Genomic hybridization of bovine class II major histocompatibility genes: 1. Extensive polymorphism of DQα and DQβ genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot analysis using human cDNA probes for DQα, DQβ, DRβ and DRβ. The presence of a DQ-like and a DR-like subregion in cattle was clearly indicated. Highly polymorphic restriction fragment patterns were obtained when genomic DNA, digested with any one of the Bam HI, Eco RI or Pvu II restriction enzymes, was hybridized with the DQα and the DQβ probe. The polymorphisms were interpreted genetically by analysing five paternal half-sib families of the Swedish Red and White breed. The material comprised, besides the bulls, 28 offspring and their dams. The analysis resolved 9 and 12 allelic variants of DQα and DQβ respectively. Thus, this investigation establishes a method for routine typing of MHC class II gene polymorphism in cattle. The results were entirely consistent with close linkage of DQα and DQβ since no recombinant was found and since alleles at these loci occurred in complete linkage disequilibrium in the material investigated. Close linkage between DQ and the blood group locus M, which has previously been found to be closely linked to the serologically defined BOLA-A locus, was also indicated. In this study DNA was isolated from frozen semen samples of dead bulls, which shows that this type of analysis will be useful in genetic investigations in cattle breeds, where artificial insemination is practised.     

Genomic hybridization of bovine class II major histocompatibility genes: 2. Polymorphism of DR genes and linkage disequilibrium in the DQ-DR region

Class II genes of the bovine major histocompatibility complex have been investigated by Southern blot analysis using human cDNA probes for DQ alpha, DQ beta, DR alpha and DR beta. In this report restriction fragment length polymorphisms of DR alpha and DR beta are described. The polymorphisms were interpreted genetically by analysing five paternal half-sib families of the Swedish Red and White Breed, comprising altogether 28 offspring. Using the restriction enzymes BamHI, EcoRI and PvuII, three DR alpha and three DR beta allelic fragment patterns were resolved. The DR alpha and DR beta genes thus appear to be much less polymorphic than the previously described DQ alpha and DQ beta genes. Also, the observed linkage disequilibrium between DR genes was less pronounced than that between DQ genes, whereas the association between DR and DQ haplotypes was very strong. The family data available indicated strongly that the DQ alpha, DQ beta, DR alpha and DR beta genes are all closely linked.     

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.     

HLA-DR beta genes vary in number between different DR specificities, whereas the number of DQ beta genes is constant

Probes isolated from DR and DQ beta cDNA and genomic clones were used in hybridizations to restriction enzyme-digested DNA from human homozygous typing cells (HTC) as well as other DR homozygous cells in order to estimate the number of beta genes in the DR/DQ class II region. Varying numbers of DR beta genes were found in HTC of different DR specificities, from possibly one in DR 8 cells to three in cells of DR 2 to 7. The DR beta genes of different specificities seem to be related to one another in a distinct fashion. In contrast, all HTC contain two DQ beta genes per chromosome. The restriction site polymorphism of DQ beta genes is considerably more extensive than that of DQ serology, although one of the genes seems to be nonpolymorphic. In addition to the two DP beta genes identified previously, a minimum of three to five DQ and DR beta genes exist in the human haploid genome.     

Differential contribution of C4 and HLA-DQ genes to systemic lupus erythematosus susceptibility

The particular histocompatability antigen (HLA) gene(s) that may confer systemic lupus erythematosus (SLE) susceptibility remains unknown. In the present study, 58 unrelated patients and 69 controls have been analyzed for their class I and class II serologic antigens, class II (DR and DQ) DNA restriction fragment length polymorphism, their deduced DQA1 and B1 exon 2 nucleotide sequences and their corresponding amino acid residues. By using the etiologic fraction () as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative SLE susceptibility locus, it has been found that the strength of association of the HLA marker may be quantified as follows: DQA1^*0501 (associated to DR3) or DQB1^*0201 (associated to DR3) > non Asp 57 DQ/Arg 52 DQ > DR3 > non Asp 57 DQ. Thus, molecular HLA DQ markers tend to be more accurate as susceptibility markers than the classical serologic markers (DR3). However, dominant or recessive non Asp 57 DQ susceptibility theories, as previously postulated for insulin-dependent diabetes mellitus, do not hold in our SLE nephritic population; indeed, three patients bear neither Arg 52 DQ nor Asp 57 DQ susceptibility factors. On the other hand, nonsusceptibility factors are included in our population in the A30B18CF130-DR3DQ2(Dw25) haplotype and not in A1B8CS01-DR3DQ2(Dw24); this distinctive association has also been recorded in type I diabetes mellitus and may reflect the existence of common pathogenic HLA-linked factors for both diseases only in the A30B18CF10DR3DQ2-(Dw 25) haplotype. Finally, the observed increase of deleted C4 genes (and not null C4 proteins) in nephritic patients shows that C4 genes are disease markers, but probably without a pathogenic role.     

Molecular mapping class II polymorphisms in the human major histocompatibility complex. II. DQ beta

The restriction fragment length polymorphisms have been determined for six restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, and Pvu II) and a DQ beta probe on 25 cell lines that are homozygous by consanguinuity at the MHC. These patterns reflect both DR haplotypes and DQ types of the cells tested. At least one non-polymorphic band is present in all the cell lines with every restriction enzyme except Hinc II. This band most probably represents DX beta hybridization. The polymorphic bands indicate that more polymorphism exists in the DQ subregion than is predicted serologically. Each DR haplotype is associated with a unique set of restriction fragments except for DR2 and DR6. The patterns are largely consistent within each DR haplotype. In addition, some bands reflect the established DQ specificities DQw1 and DQw2. Individual bands can be identified that are unique to the haplotypes DR1, DR4, DR5, and DR6 and the DQw1- and DQw2-associated haplotypes. Subdivisions of haplotypes can be identified with this probe. In particular, MVL (DR1), Akiba (DR2), QBL (DR3), FPF (DR5), and APD (DR6) have polymorphisms that distinguish them from other members of their DR haplotype.     

Polymorphism of the HLA-D region in American blacks. A DR3 haplotype generated by recombination

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a serologically undefined DQ allelic product. DNA restriction fragment analysis with the use of several unrelated individuals and an informative family has allowed us to identify unique DQ alpha- and beta-fragments associated with the DR3, DQw- haplotype. Based on fragment size, the DQ alpha genes of the DR3, DQw- and DRw8, DQw- haplotypes are similar as are the DQ beta genes of DR3, DQw-; DRw8, DQw-; and DR4, DQw- haplotypes. In addition, a DX beta gene polymorphism has been identified which is associated with some DR3 haplotypes including the American black DR3, DQw- haplotype. cDNA sequence analysis has revealed a DQw2-like alpha gene and a DQ beta gene which is similar to that previously described for a DR4, DQw- haplotype. It is postulated that recombination between DQ alpha and DQ beta genes and between the DQ and DX subregions has generated the various DR3 haplotypes and has played an important role in creating diversity in the HLA-D region.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

HLA-DR4-associated haplotypes are genotypically diverse within HLA

Biochemical diversity among products of class II HLA genes has been observed in individuals who appear to be HLA-D and DR-identical by cellular and serologic typing. We used techniques of restriction enzyme fragment analysis by Southern blotting to analyze this diversity at the level of cellular DNA. A panel of 17 HLA-DR4 homozygous cell lines (HCL) were investigated by using cDNA probes homologous to DQ beta, DQ alpha, and DR beta genes. Each probe was hybridized to cellular DNA digested with a series of different restriction endonucleases. Polymorphisms were observed with the use of the enzymes Pst I, Hind III, and Bam HI: Hybridization of cellular DNA digested with Hind III and Pst I with the DQ beta probe revealed specific polymorphisms, as did hybridization of the Pst I digest with the DQ alpha cDNA probe and the Bam HI digest with the DR beta probe. The observed differences fall into two categories: first, considerable diversity was seen between HLA-DR4 HCL that represent different HLA-D-defined haplotypes; second, diversity was also observed among HCL of the same DR4-associated HLA-D cluster. In contrast to the DQ cDNA probes, hybridization with the DR beta probe revealed relatively limited polymorphism by using a panel of different restriction endonucleases. Thus, although there is a general pattern of polymorphic restriction enzyme fragments homologous to DQ probes within an HLA-D cluster, the pattern seen for any particular cell line was not sufficiently distinct to assign an HLA-D or DR specificity.     

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha-and beta-chain cDNA probes

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.     

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLA ^c haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique genes were obtained which showed no evidence of cross-hybridization, while genes showed extensive cross-hybridization and were frequently detected in the library by more than one human gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of genes, with possible shared usage of these genes by different a loci. The data also imply that genes can readily be assigned to loci homologous to their human counterparts, but that genes will require further mapping and/or sequence analysis to confirm assignments.     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

Partial nucleotide sequence of a bovine major histocompatibility class II DRβ-like gene

Summary. A genomic clone containing a bovine DRβ-like gene, BoDRβ II , was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DRβ cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3kb 5' of the β₁ exon and 6.7kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDRβ exons sequenced. Nucleotide identities of the bovine β₁, β₂ and TM exons with the corresponding human DRβ exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DRβ-like pseudogene, BoDRβ I , were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the β₂ exons in BoDRβ I and BoDRβ II . A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the β₁ exon in both BoDRβ I and BoDRβ II .     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Restriction fragment analysis of the HLA Class II region: Application to tissue transplantation

Southern blotting and hybridization with locus specific probes is a reliable method for the determination of HLA Class II specificities and in conjunction with several other techniques is providing valuable information on the nature of Class II polymorphisms in man. In terms of the identification of Class II specificities in transplantation however, the advanced nature of DR serology means that DR restriction fragment analysis is probably limited only to those situations where serological methods have been unsuccessful.Whether DQ and DP incompatibilities can evoke rejection responsesin vivo remains to be fully ascertained although our preliminary data suggest that DQ mismatches probably have little effect upon graft outcome. Restriction fragment analysis is particularly attractive in that in enables reliable retrospective studies to be performed in conjunction with prolonged clinical follow up, and the technique will clearly continue to contribute significantly to our understanding of the importance of these molecules in transplantation.     

Polymorphisms and gross structure of glycophorin genes in common chimpanzees

Using human α glycophorin cDNA probe and six restriction enzymes, we examined the homologues of human glycophorin genes in genomic DNA of 11 unrelated chimpanzees. We show that, in contrast to the human, the chimpanzee exhibits an unusual array of nonrandomly distributed restriction fragment length polymorphisms (RFLP). No clear correlation was found between the RFLP and the V-A-B-D blood-group phenotypes of the subjects, with one possible exception. However, pairs of allelic RFLP occurring at a relatively high frequency were identified. In addition, the homology of chimpanzee glycophorin genes to the human genes was examined using as probes synthetic oligonucleotides specifying distinct regions of human glycophorin genes. We show that the glycophorin gene family in the chimpanzee consists of at least three members that are homologous to the human α, δ, and E genes (glycophorins A, B, and E) and may share a similar gross structure and overall organization. This research was supported by National Institutes of Health Grant GM 16389.     

Close association between DNA polymorphism of bovine major histocompatibility complex class I genes and serological BoLA-A specificities

Class I genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridization and by serological analysis. A large number of class I restriction fragments and an extensive polymorphism were revealed when genomic DNA samples, digested with the restriction enzyme PvuII, were hybridized with a human cDNA probe. The result indicated the presence of multiple class I genes in cattle. The extensive restriction fragment length polymorphism (RFLP) was interpreted genetically by analysing five paternal half-sib families comprising, besides the bulls, 50 offspring and their dams. No less than 21 RFLP types were distinguished in this limited sample. The class I polymorphism was also analysed using a serological test with sera corresponding to four workshop specificities (w2, w6, w10 and w16) and three locally defined specificities (SRB1, SRB2 and SRB3). There was an excellent agreement between the two typing methods since no RFLP type was associated with more than one specificity and five of the seven specificities were associated with a single RFLP type. Evidence for close genetic linkage between class I and DQ class II genes was obtained since no recombinant was found among 45 informative offspring. Linkage disequilibrium among class I, DQ class II and C4 genes was also observed. The blood group specificity M' was completely associated with the w16 class I specificity and with the haplotype I1DQ1BC4(2) in this material.     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Structural similarity of the HLA-DQ region in DQ3 and DQ4 haplotypes and structural diversity of the HLA-DQ region in HLA-DR7 haplotypes.

Genomic DNA obtained from a B lymphoblastoid cell line was digested with appropriate restriction endonuclease and hybridized with several probes specific for genes encoding HLA-DQ. Southern hybridization with a DQA1 3'untranslated (UT) region probe showed DQ2-type hybridization pattern in DR7DQ3 haplotype. On the contrary, DQB1 3'UT probe showed DQ3-type pattern in the same haplotype. Gene cloning and DNA sequencing analysis revealed a repetitive sequence, (TG)19, between DQA1 and DQB1 gene in the DR7DQ3 haplotype. These results suggest that a recombination event has occurred near this potential Z-DNA structure in the haplotype, DR7DQ3. The 3'UT region probes of DQA1 and DQB1 genes failed to detect restriction fragment length polymorphism (RFLP) differences between DR4DQ3 and DR4DQ4 haplotypes in this experiment, suggesting that the gene structure between DQA1 and DQB1 is conserved in these haplotypes.     

Mapping of the rabbit MHC reveals that class I genes are adjacent to the DR subregion and defines an insertion/deletion-related polymorphism in the class II region.

Molecular analyses of genes in the rabbit MHC (RLA) by pulsed field gel electrophoresis have shown that the relative order of class II genes (DP, DO, DQ, DR) is identical to that in humans and similar to that in the mouse. However, a major difference from either HLA or H-2 was observed at the DR end of the RLA class II complex: class I genes are located in close proximity to DR with no interposed class III sequences. A MluI fragment of 180 kb and a 210-kb SalI fragment both hybridized with the DR probe as well as with different class I probes including that for pR27, a class I gene with T cell-limited pattern of expression. Comparison of two different RLA haplotypes, A and B, indicated that the distance between the DQ and DR subregions differs by approximately 700 kb in the two haplotypes. Testing other unrelated rabbits suggested that this difference segregates within the rabbit population and presumably derives from an insertion/deletion event in different haplotypes. A further difference between the A and B haplotypes included variable distance between genes encoding DO beta and DP; the DR end of the complex and the class I genes linkage was conserved in the two haplotypes.