H2-D and H2-Q regions of 129/SvJ mice

The H2-D and -Q regions of the mouse major histocompatibility complex ( Mhc or H2) have been sequenced from strain 129/SvJ (haplotype bc), revealing a D/Q region different from all other investigated haplotypes, including the closely related b haplotype. The 300-kb class I-rich region consists of the classical class I, H2-D, and 11 non-classical class I genes. The Q region was formed by two series of tandem duplications. Comparison of the segment between the D and Q1 genes with the H2-K region provides evidence that class I genes were translocated from the K region to the D region, and gives a new explanation for the weak locus specificity of the H-Kand H2-Dalleles.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997