Physical localization and order of genes in the class I region of the bovine MHC

Fluorescence in situ hybridization (FISH) analyses were used to order 16 bacterial artificial chromosomes (BAC) clones containing loci from the bovine lymphocyte antigen (BoLA) class I and III regions of bovine chromosome 23 (BTA23). Fourteen of these BACs were assigned to chromosomal band locations of mitotic and pachytene chromosomes by single- and dual-colour FISH. Dual-colour FISH confirmed that class II DYA is proximal to and separated from BoLA class I genes by approximately three chromosome bands. The FISH results showed that tumour necrosis factor alpha (TNFA), heat shock protein 70 (HSP70.1) and 21 steroid dehydrogenase (CYP21) are closely linked in the region of BTA23 band 22 along with BoLA class I genes, and that male enhanced antigen (MEA) mapped between DYA and the CYP21/TNFA/HSP70.1 gene region. All BAC clones containing BoLA class I genes mapped distal to CYP21/TNFA/HSP70.1 and centromeric to prolactin (PRL). Myelin oligodendrocyte glycoprotein (MOG) was shown to be imbedded within the BoLA class I gene cluster. The cytogenetic data confirmed that the disrupted distribution of BoLA genes is most likely the result of a single large chromosomal inversion. Similar FISH results were obtained when BoLA DYA and class I BAC clones were mapped to discrete chromosomal locations on the BTA homologue in white-tailed deer, suggesting that this chromosomal inversion predates divergence of the advanced ruminant families from a common ancestor.     

Gene organisation, sequence variation and isochore structure at the centromeric boundary of the human MHC.

We have mapped and sequenced the region immediately centromeric of the human major histocompatibility complex (MHC). A cluster of 13 genes/pseudogenes was identified in a 175 kb PAC linking the TAPASIN locus with the class II region. It includes two novel human genes (BING4 and SACM2L) and a thus far unnoticed human leucocyte antigen (HLA) class II pseudogene, termed HLA-DPA3. Analysis of the G+C content revealed an isochore boundary which, together with the previously reported telomeric boundary, defines the MHC class II region as one of the first completely sequenced isochores in the human genome. Comparison of the sequence with limited sequence from other cell lines shows that the high sequence variation found within the classical class II region extends beyond the identified isochore boundary leading us to propose the concept of an "extended MHC". By comparative analysis, we have precisely identified the mouse/human synteny breakpoint at the centromeric end of the extended MHC class II region between the genes HSET and PHF1.     

Molecular cloning of bovine class I MHC cDNA

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Variation in T cell responses to ovalbumin in cattle: evidence for Ir gene control

Genetic control of immune responsiveness in cattle was investigated using an antigen-dependent T cell proliferation assay in vitro. Bovine T cell proliferative responses to ovalbumin were dependent upon major histocompatibility complex (MHC) class II molecules. Responses of an unrelated panel of animals to a limiting concentration of ovalbumin after a single immunization were compared. Two discrete patterns of response were observed. One group of animals had low or non-responses which were not significantly different from the preimmune levels. Another group of animals showed significant responses. After a second immunization the majority of low responders remained low responders. There was no significant correlation between bovine MHC class I BoLA haplotype and magnitude of response within this group of unrelated animals. However, the magnitude of the T cell responses by two half-sib family groups segregated with BoLA haplotypes inherited from the sire. In contrast no significant correlation with antibody responses in vivo could be demonstrated. We suggest that the observed variation in T cell response is linked to bovine MHC class II immune response (Ir) genes.     

A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb

ABSTRACT: BACKGROUND: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of the ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution. RESULTS: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by the DNA fingerprinting, BAC-end sequencing, and the sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826. CONCLUSIONS: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.     

A Radiation Hybrid Map of BTA23: Identification of a Chromosomal Rearrangement Leading to Separation of the Cattle MHC Class II Subregions

Bovine chromosome 23 (BTA23) contains the bovine major histocompatibility complex (MHC) and is thus of particular interest because of the role of MHC genes in immunity. Previous studies have shown cattle MHC class II genes to be subdivided into two distinct subregions separated by a variable genetic distance of 15–30 cM. To elucidate the genetic events that resulted in the present organization of the class II and other MHC genes, a framework radiation hybrid (RH) map of BTA23 was developed by testing DNA samples from a 5000 rad whole genome RH panel. Twenty-six markers were screened with an average retention frequency of 0.27, ranging from 0.14 to 0.42. Total length of the chromosome was 220 cR₅₀₀₀, with 4.1 cR₅₀₀₀/cM when compared to linkage data. Gene orders for the markers common to both the RH framework map and the consensus framework linkage map are identical. Large centiray intervals,D23S23–D23S7, DYA–D23S24andCYP21–D23S31,were observed compared to linkage distances. These data may indicate a much larger physical distance or suppression of recombination in the interval separating the class II subregions and also within the class I region than previously estimated. Comparison of 13 Type I genes conserved between BTA23 and the human homolog HSA6p suggests the occurrence of an inversion encompassing the centromeric half of the bovine chromosome, thus explaining the large distance between the bovine class IIa and IIb clusters. These results exemplify the power of RH mapping in solving problems in comparative genomics and evolution. Furthermore, noncongruence of the genetic and physical RH map distances indicates that caution must be observed in using either resource alone in searching for candidate genes controlling traits of economic importance.     

Structure and function of human transplantation antigens

Human transplantation antigens encoded in the major histocompatibility complex (MHC) region play a key role in regulating the immune responses. Here, we will describe the summary of our analyses on the structure and function of the human MHC molecules, HLA antigens as follows. 1) The genomic organization of the HLA antigen region was examined by cosmid cloning and pulsed-field gel electrophoresis technique. The HLA antigen region spans over at least 3,000 kb, and constitutes a multigene family. 2) Genetic polymorphisms in the HLA gene region were analyzed by Southern hybridization with restriction endonuclease digested genomic DNA using the class II cDNAs as probes (RFLP) and found to be tightly associated with each allo specificity. 3) The functional expression of the HLA class II gene product were observed after transfer of their cloned genes into the mouse fibroblast and human lymphocytes. 4) Narcolepsy is completely associated with HLA-DR2 Dw2, but no difference in the sequence of the DQ beta 1 domain could be found between narcoleptic and healthy individuals. This fact suggests that narcolepsy is not caused by mutation in the DQ beta gene. Based on results, it was inferred that one or both of the two Asps within the second variable region in the first domain of the DR beta chain is directly correlated with predisposition to narcolepsy.     

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors

Serological data identify a single major histocompatibility complex (MHC) class I locus in cattle. Molecular data, however, demonstrate the presence of at least two cattle MHC (BoLA) class I loci. To investigate the number of transcribed BoLA class I genes, we amplified cattle cDNA by using a single MHC class I-specific primer that hybridized to a conserved region of exon 4 and a non-specific 3 primer. Six BoLA class I cDNAs have been cloned and sequenced from a Bos taurus bull heterozygous for BoLA class I serological antigens, demonstrating the presence of a minimum of three loci. Sequence comparisons suggested that one of these cDNAs may be an unexpressed allele or the product of a nonclassical locus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U01186 and U01187.     

1D-IEF analysis of BoLA class I expression using allo-antisera reveals additional complexity

The use of the bovine allo-antisera in lymphocyte microcytotoxicity assays suggests that there is a single highly polymorphic class I product expressed by the BoLA system encoded by one locus. In contrast, biochemical techniques, such as 1D-IEF, reveal a complex pattern of bands for BoLA class I molecules from each animal. In order to understand the origins of this heterogeneity bovine allo-antisera were used in the immunoprecipitation step of 1D-IEF and the results compared with those from immunoprecipitation using the monoclonal antibody W6/32. By modifying existing protocols to include Gammabind G a range of bovine allo-antisera were used successfully to immunoprecpitate bovine MHC class I molecules. The results indicate that the bovine allo-antisera do not recognize all molecules previously assigned to BoLA class I serotypes by 1D-IEF. Furthermore, some of the allo-antisera immunoprecipitated molecules are not recognized by W6/32 and vice versa. This suggests that more than one polymorphic locus is expressed from the bovine MHC and that each allo-antiserum recognizes molecules encoded by different loci. Examination of the results also suggests the existence of linkage disequilibrium in the BoLA class I region.     

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.     

Characterization of recombination in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. The strong associations between genes within the 85-kb region from DQB1 to DRB1 are consistent with low frequency of recombination in this segment of DNA. Conversely, a lack of association between alleles of TAP1 and TAP2 (approximately 15 kb) has been observed, suggesting that recombination occurs here with relatively high frequency. Much of the HLA class II region has now been sequenced, providing the tools to undertake detailed analysis of recombination. Twenty-seven families containing one or two recombinant chromosomes within the 500-kb interval between the DPB1 and DRB1 genes were used to determine patterns of recombination across this region. SSCP analysis and microsatellite typing yielded identification of 127 novel polymorphic markers distributed throughout the class II region, allowing refinement of the site of crossover in 30 class II recombinant chromosomes. The three regions where recombination was observed most frequently are as follows: the 45-kb interval between HLA-DNA and RING3 (11 cases), the 50-kb interval between DQB3 and DQB1 (6 cases), and an 8.8-kb segment of the TAP2 gene (3 cases). Six of the 10 remaining recombinants await further characterization, pending identification of additional informative markers, while four recombinants were localized to other intervals (outliers). Analysis of association between markers flanking HLA-DNA to RING3 (45 kb), as well as TAP1 to TAP2 (15 kb), by use of independent CEPH haplotypes indicated little or no linkage disequilibrium, supporting the familial recombination data. A notable sequence motif located within a region associated with increased rates of recombination consisted of a (TGGA)12 tandem repeat within the TAP2 gene.     

Genetic and molecular mapping of the Hmt region of mouse.

We have mapped a new region of the mouse major histocompatibility complex (MHC) that contains the nuclear gene, Hmt, for the maternally transmitted antigen, Mta. The Hmt region of chromosome 17 lies between a recombinational breakpoint distal to Tla and another proximal to Tpx-1, thus including Pgk-2. A novel MHC class I gene fragment, R4B2, was cloned and mapped to this region as was another new class I gene, Thy19.4. Both lie proximal to Pgk-2, within the distal inversion in t-haplotypes. The presence of several other MHC class I genes in the Hmt region is predicted from analysis of the recombinants that define the region.     

Mixed lymphocyte culture studies reveal complexity in the bovine MHC not detected by class I serology

The genetic structure of the bovine major histocompatibility complex (MHC) was investigated using the lymphocyte microcytotoxicity test for class I typing and the mixed lymphocyte culture (MLC) assay for class II typing. Using locally produced alloantisera and antisera from the Third International BoLA Workshop, 14 class I BoLA-A locus alleles were identified in the study population, a single herd of approximately 700 Holstein-Friesian cattle. Eleven of these were alleles recognized in the International Workshop and three were new alleles. An MLC titration assay was employed in conjunction with class I typing to define BoLA haplotypes and identify BoLA complex homozygotes. An embryo transfer family consisting of eight full sibling cattle including one BoLA complex homozygote was produced by half sibling mating. Five other BoLA complex homozygotes were subsequently identified in the herd. Six MLC defined class II haplotypes investigated in detail were designated BoLA-D1, D2, D3, D4, D5 and D7. BoLA-D1 was associated with the class I specificity BoLA-Aw6, D2 with Aw6 and the new class I specificity Ac3, D3 with Aw6 and Aw11, D4 with Aw10, D5 with Aw31 and Aw11, and D7 with Aw20. The discovery of four groups of class I identical-class II disparate haplotypes, and three pairs of class I disparate-class II identical haplotypes indicates the presence of considerable complexity in the BoLA complex that is not detected using class I serology.     

Production of alloantisera against class II bovine lymphocyte antigens (BoLA) by cross-immunization between class I matched cattle

This paper describes the production of alloantisera directed against bovine major histocompatibility complex (MHC) (BoLA) class II antigens in animals whose MHC phenotypes had been defined by one dimensional isoelectric focusing. Animals of closely matched BoLA class I types were selected by serology and subsequently typed for class I and class II by 1D-IEF of immunoprecipitated antigens. Those with similar class I type by both methods, but differing at the class II locus, were chosen for reciprocal immunization. Cross-immunization was by two skin implantations 6 weeks apart. The resulting antisera showed low titre after the first immunization and elevated titre 3 weeks after the second immunization. The sera reacted strongly with cells expressing specific BoLA class II antigens. The pattern of reactivity correlated well with IEF class II typing on a panel of animals representing all of the class II IEF types present in the Friesian population.     

HLA cosmid clones show complete, widely spaced human class I genes with occasional clusters

To understand the organization of the human leukocyte antigen (HLA) gene region and its relationship to the transplantation antigens expressed at the cell surface we have isolated clones containing HLA class I genes from a cosmid library (Grosveld et al., Gene 13, 227, 1981) constructed with the DNA from an individual of defined haplotype. Most of the cosmids contain a single HLA gene in 30–40 kb of human DNA, indicating that human class I genes are rather widely spaced; two contain two genes and one contains three. Most of these genes appear to be complete; the double or multiple genes are found in the same orientation. Differences in restriction maps are evident but some common features are observed in particular in the 5' half of these genes.