Mapping of the SLA complex of miniature swine: Mapping of the SLA gene complex by pulsed field gel electrophoresis

The overall order of the regions of the swine major histocompatibility complex (MHC), the SLA complex, was determined by pulsed field gel electrophoresis (PFGE). It was found that the order of the regions is class II-class III-class I. A class I probe hybridized to a 420 kbMlu I and a 420 kbNot I fragment as did a class III probe forC2. None of the class II probes hybridized to these fragments. Thus, linkage of class I to class III was shown. The class IIiC2, Bf, andC4 genes were found to reside in a 190 kbNot I fragment. Linkage of class III and class II genes was shown when both the class IIiC4 and the class IiDR probes hybridized to the same 195 kbSac II and 340 kbNot I fragments. The class I probe did not hybridize to these fragments. The order of the regions, class II-class III-class I, is similar to that of human MHC genes and may have been conserved in evolution so that coordinated expression of MHC genes could be achieved.     

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.     

Mapping of the SLA complex class III region by pulsed field gel electrophoresis

The fine order of genes in the class III region of the swine major histocompatibility complex (MHC), the SLA complex, was examined by pulsed field gel electrophoresis (PFGE) and Southern blot analysis. Four genes, C2, HSP70, TNF, and CYP21, were analyzed. The CYP21, C2, and HSP70 genes were all located within a 200-kb NotI fragment. The C2, HSP70, and TNF genes cohybridized to a 420-kb SalI fragment. The TNF gene is linked to the class I region by a 390-kb NotI fragment. Combined with a previous study from our lab, the order of genes in the SLA complex is class II-class III [(CYP21/C4)-(Bf/C2/HSP70)-TNF]-class I. The size of the class III region from CYP21 to TNF is estimated to be 500 kb. This size and the order of the genes in the swine class III region are similar to those of human, mouse, goat, and rabbit, which confirms the high conservation of class III gene organization across species.     

Conservation of the central MHC genome: PFGE mapping and RFLP analysis of complement,HSP70, and TNF genes in the goat

A degree of conservation of the genes located between class II and class I [central major histocompatibility complex (MHC) genes] is apparent among mammalian species including primates and the mouse. Few others have been analyzed. The caprine MHC is of particular interest, since it has recently been observed that susceptibility to a lentivirus-induced polyarthritis (caprine arthritis) segregates with serologically defined MHC class I antigens. This arthritis resembles, in a number of respects, rheumatoid arthritis in man. Human cDNA probes were used to examine the caprine central MHC and class I and II genes by restriction fragment length polymorphism (RFLP) and by pulsed field gel electrophoresis (PFGE) in order to define the polymorphism and linkage of central MHC genes to class I and class II genes. An outbred population of dairy goats (Saanen, British Alpine, Anglo Nubian, and Toggenberg) was examined for class I and class II RFLPs. Both regions were found to be highly polymorphic. The number of fragments hybridizing to an HLA-B7 probe after Eco RI, Bam HI, Bgl II, or Hind III digestion suggests there may be 10–13 class I genes. The degree of polymorphism was comparable to that reported in the mouse. Limited polymorphism was found in the central MHC genes. The caprine C4 and CYP21 genes were duplicated and demonstrated RFLP with Bam HI, Hind III, Eco RV, and Taq I. An infrequent Taq I C2 polymorphism was found. PFGE revealed substantial conservation of both the order and linkage of the central MHC genes when compared with mous and man. C4, C2, CYP21, HSP70, and tumor necrosis factor (TNF) genes are all located within 800 kilobase (kb) of the class I loci. Distant from the class I region, the C4, C2, and CYP21 genes are linked on a short genomic segment (180 kb Not I and 190 kb Pvu I fragments). HSP70 cohybridizes with the complement genes on a 380 kb Mlu I fragment. Linkage of HSP70, TNF, and class I genes was found on a single Not I fragment (610 kb). TNF and class I cohybridize on Pvu I (730 kb) and Not I (610 kb) fragments. Conservation of a similar central MHC genomic structure across species argues for functional interaction between the central MHC genes. We postulate selection for these central MHC genes through their role as non antigen-specific regulators of immune response.     

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.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.     

Restriction fragment length polymorphism of the major histocompatibility complex of the pig

Human HLA cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the SLA major histocompatibility complex in swine. Cellular genomic DNA from 19 SLA homozygous pigs representing 13 different haplotypes was digested with restriction endonucleases Eco RI, Hind III, or Bam H1, separated by electrophoresis, and transferred onto diazobenzyloxymethyl paper by the Southern blot technique. The blots were probed with ³²P-labeled class I or beta-DR class II cDNA. Depending on the haplotypes and the endonucleases used, seven to ten restriction fragments hybridized with the class I probe, and five to seven with the beta-DR probe. Their sizes ranged from 3.4 to 22 kilobase-pairs. Few bands were common to all 13 haplotypes. With all but one haplotype, identical autoradiogram patterns were obtained from unrelated, but phenotypically SLA-identical pigs, suggesting that most of the RFLP revealed were controlled by the SLA region. Further polymorphism was found in a group of seven unrelated pigs which typed serologically as SLA A15 CI B18 homozygotes but could be divided into two subgroups, with five animals in one subgroup and two in the other, when the genomic DNA was hybridized with the class I probe. When the class 11 beta-DR probe was tested on the same seven pigs, another subdivision was seen, and this correlated with MLR data. These results demonstrate that HLA class I and class II probes can be used to identify certain well-established SLA haplotypes and to identify subclasses within at least one SLA haplotype.Abbreviations MHC major histocompatibility complex - MLR mixed lymphocyte reaction - kbp kilobase pair(s) - RFLP restriction fragment length polymorphism     

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

Restriction fragment length polymorphisms in bovine major histocompatibility complex class II ß-chain genes using bovine exon-containing hybridization probes

Restriction fragment length polymorphisms (RFLPs) have been identified in the bovine MHC class II region using five hybridization probes constructed from two bovine genomic clones. Four probes were constructed from a bovine DR beta-like gene, BoLA-DRB2. These included a probe containing the complete beta 1 exon (R2-beta 1), a probe containing the last 129 base pairs of the beta 2 exon (R2-beta 2), a probe containing intron immediately 5' of the beta 2 exon (R2-5' beta 2), and a probe containing the complete transmembrane exon (R2-TM). A fifth probe was constructed from a novel bovine beta-chain gene, BoLA-DIB, and contained the entire TM exon (I1-TM). R2-beta 1 defined very little polymorphism. R2-beta 2 hybridized to several fragments but one or two fragments hybridized much stronger on all Southern blots and it was presumed these corresponded to BoLA-DRB2 fragments. By using R2-5' beta 2 as a probe, these BoLA-DRB2 fragments were confirmed: 6.4 and 2.7-kb Eco RI alleles, 1.7- and 1.5-kb Pvu II alleles, 5.9-, 5.4-, 3.7- and 1.9-kb TaqI alleles, and a non-polymorphic 22.5-kb BamHI fragment. I1-TM identified three alleles with TaqI. To investigate the linkage between the RFLP alleles, 166 offspring of five sires were tested. Complete linkage was found for all RFLPs identified with the BoLA-DRB2 probes. However, the RFLP patterns of 13 calves out of 58 indicated recombination between BoLA-DRB2 and BoLA-DIB.     

A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

Mapping of C2, Bf, and C4 genes to the swine major histocompatibility complex (swine leukocyte antigen)

Three miniature swine lines, inbred for swine leukocyte antigen (SLA) haplotypes, a, c, and d, and a recombinant line, haplotype g, were analyzed for possible restriction fragment length polymorphisms (RFLP) by Southern blot hybridization with human C2, factor B (Bf), and C4 specific probes. The search for RFLP by using a human C2 probe failed to reveal any variants. However, a Taq I polymorphism was identified with the human Bf probe and Bam HI and Pvu II polymorphisms were identified with the human C4 probe. Overlapping restriction fragments were found with the C2 and Bf probes, which strongly suggests close linkage of C2 and Bf genes in swine. Segregation analyses of the Bf and C4 polymorphisms indicated that the polymorphic fragments followed a Mendelian pattern of inheritance. The recombinant haplotype g, which expresses class I genes of haplotype c and class II genes of haplotype d, was shown to produce an identical RFLP pattern, by using the Bf and C4 probes, as haplotype d, but different from that of haplotype c. This indicates that there is a close association of [C4-Bf-C2] and class II genes in miniature swine. Although these data do not show conclusively the location of the [C4-Bf-C2] genes, it is hypothesized that swine [C4-Bf-C2] genes are located between the class II and class I genes, as has been demonstrated in mouse and man.     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism     

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.     

Megabase scale restriction fragment length polymorphisms in the human major histocompatibility complex

Pulsed-field gel electrophoresis of human peripheral blood lymphocyte DNA was used to ascertain the extent of megabase restriction fragment length variation in the HLA region of human chromosome 6 and to determine whether previously reported diversity was due to experimental variation or DNA polymorphism. Polymorphism was found to predominate in the vicinity of the class II DR, class III complement, and the class I A genes and to be limited or absent near the class II DP genes, the TNF genes, and the class I B and C genes. Thus, the MHC region is characterized by both fine and large-scale structural diversity.