Characterization of the beta2-microglobulin gene of the horse

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.     

A second locus and new alleles in the major histocompatibility complex class II (ELA-DQB) region in the horse

More than two nucleotide sequences of the second exon of the ELA-DQB region retrieved from a single animal and two different sequences isolated from horses homozygous in the major histocompatibility complex (MHC) region by descent indicated the existence of at least two ELA-DQB loci at the genomic level. New alleles detected by polymerase chain reaction single strand conformation polymorphism (SSCP) and defined by nucleotide sequencing of the second exon of the DQB gene(s) were described. Based on the level of nucleotide sharing, at least two groups of alleles were shown to exist. The newly defined alleles belonged preferentially to one of the groups. However, their specific locus assignment was not possible from the data collected. At least one of these alleles was shown to be transcribed. No frame-shift mutations were identified among the new alleles, although one pseudoallele containing a stop codon was identified at the genomic DNA level.     

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.     

Fixed nucleotide differences on the Y chromosome indicate clear divergence between Equus przewalskii and Equus caballus

The phylogenetic relationship between Equus przewalskii and E. caballus is often a matter of debate. Although these taxa have different chromosome numbers, they do not form monophyletic clades in a phylogenetic tree based on mtDNA sequences. Here we report sequence variation from five newly identified Y chromosome regions of the horse. Two fixed nucleotide differences on the Y chromosome clearly display Przewalski's horse and domestic horse as sister taxa. At both positions the Przewalski's horse haplotype shows the ancestral state, in common with the members of the zebra/ass lineage. We discuss the factors that may have led to the differences in mtDNA and Y-chromosomal observations.     

Extensive polymorphism and geographical variation at a positively selected MHC class II B gene of the lesser kestrel (Falco naumanni)

Understanding the selective forces that shape genetic variation in natural populations remains a high priority in evolutionary biology. Genes at the major histocompatibility complex (MHC) have become excellent models for the investigation of adaptive variation and natural selection because of their crucial role in fighting off pathogens. Here we present one of the first data sets examining patterns of MHC variation in wild populations of a bird of prey, the lesser kestrel, Falco naumanni . We report extensive polymorphism at the second exon of a putatively functional MHC class II gene, Fana- DAB*1. Overall, 103 alleles were isolated from 121 individuals sampled from Spain to Kazakhstan. Bayesian inference of diversifying selection suggests that several amino acid sites may have experienced strong positive selection (ω = 4.02 per codon). The analysis also suggests a prominent role of recombination in generating and maintaining MHC diversity (ρ = 4 Nc  = 0.389 per codon, θ = 0.017 per codon). Both the Fana -DAB*1 locus and a set of eight polymorphic microsatellite markers revealed an isolation-by-distance pattern across the Western Palaearctic ( r  = 0.67; P  = 0.01 and r  = 0.50; P  = 0.04, respectively). Nonetheless, geographical variation at the MHC contrasts with relatively uniform distributions in the frequencies of microsatellite alleles. In addition, we found lower fixation rates in the MHC than those predicted by genetic drift after controlling for neutral mitochondrial sequences. Our results therefore underscore the role of balancing selection as well as spatial variations in parasite-mediated selection regimes in shaping MHC diversity when gene flow is limited.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.     

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.     

Analysis of MHC class I genes across horse MHC haplotypes

The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The nonclassical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine major histocompatibility complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Gene duplication,allelic diversity,selection processes and adaptive value of MHC class II <Emphasis Type="Italic">DRB</Emphasis> genes of the bank vole, <Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>

The generation and maintenance of allelic polymorphism in genes of the major histocompatibility complex (MHC) is a central issue in evolutionary genetics. Recently, the focus has changed from ex situ to in situ populations to understand the mechanisms that determine adaptive MHC polymorphism under natural selection. Birth-and-death evolution and gene conversion events are considered to generate sequence diversity in MHC genes, which subsequently is maintained by balancing selection through parasites. The ongoing arms race between the host and parasites leads to an adaptive selection pressure upon the MHC, evident in high rates of non-synonymous vs synonymous substitution rates. We characterised the MHC class II DRB exon 2 of free living bank voles, Clethrionomys glareolus by single-strand conformation polymorphism and direct sequencing. Unlike other arvicolid species, the DRB locus of the bank vole is at least quadruplicated. No evidence for gene conversion events in the Clgl-DRB sequences was observed. We found not only high allelic polymorphism with 26 alleles in 36 individuals but also high rates of silent polymorphism. Exceptional for MHC class II genes is a purifying selection pressure upon the majority of MHC-DRB sequences. Further, we analysed the association between certain DRB alleles and the parasite burden with gastrointestinal trichostrongyle nematodes Heligmosomum mixtum and Heligmosomoides glareoli and found significant quality differences between specific alleles with respect to infection intensity. Our findings suggest a snapshot in an evolutionary process of ongoing birth-and-death evolution. One allele cluster has lost its function and is already silenced, another is loosing its adaptive value in terms of gastrointestinal nematode resistance, while a third group of alleles indicates all signs of classical functional MHC alleles.     

Evidence of gene orthology and trans‐species polymorphism,but not of parallel evolution,despite high levels of concerted evolution in the major histocompatibility complex of flamingo species

The major histocompatibility complex (MHC) is a cornerstone in the study of adaptive genetic diversity. Intriguingly, highly polymorphic MHC sequences are often not more similar within species than between closely related species. Divergent selection of gene duplicates, balancing selection maintaining trans‐species polymorphism (TSP) that predate speciation and parallel evolution of species sharing similar selection pressures can all lead to higher sequence similarity between species. In contrast, high rates of concerted evolution increase sequence similarity of duplicated loci within species. Assessing these evolutionary models remains difficult as relatedness and ecological similarities are often confounded. As sympatric species of flamingos are more distantly related than allopatric species, flamingos represent an ideal model to disentangle these evolutionary models. We characterized MHC Class I exon 3, Class IIB exon 2 and exon 3 of the six extant flamingo species. We found up to six MHC Class I loci and two MHC Class IIB loci. As all six species shared the same number of MHC Class IIB loci, duplication appears to predate flamingo speciation. However, the high rate of concerted evolution has prevented the divergence of duplicated loci. We found high sequence similarity between all species regardless of codon position. The latter is consistent with balancing selection maintaining TSP, as under this mechanism amino acid sites under pathogen‐mediated selection should be characterized by fewer synonymous codons (due to their common ancestry) than under parallel evolution. Overall, balancing selection maintaining TSP appears to result in high MHC similarity between species regardless of species relatedness and geographical distribution.     

Interspecific hybridization increases MHC class II diversity in two sister species of newts

Our understanding of the evolutionary mechanisms generating variation within the highly polymorphic major histocompatibility complex (MHC) genes remains incomplete. Assessing MHC variation across multiple populations, of recent and ancient divergence, may facilitate understanding of geographical and temporal aspects of variation. Here, we applied 454 sequencing to perform a large-scale, comprehensive analysis of MHC class II in the closely related, hybridizing newts, Lissotriton vulgaris (Lv) and Lissotriton montandoni (Lm). Our study revealed an extensive (299 alleles) geographically structured polymorphism. Populations at the southern margin of the Lv distribution, inhabited by old and distinct lineages (southern Lv), exhibited moderate MHC variation and strong population structure, indicating little gene flow or extensive local adaptation. Lissotriton vulgaris in central Europe and the northern Balkans (northern Lv) and almost all Lm populations had a high MHC variation. A much higher proportion of MHC alleles was shared between Lm and northern Lv than between Lm and southern Lv. Strikingly, the average pairwise F(ST) between northern Lv and Lm was significantly lower than between northern and southern Lv for MHC, but not for microsatellites. Thus, high MHC variation in Lm and northern Lv may result from gene flow between species. We hypothesize that the interspecific exchange of MHC genes may be facilitated by frequency-dependent selection. A marginally significant correlation between the MHC and microsatellite allelic richness indicates that demographic factors may have contributed to the present-day pattern of MHC variation, but unequivocal signatures of adaptive evolution in MHC class II sequences emphasize the role of selection on a longer timescale.     

Major histocompatibility complex variation at three class II loci in the northern elephant seal

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.     

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal

Summary XY females (n=17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.     

Polymorphism and signature of selection in the MHC class I genes of the three-spined stickleback Gasterosteus aculeatus

The role and intensity of positive selection maintaining the polymorphism of major histocompatibility complex (MHC) class I genes in the three-spined stickleback Gasterosteus aculeatus was investigated. The highly polymorphic set of MHC class I genes found was organized in a single linkage group. Between 5 and 14 sequence variants per individual were identified by single-stranded conformation polymorphism (SSCP) analysis. Segregation analysis studied in 10 three-spined stickleback families followed the expected pattern of Mendelian inheritance. The gamete fusion in three-spined stickleback thus seems to be random with respect to the MHC class I genes. The DNA sequence analyses showed that the expressed MHC class I loci are under strong selection pressure, possibly mediated by parasites. Codons that were revealed to be under positive selection are potentially important in antigen binding. MHC class I sequences did not form significant supported clusters within a phylogenetic tree. Analogous to MHC class II genes, it was not possible to assign the class I sequences to a specific locus, suggesting that the class I genes may have been generated by recent gene duplication.     

Analysis of major histocompatibility complex class II gene in water voles using capillary electrophoresis‐single stranded conformation polymorphism

Analysis of a single strand conformation polymorphism (SSCP) using capillary electrophoresis (CE) is a novel method to study polymorphism of DNA sequences in large scale population studies. We optimized CE‐SSCP analysis to study the major histocompatibility complex (MHC) class II alpha gene (DQA) polymorphism. Short‐chain linear polyacrylamide (6%) as sieving matrix, TrisCl (pH 8.5) as buffer for sample dilution, and 27 °C, 9 kV as electrophoresis parameters were suitable for sufficient resolution of all alleles. We found that almost 25% of clones contained a PCR (polymerase chain reaction) artefact and strict criteria have to be applied when using cloning and sequencing to analyse the allelic diversity of MHC genes.     

Molecular Characterization of the Major Histocompatibility Complex Class Ia Gene in the Black-Spotted Frog, Pelophylax nigromaculata

The genes of the major histocompatibility complex (MHC) are attractive candidates for investigating the link between adaptive variation and individual fitness. We improved rapid amplification of cDNA ends to obtain the whole coding sequence of the MHC class Ia gene of the black-spotted frog (Pelophylax nigromaculata), the most common amphibian in China. We also used genome walking to characterize the partial introns adjacent to exon 3 of the MHC Ia gene. Based on the sequences obtained, we designed locus-specific primers to investigate the molecular polymorphisms of this species in southeast China. The MHC class Ia gene showed a high level of genetic diversity, indicating that this species retains a relatively high potential for survival, despite a population decline among frog species in general and many other amphibians.     

Diversity and evolutionary patterns of immune genes in free-ranging Namibian leopards (Panthera pardus pardus)

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed.