Genetic polymorphism of a bovine t-complex gene (TCP1) linkage to major histocompatibility genes

Southern blot analysis of genomic cattle DNA was carried out using murine cDNA probes representing the Tcp-1 gene of the t complex. Excellent cross-hybridization was obtained, and the probes apparently hybridized to at least two bovine TCP1 genes. Two independent restriction fragment length polymorphisms, each composed of two allelic variants, were detected; the inheritance of the restriction fragment length polymorphisms was confirmed by family data. One of the restriction fragment length polymorphisms, designated TCP1B, was evidently due to a gene duplication and was revealed with any restriction enzyme used. The duplication was found in three different cattle breeds investigated. Family segregation data indicated that TCP1B is linked to major histocompatibility complex genes. The result was consistent with close linkage to the major histocompatibility complex class II DO beta gene, whereas a fairly high recombination frequency was indicated between TCP1B/DO beta and other major histocompatibility complex genes. The result assigns TCP1B to a bovine linkage group previously comprising major histocompatibility complex class I and class II genes and blood group locus M. The similarity between this linkage group and parts of mouse chromosome 17 (t-H-2) and human chromosome 6 (TCP1-HLA) is discussed.     

Cloning of the bovine major histocompatibility complex class II genes

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Comparative genetic study of group B streptococcal strains of human and bovine origin

The presence and restriction fragment length polymorphism (RFLP) of DNA fragments hybridizing with virulence and "house keeping" gene probes were analyzed for 87 group B streptococcal (GBS) strains of human and bovine origin. Most characteristics obtained for bovine strains were similar when compared with those for human strains. The most significant degree of RFLP was discovered for the sizes of HindIII fragments containing bca gene. Human GBS strains with bac gene, encoding beta antigen with IgA binding capacity, were characterized by almost identical complex hybridization patterns with multiple gene probes. At the same time bac gene was not found among bovine GBS strains. Gene scpB that encodes C5a peptidase in all human GBS strains was detected only in 9 of 39 strains of bovine origin. These two characteristics effectively distinguished bovine GBS strains from GBS strains of human origin.     

Physical assignment of the bovine MHC class IIa and class IIb genes.

Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date. The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain. We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13. These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.     

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.     

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.     

First domain encoding sequence mediates human class II beta-chain gene cross-hybridization

HLA class II molecules are surface heterodimers which are essential in the initiation of immune responses. The amount of polymorphism expressed by the different class II molecules is largely dependent on the polymorphic structure of their beta chains. Cross-hybridization between, class II beta genes frequently hampered restriction fragment length polymorphism analysis of donor genomic DNA. In this report we show that the cross-hybridization between human class II beta genes is mediated by a region of high homology, rich in C and G residues, between the first domain encoding sequences of DP, DQ, and DR genes. The removal of the DNA segment containing this region from the fragments used as labeled probes against the corresponding fragments of the genes at other loci or against endonuclease digested genomic DNA completely eliminated or drastically reduced the cross-hybridization. Also, the RFLP patterns generated with the shortened probes were more informative and much simpler to interpret than were these generated with probes made from the original genes.     

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.     

The relationship between bovine major histocompatibility complex class II polymorphism and disease studied by use of bull breeding values

The predictive value of class II DQ and DYA polymorphisms of the bovine major histocompatibility (MHC) complex (BoLA) for the incidence of disease in dairy cattle was estimated in a sample of 196 progeny-tested AI bulls of the Swedish Red and White breed. The BoLA DQ and DYA types of the bulls were determined by analysing restriction fragment length polymorphisms (RFLPs). Breeding values of bulls for clinical mastitis, all diseases including clinical mastitis and diseases other than clinical mastitis were used as measures of disease resistance or susceptibility. The relationship between MHC polymorphism and bull breeding values for disease resistance was evaluated statistically by linear regression analysis. A significant association between the haplotype DQ1A and susceptibility to clinical mastitis was revealed. No other DQ haplotype nor the DYA locus has a significant effect on any of the disease traits studied.     

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.     

Gene duplications and sequence polymorphism of bovine class II DQB genes

The genetic diversity of bovine class II DQB genes was investigated by polymerase chain reaction amplification and DNA sequencing. The first domain exon was amplified from genomic DNA samples representing 14 class II haplotypes, defined by restriction fragment length polymorphism (RFLP) analysis. The presence of a polymorphism in the copy number of DQB genes was confirmed since two DQB sequences were isolated from certain haplotypes. Four subtypes of bovine DQB genes were found. DQB1 is the major type and was found in almost all haplotypes. DQB2 is very similar to DQB1 but was found only in the duplicated haplotypes DQ9 to 12. DQB3 and DQB4 are two quite divergent genes only present in certain duplicated haplotypes. The bovine DQB complexity thus resembles that in the human DRB region. Bovine DQB genes were found to be highly polymorphic as ten DQB1 alleles and four DQB2 alleles were identified. The observed sequence polymorphism correlated well with previously defined DQB RFLPs. Bovine and human DQB alleles show striking similarities at the amino acid level. In contrast, the frequency of silent substitutions is much higher in comparisons of DQB alleles between species than within species ruling out the possibility that any of the contemporary DQB alleles have been maintained since the divergence of humans and cattle. The frequency of silent substitutions between DQB alleles was markedly lower in cattle than in humans, in agreement with a previous comparison of human and bovine DRB alleles.     

Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes.

Synthetic nonadecanucleotides complementary to the human beta A-, beta S-, or beta C-globin sequences were used as hybridization probes to screen human genomic DNA samples for these genes. The oligonucleotides were 32P-labeled and used as probes to genotype restriction endonuclease digests of human genomic DNA. The data obtained show that hybridization with oligonucleotide probes, unlike restriction fragment length polymorphism (RFLP) analysis or direct restriction enzyme digestion, can be used to directly distinguish among the three alleles of beta-globin, beta A, beta S, and beta C, when present either in one (heterozygous) or two copies.     

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.     

Genomic analysis of the major bovine milk protein genes.

The genomic arrangement of the major bovine milk protein genes has been determined using a combination of physical mapping techniques. The major milk proteins consist of the four caseins, alpha s1 (CASAS1), alpha s2 (CASAS2), beta (CASB), and kappa (CASK), as well as the two major whey proteins, alpha-lactalbumin (LALBA) and beta-lactoglobulin (LGB). A panel of bovine X hamster hybrid somatic cells analyzed for the presence or absence of bovine specific restriction fragments revealed the genes coding for the major milk proteins to reside on three chromosomes. The four caseins were assigned to syntenic group U15 and localized to bovine chromosome 6 at q31-33 by in situ hybridization. LALBA segregated with syntenic group U3, while LGB segregated with U16. Pulsed-field gel electrophoresis confirmed genetic mapping results indicating tight linkage of the casein genes. The four genes reside on less than 200 kb of DNA in the order CASAS1-CASB-CASAS2-CASK. Multiple restriction fragment length polymorphisms were also found at the six loci in three breeds of cattle.     

Polymorphism at the ovine major histocompatibility complex class II loci

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) ( MhcOvar ) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQAP had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.     

A comparison of vertebrate interferon gene families detected by hybridization with human interferon DNA

Cloned human interferon complementary DNAs were used as hybridization probes to detect interferon alpha and beta gene families in restriction endonuclease digests of total genomic DNA isolated from a wide range of vertebrates and invertebrates. A complex interferon-alpha multigene family was detected in all mammals examined, whereas there was little or no cross-hybridization of human interferon-alpha complementary DNA to non-mammalian vertebrates or invertebrates. In contrast, human interferon-beta complementary DNA detected one or two interferon-beta genes in all mammals tested, with the exception of the cow and the blackbuck, both of which possessed a complex interferon-beta multigene family which has presumably arisen by a recent series of gene duplications. Interferon-beta sequences could also be detected in non-mammalian vertebrates ranging from birds to bony fish. Detailed restriction endonuclease mapping of DNA sequences neighbouring the interferon-beta gene in a variety of primates indicated a strong evolutionary conservation of flanking sequences, particularly on the 3' side of the gene.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

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.     

Genomic<Emphasis Type="Italic"> Eco</Emphasis>RI polymorphism and cosmid sequencing reveal an insertion/deletion and a new<Emphasis Type="Italic"> IGLV5</Emphasis> allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV)

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.