The bovine butyrophilin gene maps to chromsome 23

Chromosomal assignment of the bovine butyrophilin gene ( BTN ) was performed by analysis of DNA from somatic hybrid cell lines using the polymerase chain reaction. The gene was assigned to bovine chromosome 23 using two sets of primers specific for bovine BTN.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

BoLA antigens are associated with increased frequency of persistent lymphocytosis in bovine leukaemia virus infected cattle and with increased incidence of antibodies to bovine leukaemia virus

The association between bovine major histocompatibility system (BoLA) type and persistent lymphocytosis in cattle with antibodies to bovine leukaemia virus was examined by comparing antigen frequencies in cattle with persistent lymphocytosis to controls matched for age, sex, breed and presence of antibodies to BLV. The cattle came from nine dairy herds in south-east Queensland, Australia; six herds were Australian Illawarra Shorthorn (AIS), two herds were Jersey and one herd was Friesian. Antigen W6 and Eu28R were more common in cattle with persistent lymphocytosis than in controls. Antigen W8 was less common in AIS cattle with persistent lymphocytosis. A study of 24 offspring from one sire, heterozygous for W10 and Eu28R, showed that offspring inheriting Eu28R from the sire were significantly more likely to have antibodies to BLV than offspring inheriting the opposing W10 haplotype.     

An MspI polymorphism at the bovine growth hormone (bGH) gene is linked to a locus affecting milk protein percentage

SSCP analysis of the bovine growth hormone (bGH) gene in Israel Holstein dairy cattle uncovered five intragenic haplotypes, denoted A to E. Of these, Haplotype E differed from the others at six fragments; one of which corresponded to the polymorphic MspI site in intron III, at which haplotype E carried the disabled MspI (-) allele. Haplotype E was observed in a single sire only, carrying haplotype A as the second bGH allele. In 523 daughters of this sire genotyped for the MspI polymorphism, heterozygous (+/-) as compared to homozygous (+/+) daughters, showed a significant increasing effect on protein percentage and kg protein per year; and a decreasing effect (P < 0.10) on milk somatic cell counts (MSSC). None of the daughters were homozygous (-/-), indicating that the frequency of this allele in the general population was essentially zero. Calculated skewness (g1) values for the two daughter groups differed significantly with (+/-) daughters showing negative skewness (in the direction of lower protein percentage), and (+/+) daughters positive skewness (in the direction of higher protein percentage). The direction of skewness in each group is indicative of the presence of a QTL having an increasing effect on milk protein percentage in coupling linkage with the MspI (-) allele in this sire, but at some distance from it. Maximum likelihood estimates of the proportion of recombination (r) between the putative QTL and bGH, and the allele substitution effect at the QTL (d), were r = 0.33, a = 0.07% protein, with standard errors 0.058 and 0.009% protein, respectively.     

Evidence for BoLA-linked resistance and susceptibility to subclinical progression of bovine leukaemia virus infection

The role of the bovine major histocompatibility complex in bovine leukaemia virus (BLV) infection and disease progression was investigated in a herd of Shorthorn cattle (n = 117). The frequency of cows that were seropositive to BLV-glycoprotein antigen was 51%. Twenty-three per cent of the seropositive cows were lymphocytotic. At the herd level, relative resistance to BLV-dependent B-cell proliferation and lymphocytosis among seropositive cows was associated with bovine lymphocyte antigen (BoLA)-DA7, whereas susceptibility was associated with BoLA-DA12.3. These associations were also confirmed at the family level, where BoLA phenotypes were used as haplotypic markers. Among the offspring of one BoLA-heterozygous sire (n = 33), resistance segregated with the DA7 haplotype and susceptibility with the DA12.3 haplotype. In this sire group, maternal transmission of the BoLA-w8 allele was associated with increased susceptibility to B-cell proliferation and lymphocytosis in w8/DA12.3 heterozygotes. These data provide the first evidence that subclinical progression of BLV infection is under the control of the BoLA complex, and suggest that the BoLA system can be used to select for resistance to B-cell proliferation and the development of lymphocytosis in BLV-infected herds.     

The bovine B and C blood group systems are not likely to be the orthologues of human RH: an interesting twist in the comparative map

We tested the hypothesis that either the bovine B or C blood group system is the orthologue of human RH. A comparative linkage mapping strategy was applied, using blood typing and restriction fragment length polymorphism (RFLP) analysis of four loci linked to RH on HSA1; PGD, FGR, ALPL and FUCA1. Four sires with a total of 255 half-sib offspring were used for the linkage analysis. Strong support for linkage between ALPL, FUCA1 and FGR was obtained for all sire families (lod scores >11 for all pairwise comparisons). This new linkage group was assigned to bovine synteny group U17 based on previous somatic cell mapping of the FGR locus. The most favoured order is ALPL—FUCA1—FGR (2·18:1), with ALPL and FGR 5·4 cm and 6·3 cm , respectively, from FUCA1. The B and C blood group systems and PGD were genetically independent of each other and all other markers, indicating that neither B nor C is likely to be the bovine orthologue of human RH. However, given available comparative mapping data, there is some chance that the bovine orthologue of RH is on bovine synteny group U6. Although gene order appears to be conserved with humans, the differences in recombination rates between these three loci in cattle, humans and mice strongly suggest that it is not possible to use human map distances to predict map distances in cattle, making it imperative that bovine gene mappers continue to emphasize adding type I markers to the bovine linkage map.     

Restriction fragment length polymorphism in amplification products of the bovine PIT1 gene and assignment of PIT1 to bovine chromosome 1

A polymorphism was identified in the bovine PIT1 gene by digesting polymerase chain reaction (PCR) products with the restriction enzyme Hinfl . This polymorphism was segregating in five diverse breeds of cattle. PIT1 was sublocalized to the centromeric region of bovine chromosome 1 by linkage analysis.     

PCR-RFLP typing of the bovine myoglobin gene

We have identified substitutions in the 3₁ untranslated region of the bovine myoglobin gene, one of which affects an MboII restriction enzyme site resulting in a bi-allelic restriction fragment length polymorphism. Co-dominant inheritance of the alleles in three reference families was observed using a polymerase chain reaction—restriction fragment length polymorphism assay. The distribution of the alleles seems characteristic of cattle type—one of the alleles was not detected in purely taurine breeds. Furthermore, we mapped, using the polymerase chain reaction on a bovine–rodent somatic cell hybrid panel, the myoglobin gene to bovine chromosome five. It is therefore syntenic with γ-interferon and insulin-like growth factor in which we have not found polymorphism. The myoglobin locus therefore serves as a type one marker on bovine chromosome five.     

Somatic cell mapping, polymorphism, and linkage analysis of bovine prolactin-related proteins and placental lactogen.

The bovine prolactin gene family includes novel members expressed in the fetal placenta that are distinct from placental lactogen. In this study, we investigated the genetic organization of four members of this gene family (PRP1, PRP3, PRP6, and PRP10) as well as placental lactogen (PL). Using a bovine-rodent hybrid somatic cell panel, all five genes were assigned to bovine chromosome 23, which contains prolactin and the major histocompatibility group (BOLA). Restriction fragment length polymorphisms were detected by all probes in breeding populations with the restriction enzyme MspI, whereas no polymorphisms were detected with BamHI. EcoRI, HindIII, TaqI, and PstI produced polymorphic fragments with some but not all of the probes tested. A PRP10 polymorphism, which is apparently the result of a insertion/deletion event, detected polymorphism frequency differences between Bos indicus and Bos taurus. No recombinational events were observed with these probes and prolactin using linkage analysis involving 91 American Holsteins. The bovine prolactin gene family was incorporated into a linkage group containing CYP21. Our studies demonstrate that members of the bovine prolactin gene family have a close physical association with each other, and all members demonstrate genetic variability in the breeding population.     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

Antibody cross-reactivity between myelin oligodendrocyte glycoprotein and the milk protein butyrophilin in multiple sclerosis

The etiology of multiple sclerosis (MS) is believed to involve environmental factors, but their identity and mode of action are unknown. In this study, we demonstrate that Ab specific for the extracellular Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) cross-reacts with a homologous N-terminal domain of the bovine milk protein butyrophilin (BTN). Analysis of paired samples of MS sera and cerebrospinal fluid (CSF) identified a BTN-specific Ab response in the CNS that differed in its epitope specificity from that in the periphery. This effect was statistically significant for the Ab response to BTN(76-100) (p = 0.0026), which cosequestered in the CSF compartment with Ab to the homologous MOG peptide MOG(76-100) in 34% of MS patients (n = 35). These observations suggested that intratheccal synthesis of Ab recognizing BTN peptide epitopes in the CNS was sustained by molecular mimicry with MOG. Formal evidence of molecular mimicry between the two proteins was obtained by analyzing MOG-specific autoantibodies immunopurified from MS sera. The MOG-specific Ab repertoire cross-reacts with multiple BTN peptide epitopes including a MOG/BTN(76-100)-specific component that occurred at a higher frequency in MS patients than in seropositive healthy controls, as well as responses to epitopes within MOG/BTN(1-39) that occur at similar frequencies in both groups. The demonstration of molecular mimicry between MOG and BTN, along with sequestration of BTN-reactive Ab in CSF suggests that exposure to this common dietary Ag may influence the composition and function of the MOG-specific autoimmune repertoire during the course of MS.     

Populationwide Investigation of Two Indel Polymorphisms at the Prion Protein Gene in Polish Holstein�CFriesian Cattle

The allele, genotype, and haplotype frequencies among 837 Polish Holstein-Friesian cattle were determined at two regulatory indel polymorphisms of the PRNP gene. Allele frequencies at the 23 bp indel promoter polymorphism were 0.622 (del) and 0.378 (ins), with 0.613 and 0.387 in sires and 0.633 and 0.366 in dams. Allele frequencies at the 12 bp indel intron polymorphism were 0.527 (del) and 0.473 (ins), with 0.529 and 0.471 in sires and 0.543 and 0.456 in dams. Four haplotypes were identified in this population (23-12del, 23-12ins, 23del-12ins, and 23ins-12del). Haplotype 23-12del occurred most frequently in both sire and dam groups. Comparative analysis of Polish Holstein-Friesian and German Holstein populations revealed a similar genetic structure for the 23 bp indel polymorphism and a significantly different one for the 12 bp indel polymorphism. In allele and haplotype analysis, significant differences were observed between the Polish Holstein-Friesian population and a BSE-free German Holstein population.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

The fractionation and characterization of bovine tear proteins,especially lactoferrin

Summary Gentle procedures are described for the fractionation of bovine tear fluid by a combination of centrifugation, salt precipitation, gel filtration and ion exchange chromatography. Fractions are examined by gel electrophoretic and immunological methods. Reference patterns are compiled and compared with bovine milk and serum patterns. Properties of some of the components are determined. Lactoferrin is isolated in two separate, but closely related, fractions. A nacidic protein with a molecular weight of 23 000 daltons, is also isolated. Distinct heterogeneity is observed between individual animals, suggestive of a genetic polymorphism. A method is presented for the determination of the antibacterial activity of tear fluid and its fractions.     

Linkage mapping of the locus responsible for forelimb-girdle muscular anomaly of Japanese black cattle on bovine chromosome 26

Forelimb-girdle muscular anomaly is an autosomal recessive disorder of Japanese black cattle characterized by tremor, astasia and abnormal shape of the shoulders. Pathological examination of affected animals reveals hypoplasia of forelimb-girdle muscles with reduced diameter of muscle fibres. To identify the gene responsible for this disorder, we performed linkage mapping of the disorder locus using an inbred pedigree including a great-grand sire, a grand sire, a sire and 26 affected calves obtained from a herd of Japanese black cattle. Two hundred and fifty-eight microsatellite markers distributed across the genome were genotyped across the pedigree. Four markers on the middle region of bovine chromosome 26 showed significant linkage with the disorder locus. Haplotype analysis using additional markers in this region refined the critical region of the disorder locus to a 3.5-Mb interval on BTA26 between BM4505 and MOK2602 . Comparative mapping data revealed several potential candidate genes for the disorder, including NRAP , PDZD8 and HSPA12A , which are associated with muscular function.     

Restriction fragment length polymorphism of bovine lysozyme genes

Genetic polymorphism of bovine lysozyme (LYZ) genes was investigated by analysing restriction fragment length polymorphism (RFLP). The analysis revealed three RFLP loci designated LYZ1, LYZ2 and LYZ2. Each system included two or three allelic variants. Evidence for close genetic linkage of the three loci was found. There was also a significant linkage disequilibrium among the three loci in a sample of about 200 breeding bulls from one breed. No statistically significant association was found between LYZ RFLPs and breeding values of bulls for disease or milk production traits.     

Genetic mapping of bovine T-cell receptor complex loci

Thymic-derived lymphocytes (T cells) recognize and respond to antigens through the mediation of the T-cell receptor complex. Here we report polymorphism at each of the five loci that encode the different components of the T-cell receptor complex in cattle. These genes were mapped on the bovine genome by genetic linkage analysis in the International Bovine Reference Panel (IBRP). These mapping data provide additional type I markers for linking the bovine genetic map with the human and mouse maps and also permit investigation of the effect of T-cell receptor polymorphism on immune responsiveness and disease susceptibility.     

A QTL affecting milk yield and composition maps to bovine chromosome 20: a confirmation

As part of a whole genome scan undertaken to detect quantitative trait loci (QTL) affecting milk yield and composition, we have genotyped a granddaughter design comprising 1152 sons for six microsatellite markers spanning bovine chromosome 20. An analysis performed across families provided strong evidence (experiment-wise P -values < 0·01) for the presence of a QTL affecting primarily protein percentage towards the telomeric end of the chromosome. A founder sire, shown in a previous study to segregate for a similar QTL in the corresponding chromosome region, was characterized by 29 and 57 sons and maternal grandsons, respectively, in the present design. Sorting corresponding sons and grandsons by paternal or grandpaternal allele provided significant evidence for the segregation of a QTL on chromosome 20. Altogether these results confirm the location of a QTL affecting milk production on bovine chromosome 20.     

Identification of a DNA methylation point in the promoter region of the bovine CYP21 gene

The CYP21 (steroid 21-hydroxylase) gene is involved in the synthesis of steroid hormones. Bov-A2 is a retroposon that is common in ruminant genomes. The promoter region of bovine CYP21 contains a short interspersed nucleotide element of Bov-A2, which overlaps a putative Sp1 binding site. We looked for RFLP/HpaII polymorphism in the Bov-A2 element in bovine Zebu breeds by PCR-RFLP, and examined whether polymorphism in this element is associated with methylation. Among DNA samples from 135 Brazilian Zebu breed cattle, we identified an RFLP/HpaII polymorphism (T/C), which, based on a restriction methylation-sensitive assay employing HpaII and isoschizomer MspI enzymes (methylation-sensitive and -non-sensitive enzymes, respectively), appears to be a DNA methylation point. This is the first report of this polymorphism and on DNA methylation in the bovine CYP21 promoter region in Brazilian Zebu cattle.