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.     

A locus for familial hypertrophic cardiomyopathy is closely linked to the cardiac myosin heavy chain genes, CRI-L436, and CRI-L329 on chromosome 14 at q11-q12

We report that a gene responsible for familial hypertrophic cardiomyopathy (HC) is closely linked to the cardiac alpha and beta myosin heavy chain (MHC) genes on chromosome 14q11. We have recently shown that probe CRI-L436, derived from the anonymous DNA locus D14S26, detects a polymorphic restriction fragment that segregates with familial HC in affected members of a large Canadian family. Using chromosomal in situ hybridization, we have mapped CRI-L436 to chromosome 14 at q11-q12. Because the cardiac MHC genes also map to this chromosomal band, we have determined the genetic distances between the cardiac beta MHC gene, D14S26, and the familial HC locus. Data presented here show that these three loci are linked within 5 centimorgans on chromosome 14 at q11-q12. The possibility that defects in either the cardiac alpha or beta MHC genes are responsible for familial HC is discussed.     

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     

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.     

Analysis of a 26-kb region linked to the Mhc in zebrafish: genomic organization of the proteasome component beta/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome beta subunit genes.

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for beta subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-gamma. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.     

Evidence for multiple MHC class II β loci in New Zealand’s critically endangered kakapo, Strigops habroptilus

Immunologically important genes of the major histocompatibility complex (MHC) have been characterized in a number of avian species with the general finding of considerable variation in size and structural organization among organisms. A range of nonpasserines which represent early-diverging Neoave lineages have been described as having only one MHC class II β locus potentially leading to the conclusion that this is the ancestral condition. Here, we examine the monotypic, early-diverging, critically endangered kakapo, Strigops habroptilus, for allelic variation at MHC class II β exon 2, as part of species’ recovery efforts. We found two to four confirmed sequence variants per individual indicating the presence of more than one MHC class II β locus. Given the kakapo’s basal evolutionary status, evidence for multiple MHC class II β loci seems to counter the proposed mono-locus history of modern birds. However, MHC gene duplication, maintenance, and loss among and within bird species may confound avian relationships making it difficult to elucidate the ancestral state. This study adds essential data for disentangling the course of MHC structural evolution in birds.     

Gene conversion rapidly generates major histocompatibility complex diversity in recently founded bird populations

Population bottlenecks can restrict variation at functional genes, reducing the ability of populations to adapt to new and changing environments. Understanding how populations generate adaptive genetic variation following bottlenecks is therefore central to evolutionary biology. Genes of the major histocompatibility complex (MHC) are ideal models for studying adaptive genetic variation due to their central role in pathogen recognition. While de novo MHC sequence variation is generated by point mutation, gene conversion can generate new haplotypes by transferring sections of DNA within and across duplicated MHC loci. However, the extent to which gene conversion generates new MHC haplotypes in wild populations is poorly understood. We developed a 454 sequencing protocol to screen MHC class I exon 3 variation across all 13 island populations of Berthelot's pipit (Anthus berthelotii). We reveal that just 11-15 MHC haplotypes were retained when the Berthelot's pipit dispersed across its island range in the North Atlantic ca. 75,000 years ago. Since then, at least 26 new haplotypes have been generated in situ across populations. We show that most of these haplotypes were generated by gene conversion across divergent lineages, and that the rate of gene conversion exceeded that of point mutation by an order of magnitude. Gene conversion resulted in significantly more changes at nucleotide sites directly involved with pathogen recognition, indicating selection for functional variants. We suggest that the creation of new variants by gene conversion is the predominant mechanism generating MHC variation in genetically depauperate populations, thus allowing them to respond to pathogenic challenges.     

Full sib pens of pigs are not suitable to identify variance component of associative effect: a simulation study using Gibbs Sampling

Background

Rainbow trout have an XX/XY genetic mechanism of sex determination where males are the heterogametic sex. The homology of the sex-determining gene (SDG) in medaka to Dmrt1 suggested that SDGs evolve from downstream genes by gene duplication. Orthologous sequences of the major genes of the mammalian sex determination pathway have been reported in the rainbow trout but the map position for the majority of these genes has not been assigned.

Results

Five loci of four candidate genes (Amh, Dax1, Dmrt1 and Sox6) were tested for linkage to the Y chromosome of rainbow trout. We exclude the role of all these loci as candidates for the primary SDG in this species. Sox6i and Sox6ii, duplicated copies of Sox6, mapped to homeologous linkage groups 10 and 18 respectively. Genotyping fishes of the OSU × Arlee mapping family for Sox6i and Sox6ii alleles indicated that Sox6i locus might be deleted in the Arlee lineage.

Conclusion

Additional candidate genes should be tested for their linkage to the Y chromosome. Mapping data of duplicated Sox6 loci supports previously suggested homeology between linkage groups 10 and 18. Enrichment of the rainbow trout genomic map with known gene markers allows map comparisons with other salmonids. Mapping of candidate sex-determining loci is important for analyses of potential autosomal modifiers of sex-determination in rainbow trout.     

A meta-analysis of kidney microarray datasets: investigation of cytokine gene detection and correlation with rt-PCR and detection thresholds

Background

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results

We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion

By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.     

Extended gene map reveals tripartite motif, C-type lectin, and Ig superfamily type genes within a subregion of the chicken MHC-B affecting infectious disease

MHC haplotypes have a remarkable influence on whether tumors form following infection of chickens with oncogenic Marek's disease herpesvirus. Although resistance to tumor formation has been mapped to a subregion of the chicken MHC-B region, the gene or genes responsible have not been identified. A full gene map of the subregion has been lacking. We have expanded the MHC-B region gene map beyond the 92-kb core previously reported for another haplotype revealing the presence of 46 genes within 242 kb in the Red Jungle Fowl haplotype. Even though MHC-B is structured differently, many of the newly revealed genes are related to loci typical of the MHC in other species. Other MHC-B loci are homologs of genes found within MHC paralogous regions (regions thought to be derived from ancient duplications of a primordial immune defense complex where genes have undergone differential silencing over evolutionary time) on other chromosomes. Still others are similar to genes that define the NK complex in mammals. Many of the newly mapped genes display allelic variability and fall within the MHC-B subregion previously shown to affect the formation of Marek's disease tumors and hence are candidates for genes conferring resistance.     

The predisposition to type 1 diabetes linked to the human leukocyte antigen complex includes at least one non-class II gene

The human leukocyte antigen (HLA) complex, encompassing 3.5 Mb of DNA from the centromeric HLA-DPB2 locus to the telomeric HLA-F locus on chromosome 6p21, encodes a major part of the genetic predisposition to develop type 1 diabetes, designated "IDDM1." A primary role for allelic variation of the class II HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci has been established. However, studies of animals and humans have indicated that other, unmapped, major histocompatibility complex (MHC)-linked genes are participating in IDDM1. The strong linkage disequilibrium between genes in this complex makes mapping a difficult task. In the present paper, we report on the approach we have devised to circumvent the confounding effects of disequilibrium between class II alleles and alleles at other MHC loci. We have scanned 12 Mb of the MHC and flanking chromosome regions with microsatellite polymorphisms and analyzed the transmission of these marker alleles to diabetic probands from parents who were homozygous for the alleles of the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes. Our analysis, using three independent family sets, suggests the presence of an additional type I diabetes gene (or genes). This approach is useful for the analysis of other loci linked to common diseases, to verify if a candidate polymorphism can explain all of the association of a region or if the association is due to two or more loci in linkage disequilibrium with each other.     

Selection outweighs drift at a fine scale: Lack of MHC differentiation within a family living lizard across geographically close but disconnected rocky outcrops

The highly polymorphic genes of the major histocompatibility complex (MHC) are involved in disease resistance, mate choice and kin recognition. Therefore, they are widely used markers for investigating adaptive variation. Although selection is the key driver, gene flow and genetic drift also influence adaptive genetic variation, sometimes in opposing ways and with consequences for adaptive potential. To further understand the processes that generate MHC variation, it is helpful to compare variation at the MHC with that at neutral genetic loci. Differences in MHC and neutral genetic variation are useful for inferring the relative influence of selection, gene flow and drift on MHC variation. To date, such investigations have usually been undertaken at a broad spatial scale. Yet, evolutionary and ecological processes can occur at a fine spatial scale, particularly in small or fragmented populations. We investigated spatial patterns of MHC variation among three geographically close, naturally discrete, sampling sites of Egernia stokesii, an Australian lizard. The MHC of E. stokesii has recently been characterized, and there is evidence for historical selection on the MHC. We found E. stokesii MHC weakly differentiated among sites compared to microsatellites, suggesting selection, acting similarly at each site, has outweighed any effects of low gene flow or of genetic drift on E. stokesii MHC variation. Our findings demonstrate the strength of selection in shaping patterns of MHC variation or consistency at a fine spatial scale.