High-resolution typing for chicken BF2 (MHC class I) alleles by automated sequencing

Sequence-based typing (SBT) was developed for major histocompatibility complex (MHC) class I and class II alleles in humans. We report here the development and application of a SBT method for alleles of the chicken BF2 locus (the more polymorphic of the two MHC class I loci in chickens). Exon 2 of the BF2 gene was selectively amplified from genomic DNA using a BF2 locus-specific PCR primer. Exon 2 sequences were sufficient to identify the 21 distinct BF2 alleles described in standard B haplotypes of Leghorns and in commercial broiler-breeder lines. Sixty-six samples from MHC typed, pedigreed chickens were tested, including 50 different heterozygous combinations. BF2 sequences from all B homozygotes were successfully amplified, and all combinations of BF2 alleles in heterozygotes were co-amplified equally. The two different BF2 alleles in heterozygotes could be identified unambiguously by distinct sequence motif patterns. In tests of samples of unknown B genotype in commercial broiler-breeder flocks, we identified expected BF2 alleles as well as an allele not previously encountered in one of the lines.     

Evidence for the expression of two distinct MHC class II DRβ like molecules in the sheep

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DRβ chains are expressed in the sheep. Two anti-β chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DRβ chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 β chain. Amino-terminal sequence analysis of the α chain associated with VPM37β chain shows that this α chain is homologous to the human DRα chain strongly indicating that the β chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of posttranslational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DRP chains in the sheep.     

Molecular characterization of major and minor MHC class I and II genes in B21-like haplotypes in chickens

The major histocompatibility complex (MHC) sequences of three B21-like haplotypes deriving from very different origins including the Red Jungle Fowl Gallus Gallus gallus were compared with the MHC sequences of the standard B21 haplotype from Scandinavian White Leghorn Gallus domesticus. The present analysis reveals two cDNA sequences for B-F and two cDNA sequences for B-LB for every B21-like haplotype, including B21 itself. Contrary to expectation, no sequence polymorphism in the antigen-binding domains of the MHC genes, between the investigated haplotypes, was found. The relative level of MHC class I molecules on the surface of leukocytes measured by flow cytometry was also analysed and found to be low in Marek's Disease (MD)-resistant B haplotypes (B21 and B21-like) and high in MD-susceptible B haplotypes (B15 and B19). However, in heterozygous (resistant/susceptible) animals, the relative level was almost as high as in susceptible haplotypes.     

Polymorphic Alu insertions and their associations with MHC class I alleles and haplotypes in Han and Jinuo populations in Yunnan Province, southwest of China

The associations of polymorphic Alu insertions （POALINs） with major histocompatibility complex （MHC） class I genes enable us to better identify origins and evolution of MHC class I region haplotypes in different populations. For further studying origins and evolution of MHC class I region haplotypes in Han and Jinuo populations in Yunnan Province, we investigated frequencies of five POALINs, their associations with HLA-A and -B, the three-loci POALINs haplotype frequencies and HLA/POALIN four-loci haplotype frequencies within the alpha block of MHC class I region. We found that a strong positive association between AluHG and HLA-A＊02 is in Jinuo, but not in Yunnan Han. These results suggest that MHC class I region haplotypes of the two studied populations might derive from different progenitor haplotypes and MHC I-POALINs are informative genetic markers for investigating origins and evolution of MHC class I region haplotypes in different populations.     

Designation by restriction fragment length polymorphism of major histocompatibility complex class IV haplotypes in meat-type chickens

Major histocompatiblity complex (MHC) class IV haplotypes were identified in a population of meat-type chickens by restriction fragment length polymorphism (RFLP) analysis. Fourteen different haplotypes were designated on the basis of restriction patterns obtained from Southern blots of PvuII- or BglII-digested DNA, hybridized with the MHC class IV cDNA probe bg32.1. Digestion with each restriction enzyme yielded the same level of polymorphism among individuals. For each haplotype, 4–10 restriction fragments ranging from 0–8 to 8 kb were observed. Such a designation of meat-type chicken MHC class IV haplotypes enables a rapid recognition of previously defined haplotypes, is readily adjustable to additional, newly found restriction patterns and could prove useful in practical breeding programmes.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class IV (B-G) genotypes in meat-type chickens

Restriction fragment length polymorphism (RFLP) was used as a molecular genotyping approach to characterize differences in major histocompatibility complex class IV genes in meat-type chickens. A high level of polymorphism was observed following digestion with each of the two restriction endonucleases PvuII and BglII. Examination of DNA from 54 chickens revealed 23 polymorphic fragments. Application of RFLP techniques in the analysis of family groups should make possible the determination of B-G genotypes in the meat type chickens.     

Spatial pattern of MHC class II variation in the great snipe (Gallinago media)

The genes of the major histocompatibility complex (MHC) code for proteins involved in antigen recognition and triggering of the adaptive immune response, and are therefore likely to be under selection from parasites. These selection regimes may vary in space and time. Here we report a strong geographical structure in MHC class II B genes of a migrating bird, the great snipe (Gallinago media). Genetic differentiation in the MHC between two ecologically distinct distributional regions (Scandinavian mountain populations vs. East European lowland populations) was still present after statistically controlling for the effect of selectively neutral variation (microsatellites) using partial Mantel tests. This suggests a role for selection in generating this spatial structure and that it represents local adaptation to different environments. Differentiation between populations within the two regions was negligible. Overall, we found a high number of MHC alleles (50, from 175 individuals). This, together with a tendency for a higher rate of nonsynonymous than synonymous substitutions in the peptide binding sites, and high Tajima's D in certain regions of the gene, suggests a history of balancing selection. MHC variation is often thought to be maintained by some form of balancing selection, but the nature of this selection remains unclear. Our results support the hypothesis that spatial variation in selection regimes contributes to the high polymorphism.     

Mapping of susceptibility to Marek's disease within the major histocompatibility (B) complex by refined typing of White Leghorn chickens

The major histocompatibility (B) complex of a distinct commercial pure White Leghorn chicken line was characterized using serological, biochemical and restriction fragment length polymorphism (RFLP) typing. Line B chickens displayed a high recombination frequency within the B complex. Three recombinant haplo-types were identified. The influence of these haplotypes was determined in relation to the haplotypes B^l9 and B²¹ on their resistance to Marek's disease (MD) in an experimental infection with the virus. Offspring of sires with a recombinant haplotype in combination with B¹⁹ or B²¹, and dams, which were homozygous B¹⁹/B¹⁹ or B²¹/B²¹ were infected. The B type of the offspring had a significant effect upon survival. Animals with B complex types B²¹/B²¹, B¹³⁴/B²¹ and B²³⁴/B²¹ were relatively resistant to MD (24–32% mortality), whereas B¹⁹/B¹⁹ birds were highly susceptible (68% mortality). Animals with a recombinant halpotype B^19r21 (B-G²¹, B-F¹⁹) were equally susceptible to MD as birds with the complete B¹⁹ haplotype. In contrast to earlier publications, resistance was not inherited as a dominant trait. Apparently, B¹⁹ was associated with a dominant susceptibility. The gene(s) associated with the B complex and involved in resistance to MD were localized within the B-F/B-L region. However, the association with a presumably non-coding subregion of B-G could not be excluded.     

A probabilistic meta-predictor for the MHC class II binding peptides

Several computational methods for the prediction of major histocompatibility complex (MHC) class II binding peptides embodying different strengths and weaknesses have been developed. To provide reliable prediction, it is important to design a system that enables the integration of outcomes from various predictors. The construction of a meta-predictor of this type based on a probabilistic approach is introduced in this paper. The design permits the easy incorporation of results obtained from any number of individual predictors. It is demonstrated that this integrated method outperforms six state-of-the-art individual predictors based on computational studies using MHC class II peptides from 13 HLA alleles and three mouse MHC alleles obtained from the Immune Epitope Database and Analysis Resource. It is concluded that this integrative approach provides a clearly enhanced reliability of prediction. Moreover, this computational framework can be directly extended to MHC class I binding predictions.     

The influence of ovine MHC class II DRB1 alleles on immune response in bovine leukemia virus infection

We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions beta70/71 of the OLA-DRbeta1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. Although the number of BLV-infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5- B-cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-gamma, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4+ T-cells.     

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum)

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, transspecies evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relict of their unique historical demography, not their phylogenetic legacy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Detection of multiple β-casein (CASB) alleles by amplification created restriction sites (ACRS)

Direct sequencing of polymerase chain reaction (PCR) amplified DNA has been used to detect the DNA sequences for bovine beta-casein (CASB) A3 and B variants. Based on these sequences we have designed primers which create allele-specific restriction sites in the PCR product. Restriction analysis of PCR product generated in one reaction enable us to identify the A1, A2, A3 and B alleles of CASB rapidly without the use of radioactivity.     

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens)

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD⁴⁺ T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d _S) and non-synonymous (d _N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d _N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.     

Isolation and characterization of major histocompatibility complex (MHC) class II B genes in the Barn owl (Aves: Tyto alba)

We isolated major histocompatibility complex class II B (MHCIIB) genes in the Barn owl (Tyto alba). A PCR-based approach combined with primer walking on genomic and complementary DNA as well as Southern blot analyses revealed the presence of two MHCIIB genes, both being expressed in spleen, liver, and blood. Characteristic structural features of MHCIIB genes as well as their expression and high non-synonymous substitution rates in the region involved in antigen binding suggest that both genes are functional. MHC organization in the Barn owl is simple compared to passerine species that show multiple duplications, and resembles the minimal essential MHC of chicken.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.