The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

Molecular genotype identification of the Gallus gallus major histocompatibility complex

The chicken major histocompatibility complex (MHC) is commonly defined by serologic reactions of erythrocytes with antibodies specific to the highly polymorphic MHC class I (BF) and MHC class IV (BG) antigens. The microsatellite marker LEI0258 is known to be physically located within the MHC, between the BG and BF regions. DNA from various serologically defined MHC haplotypes was amplified by polymerase chain reaction with primers surrounding this marker. Twenty-six distinctive allele sizes were identified. Some serologically well-defined MHC haplotypes shared a common LEI0258 allele size but could be distinguished either by the addition of information from another nearby marker (MCW0371) or by small indels or single nucleotide polymorphism (SNP) differences between the alleles. The association between LEI0258 allele and serologically defined MHC haplotype was very consistent for the same haplotype from multiple sources. Sequence information for the region defined by LEI0258 was obtained for 51 different haplotypes. Two internal repeats whose lengths were 13 and 12 bp, respectively, are the primary basis for allelic variability. Allele size variation ranges from 182 to 552 bp. Four indels and five SNPs in the surrounding sequence provide additional means for distinguishing alleles. Typing with LEI0258 and MCW0371 will be useful in identifying MHC haplotypes in outbred populations of chickens particularly for the initial development of serological reagents.     

Diversity and locus specificity of chicken MHC B class I sequences

The major histocompatibility complex B (MHC B) region in a standard haplotype of Leghorn chickens contains two closely linked class I loci, B-FI and B-FIV. Few sequences of B-FI alleles are available, and therefore alleles of the two loci have not been compared with regard to sequence diversity or locus specificity. Here, we report eight new B-F alpha 1/alpha 2-coding sequences from broiler chicken MHC B haplotypes, and a unique recombinant between the two B-F loci. The new sequences were combined with existing B-F sequences from Leghorn and broiler haplotypes for analysis. On the basis of phylogenetic analysis and conserved sequence motifs, B-F sequences separated into two groups (Groups A and B), corresponding to B-FIV and B-FI locus, respectively. Every broiler haplotype had one B-F sequence in Group A and the second B-F sequence, if it existed, clustered in Group B. Group B (presumptive B-FI locus) sequences identified in broiler haplotypes resembled the human MHC class I HLA-C locus in their distinctive pattern of allelic polymorphism. Compared with B-FIV, B-FI alleles were less polymorphic and possessed a conserved locus-specific motif in the alpha1 helix, but nevertheless demonstrated evidence of diversifying selection. One B-FI alpha 1/alpha 2-coding nucleotide sequence was completely conserved in four different broiler haplotypes, but each allele differed in the exon encoding the alpha 3 domain.     

MHC class I typing in a songbird with numerous loci and high polymorphism using motif-specific PCR and DGGE

The major histocompatibility complex (MHC) has a central role in the specific immune defence of vertebrates. Exon 3 of MHC class I genes encodes the domain that binds and presents peptides from pathogens that trigger immune reactions. Here we develop a fast population screening method for detecting genetic variation in the MHC class I genes of birds. We found evidence of at least 15 exon 3 sequences in the investigated great reed warbler individual. The organisation of the great reed warbler MHC class I genes suggested that a locus-specific screening protocol is impractical due to the high similarity between alleles across loci, including the introns flanking exon 3. Therefore, we used motif-specific PCR to amplify two subsets of alleles (exon 3 sequences) that were separated with by DGGE. The motif-specific primers amplify a substantial proportion of the transcribed class I alleles (2-12 alleles per individual) from as many as six class I loci. Although not exhaustive, this gives a reliable estimate of the class I variation. The method is highly repeatable and more sensitive in detecting genetic variation than the RFLP method. The motif-specific primers also allow us to avoid screening pseudogenes. In our study population of great reed warblers, we found a high level of genetic variation in MHC class I, and no less than 234 DGGE genotypes were detected among 248 screened individuals.     

Associations of BF2 alleles with antibody titres and production traits in commercial pure line broiler chickens

Three commercial broiler pure lines were evaluated for associations of sire BF2 (major histocompatibility complex class I) alleles with progeny phenotypic traits. Significant BF2 associations with a subset of traits were observed in two lines. The BF2*21 allele was positively associated with antibody titre to infectious bursal disease virus in both lines. Other associations were line-specific.     

Class I MHC expression in the yellow baboon

MHC class I molecules play a crucial role in the immune response to pathogens and vaccines and in self/non-self recognition. Therefore, characterization of MHC class I gene expression of Papio subspecies is a prerequisite for studies of immunology and transplantation in the baboon (papio hamadryas). To elucidate MHC class I expression and variation within Papio subspecies and to further investigate the evolution of A and B loci in Old World primates, we have characterized the expressed class I repertoire of the yellow baboon (Papio hamadryas cynocephalus) by cDNA library screening. A total of nine distinct MHC class I cDNAs were isolated from a spleen cDNA library. The four A alleles and four B alleles obtained represent four distinct loci indicating that a duplication of the A and B loci has taken place in the lineage leading to these Old World primates. No HLA--C homologue/orthologue was found. In addition a single, nonclassical homologue of HLA--E was characterized. Examination of nucleotide and extrapolated protein sequences indicates that alleles at the two B loci are much more diversified than the alleles at the A loci. One of the A loci in particular appears to display very limited polymorphism in both Papio hamadryas cynocephalus and Papio hamadryas anubis subspecies. The failure to detect a homologue of HLA--C in the baboon provides additional evidence for the more recent origin of this locus in the pongidae and hominidae: Further comparative analysis with MHC sequences among the primate species reveals specific patterns of divergence and conservation within class I molecules of the yellow baboon.     

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.     

Modes of salmonid MHC class I and II evolution differ from the primate paradigm

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.     

How specific MHC class I and class II combinations affect disease resistance against infectious salmon anaemia in Atlantic salmon (Salmo salar)

The aim was to evaluate the performance of selected individual MHC class I and class II alpha (A) alleles, and combinations of these on disease resistance against infectious salmon anaemia (ISA). The material consisting of 1966 fish from seven families, contained five MHC class I alleles and four MHC class II A alleles. Which representing given class II A and class II beta (B) haplotypes, totalling 19 MHC class I and class II A genotypes. The fish were challenged with infectious salmon anaemia virus (ISAV), the virus causing ISA. Dead fish were collected daily during the challenge experiment and the survivors were collected at termination. All fish were genotyped for MHC class I and class II A. The total mortality in the material was 85.14%. For MHC class I, UBA*0201 and UBA*0301 were significantly the most resistant alleles, while UBA*0601 for class I and DAA*0301 for class II A were the significantly most susceptible alleles. The analysis of combined MHC class I and class II A genotypes detected that fish with the genotype UBA*0201/*0301;DAA*0201/*0201 were the most resistant fish with a hazard ratio (HR) at 0.750, while the fish with the genotypes UBA*0601/*0801;DAA*0501/*0501 and UBA*0201/*0301;DAA*0301/*0501 were the most susceptible fish with HR of 1.334 and 1.425. In addition, Cox regression analysis within family detected combined MHC class I and class II A genotypes that contributed significantly to resistance and susceptibility. The study confirmed the expectation of performance of individual MHC class I and class II A alleles, and also detected an effect of MHC class I and class II A in combinations.     

MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris)

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment—admittedly with limited geographic coverage and sample size—did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.     

Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

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.     

The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV)

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with β-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 6₁ and B12 from line C determine protection, although another locus from line 6₁ was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule.     

MICA genetic polymorphism and linkage disequilibrium with HLA-B in 29 African-American families

The human major histocompatibility complex (MHC) class I chain-related gene A ( MICA) is located 46 kb upstream of HLA-B and encodes a stress-inducible protein which displays a restricted pattern of tissue expression. MICA molecules interact with NKG2D, augmenting the activation of natural killer cells, CD8(+) alpha beta T cells, and gamma delta T cells. MICA allelic variation is thought to be associated with disease susceptibility and immune response to transplants. We investigated MICA allelic variations and linkage disequilibrium with HLA-A, B, and DRB1 loci on 110 parental haplotypes from 29 African-American families. PCR/sequence-specific oligonucleotide probing (SSOP) was used to define MICA polymorphisms in exons 2, 3, and 4. Ambiguous allelic combinations were resolved by sequencing exons 2, 3, and 4. Exon 5 polymorphisms were analyzed by size sequencing. For HLA-A, B and DRB1 typing, low-resolution PCR/SSOP and allelic PCR/sequence-specific priming techniques were used. Twelve MICA alleles were observed, the most frequent of which were MICA*008, MICA*004, and MICA*002, with gene frequencies of 28.2, 26.4, and 25.5%, respectively. Thirty-eight HLA-B- MICA haplotypic combinations were uncovered, 22 of which have not been reported in the HLA homozygous typing cell lines from the 10th International Histocompatibility Workshop. Significant positive linkage disequilibria were found in 8 HLA-B- MICA haplotypes. Furthermore, haplotypes bearing HLA-B*1503, *1801, *4901, *5201, *5301, and *5703 were found to segregate with at least two different MICA alleles. Our results provide new data about MICA genetic polymorphisms in African-Americans, which will form the basis for future studies of MICA alleles in allogeneic stem cell transplantation outcome.     

MHC class I characterization of Indonesian cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) are quickly becoming a useful model for infectious disease and transplantation research. Even though cynomolgus macaques from different geographic regions are used for these studies, there has been limited characterization of full-length major histocompatibility complex (MHC) class I immunogenetics of distinct geographic populations. Here, we identified 48 MHC class I cDNA nucleotide sequences in eleven Indonesian cynomolgus macaques, including 41 novel Mafa-A and Mafa-B sequences. We found seven MHC class I sequences in Indonesian macaques that were identical to MHC class I sequences identified in Malaysian or Mauritian macaques. Sharing of nucleotide sequences between these geographically distinct populations is also consistent with the hypothesis that Indonesia was a source of the Mauritian macaque population. In addition, we found that the Indonesian cDNA sequence Mafa-B7601 is identical throughout its peptide binding domain to Mamu-B03, an allele that has been associated with control of Simian immunodeficiency virus (SIV) viremia in Indian rhesus macaques. Overall, a better understanding of the MHC class I alleles present in Indonesian cynomolgus macaques improves their value as a model for disease research, and it better defines the biogeography of cynomolgus macaques throughout Southeast Asia.     

A Mechanistic Basis for the Co-evolution of Chicken Tapasin and Major Histocompatibility Complex Class I (MHC I) Proteins

MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical MHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed MHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo.     

Single locus typing of MHC class I and class II B loci in a population of red jungle fowl

In species with duplicated major histocompatibility complex (MHC) genes, estimates of genetic variation often rely on multilocus measures of diversity. It is possible that such measures might not always detect more detailed patterns of selection at individual loci. Here, we describe a method that allows us to investigate classical MHC diversity in red jungle fowl (Gallus gallus), the wild ancestor of the domestic chicken, using a single locus approach. This is possible due to the well-characterised gene organisation of the ‘minimal essential’ MHC (BF/BL region) of the domestic chicken, which comprises two differentially expressed duplicated class I (BF) and two class II B (BLB) genes. Using a combination of reference strand-mediated conformation analysis, cloning and sequencing, we identify nine BF and ten BLB alleles in a captive population of jungle fowl. We show that six BF and five BLB alleles are from the more highly expressed locus of each gene, BF2 and BLB2, respectively. An excess of non-synonymous substitutions across the jungle fowl BF/BL region suggests that diversifying selection has acted on this population. Importantly, single locus screening reveals that the strength of selection is greatest on the highly expressed BF2 locus. This is the first time that a population of red jungle fowl has been typed at the MHC region, laying the basis for further research into the underlying processes acting to maintain MHC diversity in this and other species.