Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis

The B-G antigens of the chicken major histocompatibility complex (MHC) have been analyzed by high resolution two-dimensional (2-D) gel electrophoresis. Monoclonal antibodies recognizing a widely shared B-G determinant were used for immunoprecipitating the B-G antigens from radioiodinated, detergent-solubilized erythrocyte membrane preparations. The B-G antigens produce a variety of patterns on 2-D gels. The number of polypeptides within a B-G pattern varies among haplotypes from single polypeptide arrays showing slight microheterogeneity to complex patterns which contain as many as four or five polypeptide arrays differing in relative mobility and isoelectric point. Many of the patterns, but not all, include a polypeptide of Mr =48 kd focusing near pH 6.9. At present it is not understood whether the multiple polypeptides within some B-G patterns represent the expression of multiple B-G genes or whether they are the result of modifications of single gene products during biosynthetic processing. 2-D gel analyses were also used to confirm the assignment of the same B-G haplotype in several different inbred flocks and the fate of the B-G antigens in two B system recombinant haplotypes. The 2-D gel patterns of these highly polymorphic antigens provide evidence for a complexity of the B-G locus not previously demonstrated. This technique may serve to define more objectively the diverse chicken MHC haplotypes which are now recognized and characterized only by serological techniques using alloantisera and monoclonal antibodies with varying cross-reactivities.     

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.     

The MHC haplotypes of the chicken

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as standard B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The standard haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.     

Antigens similar to major histocompatibility complex B-G are expressed in the intestinal epithelium in the chicken

A monoclonal antibody directed against the erythrocytic B-G antigens of the major histocompatibility complex (MHC) of the chicken, an antiserum raised against purified erythrocytic B-G protein, and a cDNA probe from the BeckmanB-G subregion were used to look for evidence of the expression ofB-G genes in tissues other than blood. Evidence has been found in northern hybridizations, in immunoblots, and in immunolabeled cryosections for the presence of B-G-like antigens in the duodenal and caecal epithelia. Additional B-G-like molecules may be expressed in the liver as well. The BG-like molecules in these tissues appear larger and somewhat more heterogeneous than the B-G antigens expressed on erythrocytes. Further characterization of these newly recognized B-G-like molecules may help to define a function for the enigmatic B-G antigens of the MHC. al. 1977; Miller et al. 1982, 1984; Salomonsen et al. 1987; Kline et al. 1988), and in the multiplicity of B-G restriction fragment patterns found in genomic DNA from different haplotypes (Goto et al. 1988; Miller et al. 1988; Chaussé et al. 1989). The B-G antigens have contributed, together with the B-F (class I) and B-L (class II) antigens, to the definition of over 27 B system haplotypes in experimental flocks (Briles et al. 1982). Yet the function of the B-G antigens remains entirely unknown. No mammalian counterparts have been identified, although the possibility remains that there may be similar antigens among the blood group systems of mammals. In an effort to define a function of the B-G antigens, a recently cloned B-G sequence (Miller et al. 1988; Goto et al. 1988) and antibodies to the B-G polypeptides (Miller et al. 1982, 1984) were used to examine other tissues for evidence of B-G expression.     

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.     

Restriction fragment length polymorphism analysis of the chicken B-F and B-L genes and their association with serologically defined B haplotypes

Seven serologically defined chicken haplotypes have been analysed by restriction fragment length polymorphism (RFLP) with chicken cDNA probes specific for MHC class I and II. The results demonstrate an excellent correlation between the observed RFLP banding patterns in the investigated haplotypes and the serological B-typing. In future, RFLP analysis in addition to serological B-typing may sharpen the tools in the search for recombinant chromosomes separating B-F and B-L.     

Characterization of class I bovine lymphocyte antigens (BoLA) by one-dimensional isoelectricfocusing

BoLA class I antigens were characterized in a group of British and Dutch Friesian cattle by one-dimensional isoelectric focusing (1D-IEF) and the results compared with serology using alloantisera and microcytotoxicity. For IEF analysis, non-stimulated peripheral blood mononuclear cells (PBM) were metabolically labelled with 35S methionine, detergent lysates were prepared and MHC molecules precipitated with the monoclonal antibodies (mAbs) W6/32 or B1.1G6. Staphylococcus protein A precipitated antigens were separated on a vertical slab gel under denaturing conditions. The banding patterns seen for the W6/32 precipitated molecules obtained by 1D-IEF were compared with the serological specificities. Characteristic banding patterns were observed for most serological specificities as well as workshop undefined haplotypes. These patterns were seen both in families and the outbred population. In families IEF haplotypes segregated with serotypes. Additional MHC class I products were suggested by variable banding patterns for different w10 haplotypes and when using the different mAbs. A pulse chase experiment with a w12 animal also suggested more than one expressed product. The w2 and w5 specificities were not precipitated by either W6/32 or B1.1G6 and w6.2 and w6.4 were precipitated by W6/32 but not by B1.1G6. These results show that 1D-IEF is useful for BoLA typing. For the characterization of class I antigens, however, much depends on the mAbs used.     

The major histocompatibility complex in the chicken

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

Characterization of two distinct disulfide-linked B-G molecules in the chicken

Alloantisera specific for B-G antigens recognized a complex of molecules of apparent molecular weights of 90 and 98 Kd under nonreducing conditions and molecules of 40, 44, and 48 Kd under reducing conditions on both embryo- and adult-derived peripheral red blood cells (RBC). The chicken B-G molecules produced a unique two-dimensional "diagonal" pattern. Two antisera permitted the characterization of the complex B-G molecular profile as a homodimer composed of 48-Kd subunits and as a heterodimer composed of 40- and 44-Kd subunits. A rabbit antiserum produced against B-G molecules preferentially recognized the 48-Kd reduced molecules, suggesting that the 90-Kd molecule was a homodimer composed of two 48-Kd molecules. One B-G reagent was capable of recognizing only the 98-Kd nonreduced B-G molecule that gave rise to 40- and 44-Kd molecules under reducing conditions, suggesting that the 98-Kd molecule was a heterodimer composed of 44- and 40-Kd subunits. Adult chicken B-G2 molecules produced a variety of two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) patterns depending on the characteristics of the reagent employed in the immunoprecipitation. B-G molecules were immunoprecipitated from primitive and definitive chicken RBCs but not from any nonerythroid cells tested. B-G molecules were not expressed by avian erythroblastosis virus (AEV)-transformed erythroleukemia cells, nor were they induced to appear with butyric acid-induced erythroid differentiation.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Summary. Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed.
Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed. Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

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.     

Three new MHC haplotypes in broiler breeder chickens

Six distinct serotypes of the chicken B blood group system (which encodes the major histocompatibility complex) were identified in a commercial broiler breeder line (Line C). The B serotypes were compared by B-G restriction fragment length polymorphism (RFLP) analysis, allele-specific PCR typing test for B-LBII family genes and nucleotide sequence analysis of expressed B-F and B-LBII family genes. The results indicated the existence of seven distinct B haplotypes. Nucleotide sequence analysis demonstrated that three of the Line C haplotypes encode new B-F and B-LB alleles.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

Summary. High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes ( Pvu II, Hind III, Bg /II, and Bam HI), electrophoresed for 18 or 45h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II β-chain probe (β2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B halotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.     

Biochemical confirmation of recombination within the B-G subregion of the chicken major histocompatibility complex

Analysis of the B-G antigens of eight chicken major histocompatibility complex (B) system recombinant haplotypes by high resolution two-dimensional gel electrophoresis has provided evidence for the transfer of the complete B-G subregion in seven cases. In the eighth, a partial duplication within the B-G subregion appears to have occurred. In this recombinant, the entire array of polypeptides associated with one parental allele, B-G ²³ is expressed together with nearly the entire array of B-G polypeptides of the other parental haplotype, B ². This compound polypeptide pattern corroborates the serological evidence for a partial duplication within the B-G subregion and provides indirect evidence for the existence of multiple loci within B-G and for a means by which polymorphism may be introduced into the chicken major histocompatibility complex.     

Major histocompatibility complex (MHC) related cDNA probes associated with antibody response in meat-type chickens

The major histocompatibility complex (MHC) region was examined as a set of candidate genes for association between DNA markers and antibody response. Intercross F₂ families of chickens were generated from a cross between high (HC) and low (LC) Escherichia coli _i antibody lines. Restriction fragment length polymorphism (RFLP) analysis was conducted by using three MHC-related cDNA probes: chicken MHC class IV ( B-G ), chicken MHC class I ( B-F ), and human MHC-linked Tap2 . Association between RFLP bands and three antibody response traits ( E. coli , sheep red blood cells and Newcastle disease virus) were determined by two methods: by statistically analyzing each band separately and also by analyzing all bands obtained from the three probes by using multiple regression analysis to account for the multiple comparisons. The MHC class IV probe was the highest in polymorphisms but had the lowest number of bands associated with antibody response. The MHC class I probe yielded 15 polymorphic bands of which four exhibited association with antibody response traits. The Tap2 probe yielded 20 different RFLP bands of which five were associated with antibody production. Some Tap2 bands were associated with multiple antibody response traits. The multiband analysis of the three probes' bands revealed more significant effects than the analysis of each band separately. This study illustrates the efficacy of using multiple MHC region probes as candidate markers for quantitative trait loci (QTLs) controlling antibody response in chickens.