Locus specificity of polymorphic alleles and evolution by a birth-and-death process in mammalian MHC genes

We have conducted an extensive phylogenetic analysis of polymorphic alleles from human and mouse major histocompatibility complex (MHC) class I and class II genes. The phylogenetic tree obtained for 212 complete human class I allele sequences (HLA-A, -B, and -C) has shown that all alleles from the same locus form a single cluster, which is highly supported by bootstrap values, except for one HLA-B allele (HLA-B*7301). Mouse MHC class I loci did not show locus-specific clusters of polymorphic alleles. This was considered to be because of either interlocus genetic exchange or the confusing designation of loci in different haplotypes at the present time. The locus specificity of polymorphic alleles was also observed in human and mouse MHC class II loci. It was therefore concluded that interlocus recombination or gene conversion is not very important for generating MHC diversity, with a possible exception of mouse class I loci. According to the phylogenetic trees of complete coding sequences, we classified human MHC class I (HLA-A, -B, and -C) and class II (DRB1) alleles into three to five major allelic lineages (groups), which were monophyletic with high bootstrap values. Most of these allelic groups remained unchanged even in phylogenetic trees based on individual exons, though this does not exclude the possibility of intralocus recombination involving short DNA segments. These results, together with the previous observation that MHC loci are subject to frequent duplication and deletion, as well as to balancing selection, indicate that MHC evolution in mammals is in agreement with the birth-and-death model of evolution, rather than with the model of concerted evolution.     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

 HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons. Received: 22 April 1998 / Revised: 15 July 1998     

Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci—FLAI-E, FLAI-H, and FLAI-K—are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.     

Molecular analyses of five new chimpanzee MHC class I alleles: implications for differences between evolutional mechanisms of HLA-A, -B, and -C loci.

In order to study the origin of the polymorphism of MHC class I molecules, we have cloned and sequenced five new Patr-A, -B, and -C loci alleles from two chimpanzees. Previous studies of sequence comparison between Patr and HLA class I alleles revealed that many of the sequence motifs were shared and the origin of class I molecules predated the divergence of chimpanzees and humans. These findings are confirmed by our current study. Additionally, our data suggest significant differences between mechanisms of evolution of the A, B, and C loci: (1) The B locus is characterized by frequent nucleotide substitutions, whereas the A and C loci are relatively more conserved; (2) However, unlike the A locus, the alpha2 domains of the C locus sequenced appear to produce MHC polymorphism between these species. These differences might imply the distinctive contributions of each locus during the evolutionary history.     

Contrasting Roles of Interallelic Recombination at the Hla-a and Hla-B Loci

A statistical study of DNA sequences of alleles at the highly polymorphic class I MHC loci of humans, HLA-A and HLA-B, showed evidence of both large-scale recombination events (involving recombination of exons 1-2 of one allele with exons 3-8 of another) and small-scale recombination events (involving apparent exchange of short DNA segments). The latter events occurred disproportionately in the region of the gene encoding the antigen recognition site (ARS) of the class I molecule. Furthermore, they involved the ARS codons which are under the strongest selection favoring allelic diversity at the amino acid level. Thus, the frequency of recombinant alleles appears to have been increased by some form of balancing selection (such as overdominant selection) favoring heterozygosity in the ARS. These analyses also revealed a striking difference between the A and B loci. Recombination events appear to have occurred about twice as frequently at the B locus, and recombinants at the B locus were significantly more likely to affect polymorphic sites in the ARS. At the A locus, there are well-defined allelic lineages that have persisted since prior to the human-chimpanzee divergence; but at the B locus, there is no evidence for such long-lasting allelic lineages. Thus, relatively frequent interallelic recombination has apparently been a feature of the long-term evolution of the B locus but not of the A locus.     

A novel, nonclassical MHC class I molecule specific to the common chimpanzee

All expressed human MHC class I genes (HLA-A, -B, -C, -E, -F, and -G) have functional orthologues in the MHC of the common chimpanzee (Pan troglodytes). In contrast, a nonclassical MHC class I gene discovered in the chimpanzee is not present in humans or the other African ape species. In exons and more so in introns, this Patr-AL gene is similar to the expressed A locus in the orangutan, Popy-A, suggesting they are orthologous. Patr-AL/Popy-A last shared a common ancestor with the classical MHC-A locus >20 million years ago. Population analysis revealed little Patr-AL polymorphism: just three allotypes differing only at residues 52 and 91. Patr-AL is expressed in PBMC and B cell lines, but at low level compared with classical MHC class I. The Patr-AL polypeptide is unusually basic, but its glycosylation, association with beta(2)-microglobulin, and antigenicity at the cell surface are like other MHC class I. No Patr-AL-mediated inhibition of polyclonal chimpanzee NK cells was detected. The Patr-AL gene is present in 50% of chimpanzee MHC haplotypes, correlating with presence of a 9.8-kb band in Southern blots. The flanking regions of Patr-AL contain repetitive/retroviral elements not flanking other class I genes. In sequenced HLA class I haplotypes, a similar element is present in the A*2901 haplotype but not the A*0201 or A*0301 haplotypes. This element, 6 kb downstream of A*2901, appears to be the relic of a human gene related to Patr-AL. Patr-AL has characteristics of a class I molecule of innate immunity with potential to provide common chimpanzees with responses unavailable to humans.     

Serological and molecular diversity in the cattle MHC class I region

Information on major histocompatibility complex (MHC) diversity in cattle is important to aid our understanding of immune responses and may contribute to maintenance of healthy cattle populations. Equally, understanding the mechanisms involved in generating this diversity may shed light on the complex nature of mammalian MHC evolution. The aim of this study was to assess molecular and serological variation within cattle MHC class I molecules and to study the mechanisms generating diversity. To address this aim, sequence variation was examined in 12 serologically assigned alleles from three putative loci and correlated with monoclonal antibody (mAb) binding data. The results demonstrate that both alloantisera and mAbs often fail to distinguish gene products that differ by a significant number of amino acids. Conversely, some mAbs could distinguish alleles differing by only one or two amino acids. Examination of the sequences demonstrates sharing of motifs between alleles, some encoded at distinct loci, supporting the occurrence of interlocus recombination within the cattle MHC class I region. The implications of this for MHC sequence diversity, and functional capability, are discussed.     

The MHC E locus in macaques is polymorphic and is conserved between macaques and humans

Although the functions of the molecules encoded by the classical MHC class I loci are well defined, no function has been ascribed to the molecules encoded by the non-classical MHC class I loci. To investigate the evolution and conservation of the non-classical loci, we cloned and sequenced HLA-E homologues in macaques. We isolated four E locus alleles from five rhesus monkeys and two E locus alleles from one cynomolgus monkey, which indicated that the E locus in macaques is polymorphic. We also compared the rate of nucleotide substitution in the second intron of the macaque and human E locus alleles with that of exons two and three. The rate of nucleotide substitution was significantly higher in the introns, which suggested that the E locus has evolved under selective pressure. Additionally, comparison of the rates of synonymous and non-synonymous substitutions in the peptide binding region versus the remainder of the molecule suggested that the codons encoding the amino acids in the peptide binding region had been conserved in macaques and humans over the 36 million years since macaques and humans last shared a common ancestor.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers UO2976–UO2981     

Characterisation of Major Histocompatibility Complex Class I in the Australian Cane Toad,Rhinella marina

The Major Histocompatibility Complex (MHC) class I is a highly variable gene family that encodes cell-surface receptors vital for recognition of intracellular pathogens and initiation of immune responses. The MHC class I has yet to be characterised in bufonid toads (Order: Anura; Suborder: Neobatrachia; Family: Bufonidae), a large and diverse family of anurans. Here we describe the characterisation of a classical MHC class I gene in the Australian cane toad, Rhinella marina. From 25 individuals sampled from the Australian population, we found only 3 alleles at this classical class I locus. We also found large number of class I alpha 1 alleles, implying an expansion of class I loci in this species. The low classical class I genetic diversity is likely the result of repeated bottleneck events, which arose as a result of the cane toad''s complex history of introductions as a biocontrol agent and its subsequent invasion across Australia.     

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.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

Genetic diversity of MHC class I loci in six non-model frogs is shaped by positive selection and gene duplication

Comparative studies of major histocompatibility complex (MHC) genes across vertebrate species can reveal the evolutionary processes that shape the structure and function of immune regulatory proteins. In this study, we characterized MHC class I sequences from six frog species representing three anuran families (Hylidae, Centrolenidae and Ranidae). Using cDNA from our focal species, we amplified a total of 79 unique sequences spanning exons 2-4 that encode the extracellular domains of the functional alpha chain protein. We compared intra- and interspecific nucleotide and amino-acid divergence, tested for recombination, and identified codon sites under selection by estimating the rate of non-synonymous to synonymous substitutions with multiple codon-based maximum likelihood methods. We determined that positive (diversifying) selection was acting on specific amino-acid sites located within the domains that bind pathogen-derived peptides. We also found significant signals of recombination across the physical distance of the genes. Finally, we determined that all the six species expressed two or three putative classical class I loci, in contrast to the single locus condition of Xenopus laevis. Our results suggest that MHC evolution in anurans is a dynamic process and that variation in numbers of loci and genetic diversity can exist among taxa. Thus, the accumulation of genetic data for more species will be useful in further characterizing the relative importance of processes such as selection, recombination and gene duplication in shaping MHC loci among amphibian lineages.     

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.     

Molecular cloning of orangutan and gibbon MHC class I cDNA. The HLA-A and -B loci diverged over 30 million years ago.

To investigate whether the classical HLA MHC class I loci have been preserved during evolution of the primates, we have cloned, sequenced, and expressed eight MHC class I cDNA from orangutan and gibbon lymphocytes. Both the HLA-A and -B loci are present in both of these species. In fact, lymphocytes from the orangutan expressed three HLA-B-related gene products, suggesting that the ancestral homologue of the HLA-B locus had undergone a duplication in this species. Interestingly, several amino acid motifs thought to be important in the Ag-presenting function of MHC class I molecules were preserved in the Ag-recognition sites of the orangutan and gibbon MHC class I molecules. Finally, these findings suggest that the recombination event between the HLA-A and -E loci occurred over 38 million years ago. These data indicate that the HLA-A and -B loci are extremely stable and that recombination between them is rare. Furthermore, the data presented here argue against the role of concerted evolution in the evolution of primate MHC class I molecules.     

Comparison of five tandem repeat loci between humans and chimpanzees.

Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.     

Structural diversity of the classical H-2 genes: K, D, and L.

Twenty-three class I DNA sequences, representing alleles of the H-2K, D, and L loci, were analyzed to assess patterns of nucleotide and amino acid diversity. Comparisons of the allelic and nonallelic sequences revealed locus specificity in regions encoding the leader peptides and the carboxyl-terminal segments of the Ag presenting molecules. Analyses focusing on the sequences that determine the Ag binding domains revealed weak or insignificant allelic associations, a finding that is in sharp contrast to previously observed relationships among the homologous human sequences. The amino acid positions exhibiting high diversity in the encoded glycoproteins in both mice and humans are localized primarily to the Ag binding site. In the mouse, diverse amino acids were positioned similarly in the K and D/L glycoproteins, although in humans, the A and B glycoproteins exhibit distinctive differences in their locations within the Ag binding site. The absence of locus specificity among the sequences that determine the Ag binding domains of the mouse is consistent with the hypothesis that ectopic gene conversion leads to interlocus exchange of class I sequences. Comparable interlocus exchanges among human class I genes have not played a similar role in shaping human A and B sequences. The basis of this difference between mice and humans is not clear. The nature of amino acid substitutions distinguishing class I loci in mice and humans are comparable, and the role of natural selection in determining diversity appears to be similar in the two species.