Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.     

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrats considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dCdA)₁₅, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M33306 and M33307. Address correspondence and offprint requests to: M. R. Brandon.     

Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates

Major histocompatibility complex (MHC) antigen-presenting genes are the most variable loci in vertebrate genomes. Host-parasite co-evolution is assumed to maintain the excessive polymorphism in the MHC loci. However, the molecular mechanisms underlying the striking diversity in the MHC remain contentious. The extent to which recombination contributes to the diversity at MHC loci in natural populations is still controversial, and there have been only few comparative studies that make quantitative estimates of recombination rates. In this study, we performed a comparative analysis for 15 different ungulates species to estimate the population recombination rate, and to quantify levels of selection. As expected for all species, we observed signatures of strong positive selection, and identified individual residues experiencing selection that were congruent with those constituting the peptide-binding region of the human DRB gene. However, in addition for each species, we also observed recombination rates that were significantly different from zero on the basis of likelihood-permutation tests, and in other non-quantitative analyses. Patterns of synonymous and non-synonymous sequence diversity were consistent with differing demographic histories between species, but recent simulation studies by other authors suggest inference of selection and recombination is likely to be robust to such deviations from standard models. If high rates of recombination are common in MHC genes of other taxa, re-evaluation of many inference-based phylogenetic analyses of MHC loci, such as estimates of the divergence time of alleles and trans-specific polymorphism, may be required.     

Primary structural sequence polymorphism in the human class II MHC antigen 33.1

Monoclonal antibody 33.1 defines a non-DR, class 11, human major histocompatibility complex antigen, 33.1, which appears to be distinct from other class II antigens in its cellular distribution and primary structure. To characterize the structure more fully and to determine the degree of polymorphism within 33.1, a comparative N-terminal sequence study has been undertaken using a series of ten B lymphoblastoid cell lines with different DR and MB types. The results confirm that both the and chains of 33.1 are homologues of the corresponding chains of the murine I-A antigen and indicate that while 33.1 does not appear to be identical with MB, it is closely related. Sequence analyses revealed two major variants of 33.1, corresponding to cells with specificities MB1 and MB 3, respectively. Within each MB type, other polymorphisms have been detected. Cells that are MB2 do not react with monoclonal antibody 33.1. Suggestive evidence is presented that monoclonal antibody 33.1 reacts predominantly with the chain of the antigen. The preferential expression of 33.1 on activated B cells suggests that expression of at least the 33.1 chain gene is greatly enhanced in the course of B-cell activation, but the specific function of 33.1 remains to be determined.Abbreviations used in this paper McAb monoclonal antibody - BLCL B lymphoblastoid cell lines - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - NP-40 Nonidet P-40 Fellow of the Arthritis Foundation     

Characterization and evolution of ovine MHC class II DQB sequence polymorphism

The second exons of OLA-DQB genes from 13 merino sheep were sequenced following amplification by the polymerase chain reaction or isolation from a cDNA library. Ten distinct exon 2 sequences, coding for 10 novel amino acid sequences, were characterized in these sheep. The single-strand conformation polymorphism technique was shown to be capable of discriminating between all sequences. This brings the total number of different OLA-DQB exon 2 sequences (nucleotide and amino acid) which have been characterized to 12, and demonstrates that the OLA-DQB region is highly polymorphic with 29% of nucleotide and 46% of amino acid sites showing variation. Evidence is presented that the OLA-DQB sequences belong to at least two lineages of DQB genes. Some ovine DQB sequences are more like bovine DQB counterparts than other ovine DQB sequences suggesting that the artiodactyl DQB gene and allele lineages predate the separation of the ovine and bovine species 20 million years ago.     

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.     

Characterisation of MHC class II DRB genes in the northern tree shrew (Tupaia belangeri)

Genes of the major histocompatibility complex (MHC) mainly code for proteins of the immune system of jawed vertebrates. In particular, MHC class I and II cell surface proteins are crucial for the self/non-self discrimination of the adaptive immune system and are the most polymorphic genes in vertebrates. Positive selection, gene duplications and pseudogenes shape the face of the MHC and reflect a highly dynamic evolution. Here, we present for the first time data of the highly polymorphic MHC class II DRB exon 2 of a representative of the mammalian order scandentia, the northern tree shrew Tupaia belangeri. We found up to eight different alleles per individual and determined haplotype constitution by intensively studying their inheritance. The alleles were assigned to four putative loci, all of which were polymorphic. Only the most polymorphic locus was subject to positive selection within the antigen binding sites and only alleles of this locus were transcribed.     

Characterization of pig-tailed macaque classical MHC class I genes: implications for MHC evolution and antigen presentation in macaques

MHC-dependent CD8(+) T cell responses have been associated with control of viral replication and slower disease progression during lentiviral infections. Pig-tailed macaques (Macaca nemestrina) and rhesus monkeys (Macaca mulatta), two nonhuman primate species commonly used to model HIV infection, can exhibit distinct clinical courses after infection with different primate lentiviruses. As an initial step in assessing the role of MHC class I restricted immune responses to these infections, we have cloned and characterized classical MHC class I genes of pig-tailed macaques and have identified 19 MHC class I alleles (Mane) orthologous to rhesus macaque MHC-A, -B, and -I genes. Both Mane-A and Mane-B loci were found to be duplicated, and no MHC-C locus was detected. Pig-tailed and rhesus macaque MHC-A alleles form two groups, as defined by 14 polymorphisms affecting mainly their B peptide-binding pockets. Furthermore, an analysis of multiple pig-tailed monkeys revealed the existence of three MHC-A haplotypes. The distribution of these haplotypes in various Old World monkeys provides new insights about MHC-A evolution in nonhuman primates. An examination of B and F peptide-binding pockets in rhesus and pig-tailed macaques suggests that their MHC-B molecules present few common peptides to their respective CTLs.     

Genetic diversity at class II DRB loci of the primate MHC

The evolution of polymorphism at loci encoding the beta-chains of the MHC class II DR Ag was studied in primates by DNA amplification (polymerase chain reaction). Phylogenetic analysis of 63 DRB sequences from the polymorphic second exon (first domain) of nonhuman primates and 53 human sequences indicates the presence of five DRB loci in primates, derived from a DRB1-like ancestral locus over 20 million yr ago. Many of the allelic types at the DRB1 locus predate the divergence of hominoids (5 million yr ago) and some (DR4, DR3, 5, 6) predate the divergence of Old world monkeys and hominoids (20 million yr ago). The DRB3 locus appears to have arisen before the divergence of hominoids on an ancestral DRB1 lineage. The DRB2 and DRB5 loci were generated more than 20 million yr ago and the DRB4 locus more than 5 million yr ago. The DRB2 locus, a pseudogene in humans, is polymorphic in the nonhuman primates.     

Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.     

Insights into the complex associations between MHC class II DRB polymorphism and multiple gastrointestinal parasite infestations in the striped mouse

Differences in host susceptibility to different parasite types are largely based on the degree of matching between immune genes and parasite antigens. Specifically the variable genes of the major histocompatibility complex (MHC) play a major role in the defence of parasites. However, underlying genetic mechanisms in wild populations are still not well understood because there is a lack of studies which deal with multiple parasite infections and their competition within. To gain insights into these complex associations, we implemented the full record of gastrointestinal nematodes from 439 genotyped individuals of the striped mouse, Rhabdomys pumilio. We used two different multivariate approaches to test for associations between MHC class II DRB genotype and multiple nematodes with regard to the main pathogen-driven selection hypotheses maintaining MHC diversity and parasite species-specific co-evolutionary effects. The former includes investigations of a 'heterozygote advantage', or its specific form a 'divergent-allele advantage' caused by highly dissimilar alleles as well as possible effects of specific MHC-alleles selected by a 'rare allele advantage' (= negative 'frequency-dependent selection'). A combination of generalized linear mixed models (GLMMs) and co-inertia (COIA) analyses made it possible to consider multiple parasite species despite the risk of type I errors on the population and on the individual level. We could not find any evidence for a 'heterozygote' advantage but support for 'divergent-allele' advantage and infection intensity. In addition, both approaches demonstrated high concordance of positive as well as negative associations between specific MHC alleles and certain parasite species. Furthermore, certain MHC alleles were associated with more than one parasite species, suggesting a many-to-many gene-parasite co-evolution. The most frequent allele Rhpu-DRB*38 revealed a pleiotropic effect, involving three nematode species. Our study demonstrates the co-existence of specialist and generalist MHC alleles in terms of parasite detection which may be an important feature in the maintenance of MHC polymorphism.     

Extensive MHC class II DRB3 diversity in African and European cattle

Genetic deversity at the highly polymorphic BoLA-DRB3 locus was investigated by DNA sequence analyses of 18 African cattle from two breeds representing the two subspecies of cattle, Bos primigenius indicus and Bos primigenius taurus. Yhe polymorphism was compared with that found in a sample ofd 32 European cattle from four breeds, all classified as B. p. taurus. Particularly extensive genetic diversity was found among African cattle, in which as many as 18 alleles were recognized in this small random sample of animals from two breeds. The observed similarity in allele frequency distribution between the two African populations, N'Dama and Zebu cattle, is consistent with the recent recognition of gene flow between B. p. indicus and B. P taurus cattle in Africa. A total of 30 DRB3 alleles were documented and as many as 26 of these were classified as major allelic types showing at least five amino acid substitutions compared with other major types. The observation of extensive genetic diversity at MHC loci in cattle, as well as in other farm animals, provides a compelling argument against matin-type preferences as a primary cause in maintaining major histocompatibility complex diversity, since the reproduction of these animals has been controlled by humans for many generations.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been given the accession numbers X87641-X87670     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.