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Topic:DNA
DNA in horses refers to the genetic material that carries the hereditary information necessary for the growth, development, functioning, and reproduction of equine species. It consists of sequences of nucleotides that encode the genetic instructions used in the development and functioning of horses. DNA analysis in horses can provide insights into genetic diversity, lineage, and breed characteristics. It is also utilized in identifying genetic disorders, understanding hereditary traits, and assisting in selective breeding programs. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and applications of DNA analysis in equine genetics and breeding.
Restriction endonuclease analysis of equine herpesvirus-1 isolates recovered in Ontario, 1986-1992, from aborted, stillborn, and neonatal foals. Ninety-two equine herpesvirus type 1 isolates were recovered from aborted, stillborn, or neonatal foals from Ontario, Canada, from 1986 to 1992. From this total, 32 strains were randomly chosen for further study. Four or 5 isolates from each winter were selected, each from a different premises, and characterized by restriction enzyme analysis using BamHI, KpnI, BglII, HindIII, and EcoRI. Additional isolates from 2 premises and from a zebra foal were also assessed. For the strains isolated in 1986 and 1989-1992, the DNA pattern of 18 strains was similar to that of type 1P (Kentucky D) for BamHI...
Genetical and physical assignments of equine microsatellites–first integration of anchored markers in horse genome mapping. Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one o...
Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and sub-specific typing. The 16S-23S RNA gene intergenic spacers of isolates of Streptococcus equi (n = 5), S. zooepidemicus (n = 5), S. equisimilis (n = 3) and S. dysgalactiae (n = 2) were sequenced and compared. There were distinct regions within the spacer, arranged in the order 1-9 for all S. equi and one S. zooepidemicus isolate and 1,2 and 4-9 for the remaining isolates. Region 4 was identical to the tRNA(ala) gene found in the 16S-23S intergenic spacers of other streptococci. Regions 1, 5, 6 and 7 had distinct variations, each conserved in different isolates. However, amongst the intergenic spacers there were d...
Nested polymerase chain reaction for detection of Ehrlichia risticii genomic DNA in infected horses. A nested polymerase chain reaction was developed for amplifying a 529-bp segment of the 16S ribosomal RNA gene of Ehrlichia risticii from equine buffy coat cells. Confirmation of identity of the amplified bands was accomplished by Southern hybridization and DNA sequencing. The study indicated a detection limit of > 10 copies of the target gene, and specificity for E. risticii as based on a panel of test rickettsiae. Ticks (Ixodes pacificus) collected in an area of northern California enzootic for equine monocytic ehrlichiosis were found to be negative for E. risticii DNA.
Partial cloning of prohibitin cDNA from canine, feline, bovine, equine, and rabbit liver mRNA by RT-PCR. Prohibitin is the protein which has an inhibitory function in cell growth, and its gene is suggested to be one of putative tumor suppressor genes. In this report, we described a partial cloning of prohibitin cDNAs from canine, feline, bovine, equine, and rabbit liver mRNAs by RT-PCR, and their homology analysis. The sequences of these RT-PCR products were compared with each other as well as those reported for human and rat. The homology in this region of prohibitin cDNA was approximately 90%, and the amino acid sequence of each RT-PCR product shared more than 95% identity. Therefore, it is con...
Gazelle herpesvirus 1: a new neurotropic herpesvirus immunologically related to equine herpesvirus 1. A herpesvirus was isolated from Thomson's gazelle (Gazella thomsoni) kept at a zoological garden in Japan during an outbreak of epizootic acute encephalitis. The virus, gazelle herpesvirus 1 (GHV-1), was serologically related to equine herpesvirus 1 (EHV-1). However, DNA fingerprints of GHV-1 were different from those of EHV-1 and other equine herpesviruses. Southern hybridization with probes of cloned BamHI fragments derived from UL and US segments of EHV-1 revealed differences in the DNA restriction profiles throughout the entire genome. Nucleotide sequences were determined for a conserved r...
Dose-response of X-irradiated human and equine lymphocytes. We have investigated and compared DNA damage and cell killing induced in human and equine lymphocytes after in vitro X-irradiation. Our data show that the cytogenetic and the lethality effects are both greater in equine lymphocytes, but that the difference is wider for lethality. The ratios between doses inducing the same effect are 1.3, 1.7 and 9.4 for the number of binucleated cells with micronuclei, micronucleus frequency in binucleated cells and DNA synthesis inhibition, respectively. The very different radiosensitivity observed for the two mammalian species encourages us to use their lymp...
Nucleotide sequence of equine MxA cDNA. A 2.6 kb cDNA species has been isolated from a cDNA library prepared from interferon-alpha stimulated equine peripheral blood leucocytes and the nucleotide sequence determined. The cDNA has a single open reading frame potentially encoding a 660 amino acid polypeptide showing a high degree of homology with known mammalian Mx proteins, including the possession of three consensus GTP-binding motifs. The protein has a calculated pI = 6.1 and in accordance with proposed nomenclature we have designated it equine MxA.
Biochemical and site-specific effects of insulin-like growth factor I on intrinsic tenocyte activity in equine flexor tendons. To examine the site-specific and dose-dependent effects of insulin-like growth factor I (IGF-I) on normal equine tendon in vitro. Methods: Superficial digital flexor tendon explants derived from a euthanatized 3-year-old horse. Methods: Explants in culture were treated with 0, 100, 250, or 500 ng of IGF-I/ml for 14 days with an end-stage radiolabel of 20 microCi of [3H]proline/ml or 5 microCi of [3H]thymidine/ml. The tendon tissues were then analyzed biochemically for hydroxyproline content by reverse-phase high-performance liquid chromatography, DNA content by fluorometry, and glycosaminoglyc...
Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1. To determine the effects of transforming growth factor-beta 1 (TGF-beta 1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes. Methods: Articular cartilage obtained from multiple joints of a 4-month-old foal. Methods: Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 x 10(6) chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-beta 1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine seru...
Distribution and relevance of equine herpesvirus type 2 (EHV-2) infections. Equine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolat...
Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes. Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was reco...
Identification, cloning and sequence analysis of the equine adenovirus 1 hexon gene. Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents. Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
Direct detection of equine herpesvirus DNA in tissues of aborted equine fetuses. Restriction endonuclease analysis of equine herpesviruses 1 (EHV-1) and 4 has been investigated using cultured cells infected with these viruses. The DNA cleavage patterns of these viruses were observed in the intracellular DNA after digestion with Eco RI and electrophoresis. This procedure was applied to the diagnosis of equine herpesvirus infection in aborted equine fetuses. The characteristic Eco RI restriction pattern of EHV-1 DNA was directly detectable in the emulsion of lungs collected from aborted equine fetuses.
A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. The melanocyte-stimulating hormone receptor gene (MC1R) is the major candidate gene for the chestnut coat color in horses since it is assumed to be controlled by an allele at the extension locus. MC1R sequences were PCR amplified from chestnut (e/e) and non-chestnut (E/-) horses. A single-strand conformation polymorphism was found that showed a complete association to the chestnut coat color among 144 horses representing 12 breeds. Sequence analysis revealed a single missense mutation (83Ser-->Phe) in the MC1R allele associated with the chestnut color. The substitution occurs in the second ...
A 19 kDa protein secreted by the endometrium of the mare is a novel member of the lipocalin family. Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 resid...
Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1). Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.
Modulation of matrix metalloprotease 13 (collagenase 3) gene expression in equine chondrocytes by interleukin 1 and corticosteroids. To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin 1 beta (rhIL-1 beta) and corticosteroids. Methods: Equine chondrocytes in monolayer culture were stimulated with rhIL-1 beta. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxi...
The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs. The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5'-CACACCCA and (b) 5'-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA gene...
Equine motor neuron disease is not linked to Cu/Zn superoxide dismutase mutations: sequence analysis of the equine Cu/Zn superoxide dismutase cDNA. The cDNA encoding the equine copper/zinc superoxide dismutase (SOD1) was cloned from leukocyte total RNA from healthy horses and its nucleotide (nt) sequence was determined. We further sequenced the SOD1 gene from 16 horses diagnosed with equine motor neuron disease (EMND) and eight unrelated, clinically normal horses to determine if this disease, similar to amyotrophic lateral sclerosis (ALS) in humans, is linked to SOD1 mutations. The 465-bp SOD1 coding region in the horse encodes 153 amino acid (aa) residues. Equine SOD1 exhibited 81.8 and 79.9% sequence identity to the human homolog at the...
Identification of an alternatively spliced transcript of equine interleukin-1 beta. Using lipopolysaccharide (LPS)-stimulated equine peripheral blood mononuclear cell (PBMC) cDNA as a template, we performed polymerase chain reaction (PCR) amplification with equine interleukin-1 beta (IL-1 beta) specific primers. Electrophoresis of the PCR product on agarose gel revealed an additional smaller fragment that hybridized with an equine IL-1 beta cDNA probe. Sequencing of this fragment demonstrated that it was shorter than normal equine IL-1 beta cDNA by 162 nucleotides, which corresponded to exon 5 of the human and murine IL-1 beta genes. The deletion of 162 nucleotides did not re...
PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA. The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes (AciI, BamHI, RsaI). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examine...
Analysis of the equine tumor suppressor gene p53 in the normal horse and in eight cutaneous squamous cell carcinomas. Wild type equine p53 was amplified between exons 2 and 9 by the polymerase chain reaction using primers designed from conserved regions in other species. An 828 base pair region, corresponding to codons 25-313 of human p53, was sequenced in both directions. Human and equine amino acid sequences were 87% homologous in this region and 96% homologous in conserved domains II-V. Of eight equine cutaneous or mucocutaneous squamous cell carcinomas directly sequenced from exons 5-8, two had p53 point mutations resulting in single amino acid substitutions.
Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts. DNA content, ploidy level, cell size and nuclear number were investigated in 54 mammalian hearts from nine species. DNA content was determined biochemically and ploidy level of cells was studied by the means of Feulgen cytophotometry. Nuclear number was calculated by a new method, while cell size was determined by using ocular micrometry. In most mammals diploid cell nuclei predominate. Higher ploidy levels were found in the human and the pig hearts. The total amount of DNA correlated with the myocardial weight. Eight million heart muscle cell nuclei were found in mice (myocardial weight 160 m...
Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus. Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR...
Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP. Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosom...
Development of a diagnostic DNA probe to detect Setaria digitata: the causative parasite of cerebrospinal nematodiasis in goats, sheep and horses. Two repetitive sequences (IpSdM and IpSdS) have been cloned and sequenced from the genome of Setaria digitata. When IpSdM (214 bp) and IpSdS (201 bp) were aligned, a high degree of homology (85%) was observed, indicating that they belong to the same family of repeats. IpSdM represents a complete repeating element while IpSdS consists of two partial repeating elements arranged in tandem. The elements are present in about 10 000 copies comprising 2.8% of the S. digitata genome. As a diagnostic probe IpSdM detects as little as 100 pg DNA of both S. digitata and S. labiato-papillosa. It can also d...
