Molecular characterization of major histocompatibility complex class II alleles in the common frog, Rana temporaria

Major histocompatibility complex (MHC) class II genes, which play a major role in the immune system response, are some of the most polymorphic genes in vertebrates. We developed polymerase chain reaction primers for part of the second exon of an expressed MHC class II gene in the common frog, Rana temporaria. We genotyped this locus in five frog populations in southeast England and detected eight alleles in 215 individuals. Five or six alleles were detected in each population with a maximum of two alleles per individual, indicating that only a single locus was amplified. We also inferred the possible existence of a null allele. There were 23 variable nucleotide sites (out of 136) and 13 variable amino acid sites (out of 44), many of which corresponded to amino acids involved in antigen recognition. We detected a significant excess of nonsynonymous substitutions at antigen binding sites, indicating that this gene is under positive selection. The level of variation we found was similar to that in other amphibian MHC class II loci, such as those in Bombina bombina, Xenopus laevis and Ambystoma tigrinum.     

Considerable MHC diversity suggests that the functional extinction of baiji is not related to population genetic collapse

To further extend our understanding of the mechanism causing the current nearly extinct status of the baiji (Lipotes vexillifer), one of the most critically endangered species in the world, genetic diversity at the major histocompatibility complex (MHC) class II DRB locus was investigated in the baiji. Nine highly divergent DRB alleles were identified in 17 samples, with an average of 28.4 (13.2%) nucleotide difference and 16.7 (23.5%) amino acid difference between alleles. The unexpectedly high levels of DRB allelic diversity in the baiji may partly be attributable to its evolutionary adaptations to the freshwater environment which is regarded to have a higher parasite diversity compared to the marine environment. In addition, balancing selection was found to be the main mechanisms in generating sequence diversity at baiji DRB gene. Considerable sequence variation at the adaptive MHC genes despite of significant loss of neutral genetic variation in baiji genome might suggest that intense selection has overpowered random genetic drift as the main evolutionary forces, which further suggested that the critically endangered or nearly extinct status of the baiji is not an outcome of genetic collapse.     

High similarity at three MHC loci between the baiji and finless porpoise: trans-species or convergent evolution?

Two DRA alleles and six MHC-I alleles were identified from a group of 15 baiji (Lipotes vexillifer), the most threatened cetacean in the world. Little sequence variation was detected at the DRA locus but extensive variation at the MHC-I locus. In combination with data at the DQB locus previously reported, three MHC loci exon 2 of the baiji all revealed striking similarity with those of the finless porpoise. Especially, some identical alleles shared by both species at the MHC-I and DQB loci suggested the convergent evolution as a consequence of common adaptive solutions to similar environmental pressures in the Yangtze River. As for DRA locus, the identity alleles were shared not only by baiji and finless porpoise but by some other cetacean species of the families Phocoenidae and Delphinidae, suggesting trans-species evolution on this gene.     

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.     

Parasite burden and constitution of major histocompatibility complex in the Malagasy mouse lemur, Microcebus murinus

We investigated the importance of the major histocompatibility complex (MHC) constitution on the parasite burden of free-ranging mouse lemurs (Microcebus murinus) in four littoral forest fragments in southeastern Madagascar. Fourteen different MHC class II DRB-exon 2 alleles were found in 228 individuals with high levels of sequence divergence between alleles. More nonsynonymous than synonymous substitutions in the functional important antigen recognition and binding sites indicated selection processes maintaining MHC polymorphism. Animals from the four forest fragments differed in their infection status (being infected or not), in the number of different nematode morphotypes per individual (NNI) as well as in the fecal egg counts (FEC) values. Heterozygosity in general was uncorrelated with any of these measures of infection. However, a positive relationship was found between specific alleles and parasite load. Whereas the common allele Mimu-DRB*1 was more frequently found in infected individuals and in individuals with high NNI and FEC values (high parasite load), the rare alleles Mimu-DRB*6 and 10 were more prevalent in uninfected individuals and in individuals with low NNI and FEC values (low parasite load). These three alleles associated with parasite load had unique amino acid motifs in the antigen binding sites. This distinguished them from the remaining 11 Mimu-DRB alleles. Our results support the hypothesis that MHC polymorphism in M. murinus is maintained through pathogen-driven selection acting by frequency-dependent selection. This is the first study of the association of MHC variation and parasite burden in a free-ranging primate.     

Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations

We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most single-base substitutions when carried out on products amplified with a primer to which a GC clamp has been added. Although DGGE has been primarily used for the detection of limited numbers of single-base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with greater levels of sequence variation. The methodology described was developed to identify the number and distribution of MHC class I alpha 1 alleles among chinook salmon (Oncorhynchus tshawytscha) populations. DGGE detects 28 of 31 identified alpha 1 sequences, which differ by between 1 and 16 nucleotides and a two-codon indel. By creating a network of control alleles, 22-23 of the MHC alleles can be resolved rapidly and accurately by a single gel run condition, and 27 alleles can be resolved by two gel run conditions. This techniques has been used in surveys scoring alleles from two MHC markers (class I alpha 1 and alpha 2) in 20,000 individuals of chinook and coho (O. kisutch) salmon. A single person in our laboratory now analyzes 160 salmon from one MHC locus per day with DGGE.     

Allelic polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of Cynoglossus semilaevis (Cynoglossidae)

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and evolutionary mechanism of MHC genes have been investigated in many mammals, but much less is known in teleosts. We examined the polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of the half-smooth tongue sole (Cynoglossus semilaevis); 23 alleles were found in this species. Gene duplication manifested as three to six distinct sequences at each domain in the same individuals. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the PBR domain, suggesting balancing selection for maintaining polymorphisms at the MHC II DAB locus. Many positive selection sites were found to act very intensely on antigen-binding sites of MHC class II DAB gene.     

Patterns of polymorphism in the MHC class II of a non-passerine bird,the great snipe (<Emphasis Type="Italic">Gallinago media</Emphasis>)

The genomic organisation of the major histocompatibility complex (MHC) seems to vary considerably between different bird species. In order to understand this variation it is important to gather information from different species. We have, for the first time, investigated MHC class II polymorphism in a wader species, the great snipe (Gallinago media). Eleven alleles were found in five sequenced individuals; these come from at least three different loci, but RFLP data suggest that a larger number of genes may be present. For MHC genes, amino acid substitutions followed the, for MHC genes, general pattern of high non-synonymous substitution rates in peptide-binding regions, suggesting that the sequenced alleles may be expressed. The number of genes, lengths of introns and exon sequences of the great snipe MHC seem to be intermediate between those of chicken and passerine birds.     

Molecular cloning of cDNA that encode MHC class I molecules from a New World primate (Saguinus oedipus). Natural selection acts at positions that may affect peptide presentation to T cells

To investigate the evolutionary pressures that drive the generation of polymorphism in primate MHC class I molecules, three cDNA that encode MHC class I alleles from a New World monkey, the cotton-top tamarin (Saguinus oedipus), were cloned and sequenced. These tamarin MHC class I alleles contained amino acid substitutions not found in any of the previously sequenced human MHC class I alleles. Moreover, the majority of these unique amino acid substitutions was located in the Ag recognition site at positions that have been shown to be critical in the presentation of viral peptides to T cells in mice and humans. These data suggest that selective pressures on MHC class I molecules preferentially act on the Ag recognition site and that the peptide binding or presenting functions of these molecules may drive the generation of MHC class I polymorphism. The novel Ag recognition sites of the tamarin MHC class I molecules, in addition to their restricted polymorphism, might account for the unusual susceptibility of the cotton-top tamarin to human pathogens.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Contrasting patterns of variation in MHC loci in the Alpine newt

Major histocompatibility complex (MHC) genes are essential in pathogen recognition and triggering an adaptive immune response. Although they are the most polymorphic genes in vertebrates, very little information on MHC variation and patterns of evolution are available for amphibians, a group known to be declining rapidly worldwide. As infectious diseases are invoked in the declines, information on MHC variation should contribute to devising appropriate conservation strategies. In this study, we examined MHC variation in 149 Alpine newts ( Mesotriton alpestris ) from three allopatric population groups in Poland at the northeastern margin of the distribution of this species. The genetic distinctiveness of the population groups has previously been shown by studies of skin graft rejection, allozymes and microsatellites. Two putative expressed MHC II loci with contrasting levels of variation and clear evidence of gene conversion/recombination between them were detected. The Meal-DAB locus is highly polymorphic (37 alleles), and shows evidence of historical positive selection for amino acid replacements and substantial geographical differentiation in allelic richness. On the contrary, the Meal-DBB locus exhibits low polymorphism (three alleles differing by up to two synonymous substitutions) and a uniform distribution of three alleles among geographical regions. The uniform frequencies of the presumptively neutral Meal-DBB alleles may be explained by linkage to Meal-DAB . We found differences in allelic richness in Meal-DAB between regions, consistent with the hypothesis that genetic drift prevails with increasing distance from glacial refugia. Pseudogene loci appear to have evolved neutrally. The level of DAB variation correlated with variation in microsatellite loci, implying that selection and drift interplayed to produce the pattern of MHC variation observed in marginal populations of the Alpine newt.     

High levels of MHC class II allelic diversity in lake trout from Lake Superior

Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (dN) exceeded the level of synonymous substitution (dS). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.     

Genetic markers of adaptive processes in the Far Eastern pink salmon <Emphasis Type="Italic">Oncorhynchus gorbuscha</Emphasis>: Allelic diversity at the locus of major histocompatibility complex MHC I-<Emphasis Type="Italic">A1</Emphasis>

To clarify allelic diversity at the locus of major histocompatibility complex MHC class I-A1 in the Far Eastern pink salmon Oncorhynchus gorbuscha, sequencing of the electrophoretic alleles isolated from the gel (DGGE alleles) was performed. In 47 individuals, the genotypes of which consisted of ten DGGE alleles, 18 MHC I-A1 nucleotide sequences were revealed, and thus, eight cryptic alleles not detected by electrophoresis were identified. Eleven of these alleles were identified earlier in pink salmon from Hokkaido, Alaska, and British Columbia, and seven, possibly, were unique to the populations from some Far Eastern regions. Six of the previously determined DGGE alleles corresponded to more than one nucleotide sequence. However, the sequences attributed to the same DGGE allele differed on average by less than 1 nucleotide. These findings point to sufficient sensitivity of the DGGE method, although the genetic diversity and differentiation estimates obtained with it will obviously be somewhat underestimated. Considerable predominance of nonsynonymous substitutions over the synonymous ones in the codons of the MHC I-A1 antigen-binding site confirms the presence of positive selection aimed at providing the population resistance to local spectrum of pathogens. Refinement of the allelic composition of the adaptively important MHC genetic marker will contribute to more complete understanding of the adaptive genetic structure of pink salmon as an important element of the overall population structure of the species.     

尼罗鳄MHCⅡ类B基因第二外元的序列变异分析

利用一对简并引物扩增了尼罗鳄MHCⅡ类分子B基因第二外元的部分片段,并对PCR产物进行了克隆和测序,结果得到8种不同的序列,序列长度为166 bp;经分析,序列中有56个变异位点,核苷酸的非同义替换多于同义替换,造成30个位点氨基酸的改变,氨基酸的替换趋于集中在假定的抗原结合位点附近.核苷酸和氨基酸序列与已报道的扬子鳄和密河鳄的MHCⅡ类B基因第二外元序列有较高的同源性,利用PAUP4.0软件构建的NJ树显示,鳄类的MHCⅡ类B基因存在跨种多态性现象.     

Construction and Application of a Korean Reference Panel for Imputing Classical Alleles and Amino Acids of Human Leukocyte Antigen Genes

Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans.     

Evolution of MHC class IIB in the genome of wild and ornamental guppies, Poecilia reticulata

This is the first study to quantify genomic sequence variation of the major histocompatibility complex (MHC) in wild and ornamental guppies, Poecilia reticulata. We sequenced 196-219 bp of exon 2 MHC class IIB (DAB) in 56 wild Trinidadian guppies and 14 ornamental strain guppies. Each of two natural populations possessed high allelic richness (15-16 alleles), whereas only three or fewer DAB alleles were amplified from ornamental guppies. The disparity in allelic richness between wild and ornamental fish cannot be fully explained by fixation of alleles by inbreeding, nor by the presence of non-amplified sequences (ie null alleles). Rather, we suggest that the same allele is fixed at duplicated MHC DAB loci owing to gene conversion. Alternatively, the number of loci in the ornamental strains has contracted during >100 generations in captivity, a hypothesis consistent with the accordion model of MHC evolution. We furthermore analysed the substitution patterns by making pairwise comparisons of sequence variation at the putative peptide binding region (PBR). The rate of non-synonymous substitutions (dN) only marginally exceeded synonymous substitutions (dS) in PBR codons. Highly diverged sequences showed no evidence for diversifying selection, possibly because synonymous substitutions have accumulated since their divergence. Also, the substitution pattern of similar alleles did not show evidence for diversifying selection, plausibly because advantageous non-synonymous substitutions have not yet accumulated. Intermediately diverged sequences showed the highest relative rate of non-synonymous substitutions, with dN/dS>14 in some pairwise comparisons. Consequently, a curvilinear relationship was observed between the dN/dS ratio and the level of sequence divergence.     

Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II β1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a “gene walking” technique to obtain intron 2 sequences that flanked MHC class IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the β1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.     

Maintenance of DQB1 polymorphisms in primates.

To understand the evolution of the class II major histocompatibility complex (MHC) DQB1 locus in primates, the second exons of seven DQB1 alleles from five non-human primate species were amplified by polymerase chain reaction. Comparisons of these and other primate sequences show that no between-species diversity is greater than within-species diversity, suggesting maintenance of DQB1 alleles through the history of Old-World primates. There is a preponderance of nonsynonymous nucleotide substitutions at antigen-binding-site codons; this pattern is in marked contrast to what is seen at the closely related, presumably nonfunctional DQB2 gene. The results support the hypothesis that DQB1 polymorphism is maintained by overdominant selection relating to antigen presentation.     

中国地方鸡种MHC B-LBⅡ新等位基因的遗传多态性研究

为研究鸡MHC B-LBⅡ基因的遗传多态性,首先在8个中国地方鸡种(藏鸡、仙居鸡、北京油鸡、固始鸡、斗鸡、丝羽乌骨鸡、白耳鸡和狼山鸡)B-LBⅡ基因第二外显子扩增了一长度为 175 bp 的 DNA 片段并进行 SSCP 基因型分析;在8 个地方鸡种共 467 个个体中检测到 37 个 PCR-SSCP 基因型;从被检样品中筛选出不同基因型的个体,并在其 B-LBⅡ基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,通过克隆和测序获得了该片段的核苷酸序列。经序列分析,在前述地方鸡种被筛选出的 30 个无血缘关系的个体中发现了 31 个 B-LBⅡ新等位基因,并参照哺乳动物 MHC II 类 B 等位基因命名规则进行了命名。对这 31 个 B-LBⅡ新等位基因长度为 374 bp 的 DNA 片段进行比对表明,在其第二外显子序列上共有 68 个多态性变异位点,其中简约性信息位点 51 个,单变异位点 17 个,具有丰富的遗传多态性。在这些多态性变异位点中,出现在遗传密码子第一和第二位上的碱基替换率分别为 36.76% 和 35.29%。等位基因序列间的相似性估测为 90.6%-99.5%;B-LBⅡ基因第二外显子的错义替换率和同义替换率分别为 14.64±2.67%和 2.92±0.94%。结果表明,B-LBⅡ基因的丰富遗传多态性主要是由基因重组和平衡选择效应所引起的。对 B-LBⅡ等位基因第二外显子所编码的 B-LBⅡ分子β1 结构域氨基酸序列比对发现,31 个 B-LBⅡ新等位基因属于 26 个等位基因主型;在β1结构域氨基酸序列的 33个变异位点上,存在 6 个同义替换和 27 个错义替换。分析认为,那些发生在多肽结合位点上的氨基酸错义替换与鸡 MHC B-LBⅡ分子的免疫特异性有关。该结果可为鸡的抗病育种研究提供分子生物学依据。     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.