Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques?? major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC?Cpeptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.     

Comprehensive identification of MHC class II alleles in a cohort of Chinese rhesus macaques

Rhesus macaque is a very important animal model for various human diseases, especially for AIDS and vaccine research. The susceptibility and/or resistance to some of these diseases are related to the major histocompatibility complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, Mamu-DPB1, Mamu-DQB1, and Mamu-DRB alleles were investigated in 30 Chinese rhesus macaques through gene cloning and sequencing. A total of 66 alleles were identified in this study, including 14 Mamu-DPB1, 20 Mamu-DQB1, and 30 Mamu-DRB alleles as well as 2 high-frequency Mamu-DPB1 alleles. Interestingly, one of the high-frequency Mamu-DPB1 alleles had been undocumented in earlier studies. Eleven of the other alleles, including four Mamu-DPB1, three Mamu-DQB1, and four Mamu-DRB alleles were also novel. Importantly, like MHC-DRB, more than two Mamu-DPB1 sequences per animal were detected in 13 monkeys, which suggested that they might represent gene duplication. Our data also indicated quite a few differences in the distribution of MHC class II alleles between the Chinese rhesus macaques and the previously reported Indian rhesus macaques. To our knowledge, our results revealed comprehensively the combination of MHC II alleles. This information will not only promote the understanding of Chinese rhesus macaque MHC polymorphism but will also facilitate the use of Chinese rhesus macaques in studies of human disease.     

Comprehensive identification of high-frequency and co-occurring Mafa-DPA1, Mafa-DQA1, Mafa-DRA, and Mafa-DOA alleles in Vietnamese cynomolgus macaques

High-frequency alleles and/or co-occurring human leukocyte antigen alleles across loci appear to be more important than individual alleles as markers of disease risk and have clinical value as biomarkers for targeted screening or the development of new disease therapies. To better elucidate the major histocompatibility complex (MHC) background and to facilitate the experimental use of cynomolgus macaques, Mafa-DPA1, Mafa-DQA1, Mafa-DRA, and Mafa-DOA alleles were characterized, and their combinations were investigated in 30 Vietnamese macaques by gene cloning and sequencing. A total of 26 Mafa-DPA1, 18 Mafa-DQA1, 9 Mafa-DRA, and 15 Mafa-DOA alleles, including 7 high-frequency alleles, were identified in this study, respectively. In addition, 15 Mafa-DQA1, 17 Mafa-DPA1, 15 Mafa-DOA, and 2 Mafa-DRA alleles represented novel sequences that had not been documented in earlier studies. Our results also showed that the Vietnamese macaques might be valuable because no less than 30 % of the test animals possessed Mafa-DRA*01:02:01 (90 %), -DQA1*26:01:03 (37 %), -DOA*01:02:07 (34 %), and -DQA1*01:03:03 (30 %). We previously reported that the combinations of MHC class II alleles, including the combination of DOA*01:02:07-DPA1*02:09 and DOA*01:02:07-DQA1*01:03:03, were detected in 17 and 14 % of the animals, respectively. Interestingly, more than two Mafa-DQA1 and Mafa-DPA1 alleles were detected in one animal in this study, which suggested that they might be caused by a chromosomal duplication. If our findings can be validated by other studies, it will further enrich the number of known Mafa-DPA1 and Mafa-DQA1 polymorphisms. Our results identified the co-occurring MHC alleles across loci in a cohort of Vietnamese cynomolgus macaques, which emphasized the value of this species as a model for biomedical research.     

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.     

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe‐DRA*02, Mobe‐DQA1*01 and Mobe‐DQA2*05 alleles, which may be important for pathogen resistance. A Ewens–Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.     

Evolutionary stability of MHC class II haplotypes in diverse rhesus macaque populations

A thoroughly characterized breeding colony of 172 pedigreed rhesus macaques was used to analyze exon 2 of the polymorphic Mamu-DPB1, -DQA1, -DQB1, and -DRB loci. Most of the monkeys or their ancestors originated in India, though the panel also included animals from Burma and China, as well as some of unknown origin and mixed breeds. In these animals, mtDNA appears to correlate with the aforementioned geographic origin, and a large number of Mamu class II alleles were observed. The different Mamu-DPB1 alleles were largely shared between monkeys of different origin, whereas in humans particular alleles appear to be unique for ethnic populations. In contrast to Mamu-DPB1, the highly polymorphic -DQA1/DQB1 alleles form tightly linked pairs that appear to be about two-thirds population specific. For most of the DQA1/DQB1 pairs, Mamu-DRB region configurations present on the same chromosome have been ascertained, resulting in 41 different -DQ/DRB haplotypes. These distinct DQ/DRB haplotypes seem to be specific for monkeys of a determined origin. Thus, in evolutionary terms, the Mamu-DP, -DQ, and -DR regions show increasing instability with regard to allelic polymorphism, such as for -DP/DQ, or gene content and allelic polymorphism, such as for -DR, resulting in population-specific class II haplotypes. Furthermore, novel haplotypes are generated by recombination-like events. The results imply that mtDNA analysis in combination with Mhc typing is a helpful tool for selecting animals for biomedical experiments.The sequences reported in this paper have been deposited in the EMBL database (accession nos. AJ534296–AJ534304, AJ 564564, and AJ557455–AJ557511)     

Genotyping and segregation analyses indicate the presence of only two functional MIC genes in rhesus macaques

MIC molecules are stress-inducible ligands of the activating receptor NKG2D, which is expressed on natural killer cells and subsets of T lymphocytes. In rhesus macaques (Macaca mulatta), three different MIC sequences (MIC1, MIC2, MIC3) have been described that are closely related to but, according to phylogenetic analysis, do not represent orthologues of the human MICA and MICB genes. Although a single haplotype of the rhesus macaque Mhc (Mamu) has been completely sequenced, it remained unknown so far whether these three sequences are derived from two or three Mamu-MIC genes. We genotyped a cohort of 115 rhesus macaque individuals for the presence of MIC1, MIC2, and MIC3 sequences and analysed the segregation in families. All individuals were positive for MIC2, whereas only 66.1 and 80.9 % were positive for MIC1 and MIC3, respectively. MIC1 and MIC3 sequences segregated in offspring, indicating that they behave as alleles. Thus, we conclude that two MIC genes are present in the rhesus macaque Mhc, which we propose to designate as Mamu-MICA (MIC1 and MIC3) and Mamu-MICB (MIC2). “MIC1” and “MIC3” are regarded as divergent allelic lineages of the Mamu-MICA gene. Mamu-MIC genotyping of DNA of a cohort of 68 experimentally simian immunodeficiency virus (SIV)-infected rhesus macaques revealed no significant association of either of the two Mamu-MICA allelic lineages with differences in progression to AIDS-like symptoms. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorize users. Anne Averdam and Sandra Seelke contributed equally.     

Diversity of MHC class I genes in Burmese-origin rhesus macaques

Rhesus macaques (Macaca mulatta) are widely used in developing a strategy for vaccination against human immunodeficiency virus by using simian immunodeficiency virus infection as a model system. Because the genome diversity of major histocompatibility complex (MHC) is well known to control the immune responsiveness to foreign antigens, MHC loci in Indian- and Chinese-origin macaques used in the experiments have been characterized, and it was revealed that the diversity of MHC in macaques was larger than the human MHC. To further characterize the diversity of Mamu-A and Mamu-B loci, we investigated a total of 73 different sequences of Mamu-A, 83 sequences of Mamu-B, and 15 sequences of Mamu-I cDNAs isolated from Burmese-origin macaques. It was found that there were one to five expressing genes in each locus. Among the Mamu-A, Mamu-B, and Mamu-I sequences, 44 (60.2%), 45 (54.2%), and 8 (53.3%), respectively, were novel, and most of the other known alleles were identical to those reported from Chinese- or Indian-origin macaques, demonstrating a genetic mixture between the geographically distinct populations of present day China and India. In addition, it was found that a Mamu haplotype contained at least two highly transcribed Mamu-A genes, because multiple Mamu-A1 cDNAs were obtained from one haplotype. These findings further revealed the diversity and complexity of MHC locus in the rhesus macaques.     

Mamu-B genes and their allelic repertoires in different populations of Chinese-origin rhesus macaques

Since rhesus monkeys of Chinese origin have gained greater utilization in recent years, it is urgent to investigate the major histocompatibility complex (MHC) immunogenetics of Chinese rhesus macaques. In this study, we identified 81 Mamu-B sequences using complementary DNA cloning and sequencing on a cohort of 58 rhesus monkeys derived from three local populations of China. Twenty of these Mamu-B alleles are novel and four of them represent new lineages. Although more alleles are shared among different populations than Mamu-A locus, the Mamu-B allelic repertoires found in these three populations of Chinese macaques are largely independent, which underscores the MHC polymorphism among different populations of Chinese rhesus macaques. Our results are an important addition to the limited MHC immunogenetic information available for rhesus macaques of Chinese origin.     

Comparison of the frequencies of major histocompatibility (MHC) class-II DQA1 and DQB1 alleles in Indian and Chinese rhesus macaques (Macaca mulatta)

The major histocompatibility complex (MHC) comprises related gene families, some of which are highly polymorphic, whose protein products mediate immune response. Rhesus macaques (Macaca mulatta) are a vital animal model for research in human diseases and are native to regions extending from Afghanistan in the west to the Eastern Plains of China and from Peking to the north, southward through islands of Southeast Asia. The distributions of MHC class-II Mamu DQA1 and Mamu DQB1 alleles in two groups of domestically bred rhesus macaques of Indian and Chinese origin and the Mamu DQA1 genotypes of a small number of Burmese rhesus macaques were compared. Major allelic differences were observed between the Indian and Chinese rhesus macaques, and gene diversity decreased from east to west. These and other intra-specific genetic differences among regional populations of rhesus macaques might influence the outcome of biomedical research in which they are used as subjects, and illustrate the importance of completely genetically characterizing subjects used as animal models in biomedical research.     

MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as important animal models for biomedical research, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphism or diversity of the polygenetic Mafa class I A loci is limited in comparison to the more commonly studied rhesus macaque Mafa class I A loci. Therefore, in this paper, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-A1 among three native Southeast Asian populations (Indonesian, Vietnamese, and Filipino) and to investigate how the allele differences between macaques and humans might have evolved to affect their respective immune responses, we identified 83 Mafa-A loci-derived alleles by DNA sequencing of which 66 are newly described. Most alleles are unique to each population, but seven of the most frequent alleles were identical in sequence to some alleles in other macaque species. We also revealed (1) the large and dynamic genetic and structural differences and similarities in allelic variation by analyzing the population allele frequencies, Hardy-Weinberg’s equilibrium, heterozygosity, nucleotide diversity profiles, and phylogeny, (2) the difference in genetic structure of populations by Wright’s FST statistic and hierarchical analysis of molecular variance, and (3) the different demographic and selection pressures on the three populations by performing Tajima’s D test of neutrality. The large level of diversity and polymorphism at the Mafa-A1 was less evident in the Filipino than in the Vietnam or the Indonesian populations, which may have important implications in animal capture, selection, and breeding for medical research.     

The most common Chinese rhesus macaque MHC class I molecule shares peptide binding repertoire with the HLA-B7 supertype

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques.     

Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.     

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.     

Mamu-DQA1 allele and genotype frequencies in a randomly sampled breeding colony of rhesus macaques (Macaca mulatta)

We studied the allelic and genotypic distribution of the major histocompatibility class-II locus DQA1 observed in a random sample of Indian rhesus macaques (Macaca mulatta) from a major breeding facility in the United States. The DNA was isolated from whole blood samples collected between 1991 and 1994 from 65 Indian rhesus monkeys. Polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), which involves use of specific amplification of DQA1 exon 2 and subsequent restriction digestion of the 242-base pair fragment, was used to genotype the animals for the 20 known macaque (Mamu)-DQA1 alleles. Frequencies for four alleles (DQA1*240x, *2502, *2503 and *0102) differed significantly from those reported in a smaller sample of rhesus macaques from the German Primate Center. The modest genetic survey of Mamu-DQA1 genotypes presented here will be particularly useful in designing epidemiologic studies that investigate associations between immunogenetic background and disease susceptibility in macaque models of human disease.     

Extensive sharing of MHC class II alleles between rhesus and cynomolgus macaques

In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.     

猕猴MHC-DPB1基因外显子2多态性研究

猕猴(Macaca mulatta)是最理想的医学实验灵长类动物, 且为国家二级保护动物。为了解中国猕猴主要组织相容复合体(Major histocompatibility complex, MHC)基因的遗传多态性背景, 为它们在生物医学研究中的应用及其遗传资源的保护提供一定的科学依据, 文章采用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和克隆测序技术分析了106个四川野生猕猴MHC-DPB1基因的exon 2, 共检测到21个Mamu-DPB1等位基因, 其中有15个为本研究中首次发现的新等位基因; 从整个大的猕猴群体(106个个体)来看, 等位基因频率最高的是Mamu-DPB1*30(0.1120); 单独从不同地理群体来看, 最高等位基因频率分别为: 小金-DPB1*30 (0.1120), 黑水-DPB1*04 (0.1702), 巴中-DPB1*32 (0.1613), 汉源-DPB1*30(0.1120), 九龙-DPB1*04(0.1139); 氨基酸序列比对发现, 猕猴Mamu-DPB1等位基因编码的氨基酸序列中, 有12个氨基酸残基变异位点表现出物种特异性, 其中有9个位于新发现的15个Mamu-DPB1等位基因氨基酸序列中; 不同物种来源的DPB1等位基因系统发生树表明, 猕猴与其近缘物种食蟹猴(Macaca fascicularis)的DPB1等位基因间存在着跨种多态(Trans-species polymorphism)现象。研究还表明, MHC-DPB1等位基因在中国猕猴群体和先前为主要研究对象的印度猕猴群体间具有较大的差异。     

Survey of major histocompatibility complex class II diversity in pig-tailed macaques

Pig-tailed macaques (Macaca nemestrina) serve as important models for human infectious disease research. Major histocompatibility complex (MHC) class II molecules are important to this research since they present peptides to CD4+ T cells. Despite the importance of characterizing the MHC-II alleles expressed in model species like pig-tailed macaques, to date, less than 150 MHC-II alleles have been named for the six most common classical class II loci (DRA, DRB, DQA, DQB, DPA, and DPB) in this population. Additionally, only a small percentage of these alleles are full-length, making it impossible to use the known sequence for reagent development. To address this, we developed a fast, high-throughput method to discover full-length MHC-II alleles and used it to characterize alleles in 32 pig-tailed macaques. By this method, we identified 128 total alleles across all six loci. We also performed an exon 2-based genotyping assay to validate the full-length sequencing results; this genotyping assay could be optimized for use in determining MHC-II allele frequencies in large cohorts of pig-tailed macaques.     

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine Λgt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class IIA sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.