Assignment of eight loci to bovine syntenic groups by use of PCR: extension of a comparative gene map

The polymerase chain reaction (PCR) has been combined with hybrid somatic cell technology to extend the bovine physical map. Eight bovine loci—glycoprotein hormone alpha (CGA), coagulation factor X (F10), chromogranin A (CHGA), low-density lipoprotein receptor (LDLR), human prochymosin pseudogene (CYM), oxytocin (OXT), arginine-vasopressin (ARVP), and cytochrome oxidase c subunit IV pseudogene (COXP)—were assigned to bovine syntenic groups with this approach. CGA was assigned to bovine syntenic group U2, F10 to U27, CHGA to U4 [bovine Chromosome (Chr) 21], LDLR to U22, CYM to U6, OXT and ARVP to U11, and COXP to U3 (bovine Chr 5). Seven of these genes, CGA, F10, CHGA, LDLR, OXT, ARVP, and CYM, further delineate regions of chromosomal conservation on human Chrs 6, 13, 14, 19, 20, 20, and 1, respectively. CHGA, OXT, and ARVP are unmapped in the mouse. Comparative mapping predicts the mouse CHGA will map to Chr 12, and mouse OXT and ARVP will map to mouse Chr 2. Furthermore, human CYM is predicted to be sublocalized to 1p32-q21. The primers developed for these eight loci will be useful for the development of hybrid somatic cell panels in the future as well as establishing a collection of bovine expressed sequence tags.     

Study on the Relationship Between TSHR Gene and Thyroid Diseases

Thyroid stimulating hormone receptor (TSHR) is thought to play a critical role in the pathogenesis of certain thyroid diseases, including Graves' disease (GD), multinodular thyroid goiter (MTG), and Hashimoto's thyroiditis (HT). In order to understand whether single nucleotide polymorphisms in the TSHR gene contribute to thyroid diseases, we have conducted a case-control study in which, we examined 8 TSHR gene single-nucleotide polymorphisms in introns 1, 4, 5, 6 and exons 7 and 8, respectively, among patients with thyroid diseases. These included one family with GD (3 patients and 9 healthy members); 60 patients with familiar thyroid diseases (30 with GD, 20 with MTG, and 10 with HT patients), 48 sporadic patients with GD and 96 healthy control individuals. Direct sequencing of all 10 exons and part of introns of TSHR gene, in these patients as well as healthy controls revealed eight polymorphisms. A novel polymorphism in exon 8 AGA(Arg) → CGA(Arg). However, there were no significant differences between patients and controls in the incidence of these polymorphisms. These results suggest that the polymorphisms (polymorphism in intron 1 at 81 bp upstream of exon 2; polymorphism in intron 4 at 135 bp upstream of exon 5; polymorphism in intron 4 at 365 bp upstream of exon 5; polymorphism in intron 5 at 69 bp upstream of exon 6; means polymorphism in intron 6 at 13 bp downstream of exon 6; polymorphism in intron 6 at 187 bp upstream of exon 7; E7+16: polymorphism in 16 bp of exon 7; polymorphism in 40 bp of exon 8) of the TSHR gene may not contribute to the pathogenesis of thyroid diseases.     

Polymorphisms in follicle stimulation hormone receptor, inhibin alpha, inhibin bata A, and prolactin genes, and their association with sperm quality in Chinese Holstein bulls

Candidate genes follicle stimulation hormone receptor (FSHR), inhibin alpha (INHA), inhibin bata A (INHBA) and prolactin (PRL) were investigated for their association with sperm quality traits of semen volume per ejaculate (VOL), sperm concentration (SCON), motility (MOT), sperm motility in frozen semen (FMOT), acrosome integrity rate (AIR) and abnormal sperm rate (ASR) in a total of 123 normal mature Holstein bulls. Three reported single nucleotide polymorphisms (SNPs) of FSHR A-234500T (rs43676359), IHNA A192G (rs41257116), and IHNBA C7639T (rs43408735), and 3 novel SNPs (G7550A, C7661T, and T8370C) in exons 4 and 5 of bovine PRL gene (NC 007324) were analyzed. Analysis of variance revealed that FSHR A-234500T and INHBA C7639T polymorphisms significantly associated with VOL (P<0.05) and SCON (P<0.05), and the polymorphism of INHBA C7639T also had significant effects on MOT (P<0.05). Furthermore, the INHA A192G polymorphism significantly associated with AIR (P<0.05). No significant differences were found between the polymorphisms of PRL gene and sperm quality traits.     

Sequence polymorphism in the bovine major histocompatibility complex DQB loci

Polymorphism in DQB sequences of the bovine major histocompatibility complex was investigated in 22 British Friesian cattle. The first domain exon was amplified, cloned and sequenced. Eight different sequences were identified, six of which had not been identified previously. The high proportion of novel sequences suggests that additional polymorphisms within the DQB loci remain to be discovered in this breed. One sequence was present in at least 21 of the 22 cattle. This sequence, or a closely related sequence, has also been found in American Holstein Friesian, Swedish Red and White and Japanese Black cattle. The remarkably high sequence conservation suggests that the bovine DQB region may contain a locus with a low level of polymorphism and be more similar to the human DQB region than previously supposed. One sequence with three widely spaced frameshift insertions appeared to be a pseudogene.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.     

Two α(1,2) fucosyltransferase genes on porcine Chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci

The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the α(1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307–ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined. Received: 17 February 1997 / Accepted: 30 May 1997     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type I loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type I loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere- PRNP-(SODIL/AVP/OXT)-(BL42/GNAS1)-HCK-CSSM30 . The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.     

Absence of the functional Myosin heavy chain 2b isoform in equine skeletal muscles

Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b genes on the phylogenetic tree of the mammalian MyHC genes. These results suggest that an ancestral MyHC-2b gene had lost its function and changed to a pseudogene during the course of horse history. The MyHC genes in some ungulates were analyzed through the PCR amplifications using the MyHC isoform-specific primers to confirm the presence of the MyHC-2b and -2x genes. The exon coding the 3' untranslated region of the MyHC-2x was successfully amplified from the all ungulates examined; however, that of the MyHC-2b gene was amplified only from horses, pigs and lesser mouse deer. The PCR analyses from rhinoceros, sika deer, moose, giraffes, water buffalo, bovine, Japanese serow and sheep genes implied the absence of the MyHC-2b-specific sequence in their genomes. These results suggest that the MyHC-2b gene independently lost its function in some ungulate species.     

Demonstration of oxytocin release by bovine luteal cells utilizing the reverse hemolytic plaque assay.

Corpora lutea (CL) of a number of species produce oxytocin (OXT). In the present experiments we studied basal, prostaglandin (PG) F2 alpha-stimulated and ascorbate-stimulated OXT release from individual bovine luteal cells utilizing the reverse hemolytic plaque assay (RHPA). Using a mixture of C- and N-terminus-specific antisera against OXT, we were able to demonstrate OXT plaque formation by individual luteal cells. CL consist of two steroidogenic cell types: large luteal cells (LLC), believed to derive from granulosa cells and to produce and secrete OXT, and small luteal cells (SLC), thought to derive from theca cells. To distinguish between these two cell types, we designated cells greater than 20 microns as LLC and those less than 20 microns as SLC. On the basis of this morphological parameter, OXT release from both LLC and SLC was demonstrable. After an incubation period of 15 h, 7% of both cell types formed OXT plaques. PGF 2 alpha and ascorbate increased the size of plaques surrounding both LLC and SLC to more than 200% and 240%, respectively (basal plaque size = 100%). The number of plaque-forming cells increased only slightly in the presence of either PGF 2 alpha or ascorbate in comparison to basal conditions. We suggest that the RHPA can be used to demonstrate peptide release from luteal cells. It is concluded that LLC may be subdivided into functional subclasses because less than 10% of bovine luteal cells release OXT. Known OXT secretagogues increased the amount of OXT released. It appears that not only LLC but also SLC secrete this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.     

Mutation analysis reveals an insertional hotspot in exon 4 of the LDL receptor gene

Mutation analysis of the low density lipoprotein receptor (LDLR) gene revealed a novel 8-bp duplication after nucleotide 681 in a Costa Rican patient with familial hypercholesterolaemia. The frameshift caused by this mutation results in a premature termination codon in the EGF precursor homology domain of the mature LDLR, whereby a truncated protein of the first 206 residues with an additional 39 abnormal residues would be created. The insertion overlaps with previously described duplications of 18 bp and 21 bp, thus revealing an insertional hotspot in exon 4 of the LDLR gene. We propose that the structural features of this region of the LDLR gene contribute significantly to genetic instability and the subsequent DNA duplication via an endogenous sequence-directed mechanism of mutagenesis. Received: 10 November 1995 / Revised: 15 March 1996     

DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of DNA at seven loci of the nuclear genome]

Seven DNA markers from five genes and one chromosomal region were analysed in Mongolian population using the polymerase chain reaction. The frequencies of alleles of the polymorphisms detected with HindIII in the HBG-2, AvaII in the HBB, MspI and XbaI in the Apo-B, PstI in the D7S8, HincII in the LDLR and allele frequency of the minisatellite fragment in the AT-3 have been determined. The results of the RELP for Apo-B(MspI), LDLR, D7S8 and AT-3 are obtained for the first time among Mongoloids. DNA markers studied demonstrated high level of polymorphisms in the population of Mongolia, except for XbaI and MspI restriction sites at the Apo-B locus. The data obtained for Mongolian population and the literature data were compared.     

ZBTB38 gene polymorphism associated with body measurement traits in native Chinese cattle breeds

Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. In this study, we investigated the association between genetic polymorphisms of the zinc finger and BTB domain containing 38 gene (ZBTB38) and body measurement traits in native Chinese cattle. Using direct DNA sequencing in 423 individuals of 8 different cattle subpopulations, 9 novel polymorphisms were identified for genotyping within 647 bp region of exon 1 of ZBTB38. Linkage disequilibrium and association analysis revealed that two coding exon polymorphisms (2323 G>A and 2325 C>T polymorphisms), missense mutations valine GTC(T) to isoleucine ATC(T), were associated with body length (BL), withers height (WH) and rump length (RL). Furthermore, the analysis of 2323 G>A and 2325 C>T SNP markers shows that there are significant effects on the BL (P = 0.0488), WH (P = 0.0044) and RL (P = 0.0314) in the total population. These results clearly suggest that the ZBTB38 gene is among the target genes for body measurement traits in bovine breeding, and provide data for establishment of an animal model using cattle to study big animal body type.     

Linkage relationships among 20 genetic markers in cattle. Evidence for linkage between two pairs of blood group systems: B-Z and S-F/V respectively

Five bovine paternal half-sib pedigrees for a total of 527 individuals were typed for six blood group systems: A, B, F/V, L, S, Z; for nine biochemical polymorphisms: ADA, MPI, PGM-3(slow), NP, Gc, Pi2, Tf, Ptf1 and Ptf2; and for restriction fragment length polymorphisms at five autosomal loci: Tg, GH, LDLr, BoLA-DQ and BoLA-DY. Two of the pedigrees were informative for segregation at the 'muscular hypertrophy' locus, and one was informative at the coat colour determining 'roan' locus. Linkage analysis was performed between all markers. Linkage was demonstrated between the S and F/V blood group systems (z = 3.11), adding one locus to the previously identified linkage group VII (LGVII) [Pi-2 and S], the most likely order being Pi2-S-F/V with maximum likelihood recombination rates of 0.208 and 0.211. Also shown to be linked were the blood group systems B and Z (z = 5.7, theta = 0.245). We confirmed the observation previously made by Andersson et al. (1988) of a high recombination rate between class II genes DQ and DY, suggesting either a larger physical distance between those genes than expected from comparative data, or the presence of a 'recombinational hotspot' in the bovine major histocompatibility complex. No linkage was found either with the 'muscular hypertrophy' locus, or with the 'roan' locus. However, these two loci could be excluded from respectively 1.7 and 2.5 Morgans of the bovine genome.