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Topic:Molecular biology
Molecular biology in horses involves the study of molecular processes and genetic mechanisms that underpin equine physiology and health. This field encompasses the analysis of DNA, RNA, proteins, and other biomolecules to understand gene expression, genetic variation, and cellular functions in horses. Techniques such as genomic sequencing, gene expression profiling, and molecular diagnostics are employed to explore topics like hereditary diseases, performance traits, and immune responses in equines. This page assembles peer-reviewed research studies and scholarly articles that investigate the molecular biology of horses, focusing on genetic research, molecular techniques, and their applications in equine science.
Structure and expression of the equine infectious anemia virus transcriptional trans-activator (tat). Equine infectious anemia virus (EIAV) encodes a tat gene which is closely related to the trans-activators encoded by the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat message is composed of three exons; the first two encode tat and the third may encode rev.. Interestingly, EIAV tat translation is initiated at a non-AUG codon in the first exon of the message, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, ...
Rapid determination of methandrostenolone in equine urine by isotope dilution liquid chromatography-tandem mass spectrometry. Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundan...
Primary structure of horse serotransferrin glycans. Demonstration that heterogeneity is related to the number of glycans and to the presence of N-acetylneuraminic acid and N-acetyl-4-O-acetylneuraminic acid. Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400-MHz 1H-NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b varian...
Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein. A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane le...
Concentration and degree of polymerization of hyaluronate in equine synovial fluid. In addition to its well-known physicochemical properties, hyaluronate (HA) has recently been shown to have important biological and pathophysiologic regulatory effects on granulocytes, monocytes, fibroblasts, and endothelial cells, as well as on the healing of wounds and various joint disorders. Many of these effects depend on or are reflected in the concentration and degree of polymerization of HA. Therefore, high-performance liquid chromatography with size-exclusion column was used to characterize the concentration and degree of polymerization of HA in equine synovial fluid (SF). The mean (+...
Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers. Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers ca...
Androgen and 19-norandrogen aromatization by equine and human placental microsomes. The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone oc...
Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme. The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformatio...
Molecular confirmation of an abortigenic strain of equine herpesvirus 1 (subtype 1) in a pregnant mare study. Four pregnant mares were inoculated intranasally and/or intravenously with equine herpesvirus 1 (EHV-1), subtype 1 during the third trimester of gestation. One mare aborted on postinfection day 15, one mare delivered a sick, weak full term foal, and two mares delivered healthy, full term foals. EHV-1, subtype 1 was isolated from several tissues of the aborted fetus and from the thymus of the sick foal. DNA restriction endonuclease patterns of the recovered EHV-1 viruses were identical to those of the EHV-1 challenge strain, documenting the origin of the abortigenic viruses.
Difference in content ratio of components among horse serum transferrin variants. Each of five genetic variants of horse serum transferrin (Tf), D, F, H, O, and R, was separated into two bands by polyacrylamide gel isoelectric focusing (PAGIEF). The more acidic band, termed component a, was more abundant than the other one, termed component b, in all variants. Components a and b of TFO variant were immunologically indistinguishable from each other by double immunodiffusion test. Determination of the content ratio of component a to component b in each variant revealed that the variants were classified into two groups: one group (D, F, and H) had a relatively high ratio withi...
Isolation and characterization of equine microvascular endothelial cells in vitro. The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consiste...
Regulation of ovarian function by catecholestrogens: current concepts. Development of the ovarian follicle(s) destined for ovulation appears to be a process in which antral follicles undergo a recruitment, selection and subsequent dominance phase. Several intraovarian or autocrine/paracrine regulatory mechanisms have been evoked to explain these processes. One of these potential autocrine/paracrine regulators is a catecholestrogen, 2-hydroxy-estradiol (2-OH-E2). Evidence implicating 2-OH-E2 as an autocrine/paracrine regulator of follicular function is reviewed. Studies have shown 2-OH-E2 to be present in nanomolar concentrations in fluid of human and equine folli...
Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse. The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
Electron microscopy of gold-labeled human and equine chromosomes. We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. ...
DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene. The immediate early (IE) proteins of herpesviruses are important regulatory factors which control the expression of genes at the transcriptional level. We report the DNA sequence of the immediate early gene of the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This sequence is shown to be extremely rich in guanine and cytosine, resulting in a highly biased codon usage. The IE gene region possesses 38 open reading frames (ORFs) greater than 300 bp in length, 11 of which have coding regions of at least 100 amino acids (aa) following potential translation initiator codons. The largest ORF co...
DNA restriction analysis of equine adenovirus serotype I. The three equine adenovirus strains isolated in different locations showed a similar cleavage pattern with HindIII and the DNA homology among the strains was confirmed by Southern blot hybridization. The three strains revealed differences in cleavage patterns with BamHI, EcoRI and PstI, suggesting the presence of DNA polymorphisms among equine adenoviruses.
Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes. A segment of the ribosomal RNA gene of Schistosoma mansoni and a DNA fragment specific to Echinococcus granulosus, cloned in plasmids, have been used as DNA probes to assess the extent of genetic variability within E. granulosus and some distinct strains have been identified. The DNA analysis, involving restriction endonuclease digestion and Southern blot hybridization with the probes, did not demonstrate any significant genetic variation within the U.K. horse/dog or sheep/dog strains but confirmed the distinctiveness of the two strains shown in previous studies. The sheep/dog strain was shown...
Identification of carbohydrate-binding proteins in mammalian spermatozoa (human, bull, boar, ram, stallion and hamster) using [125I]fucoidin and [125I]neoglycoprotein probes. A protein-carbohydrate recognition system is thought to be involved in the early stages of fertilization in mammals. In this investigation carbohydrate-binding proteins have been identified in extracts of human, bull, boar, ram, stallion and hamster spermatozoa using [125I]fucoidin and [125I]neoglycoproteins (BSA-fucose and BSA-mannose) to probe Western blots. Results show that proacrosin is the major protein species recognized in extracts of human, bull, boar and ram spermatozoa. In hamster and stallion spermatozoa, carbohydrate-binding activity was associated with several low molecular weigh...
The primary structure of equine serum amyloid A (SAA) protein. The complete amino acid sequence of equine serum amyloid A (SAA) was elucidated. The protein consists of 110 amino acid residues and contains an 8-amino acid residue insertion tentatively located between positions 69 and 70, as compared with human SAA. Microheterogeneities were detected at positions 16, 44, and 59, compatible with the existence of more than one SAA gene in the horse. This corresponds to the situation in man and mouse. Pronounced homology with SAA from man and several animal species was observed, thus confirming the conserved structure of this acute phase reactant and apoprotei...
Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates. Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
Structure of the alpha 1 subunit of horse Na,K-ATPase gene. Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.
DNA polymorphism analysis of hereditary multiple exostoses in horses. Genomic DNA polymorphisms obtained by restriction fragment-length polymorphism from healthy horses and horses with hereditary multiple exostoses were analyzed. These DNA were digested by 12 restriction enzymes and were hybridized against 6 isotopically labeled oncogene probes. Hybridization was not detected with the viral oncogene, v-ras, which indicated this oncogene was absent in the equine genome. Oncogenes (c-raf-1, c-fes, c-myb, c-myc, and c-sis) were present and had similar hybridization patterns and signal intensities in DNA from healthy horses and horses with hereditary multiple exosto...
The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase. A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the ...
Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy. Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed s...
The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83. Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5'- and 3'-noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser(TRD) to Thr(TC-83) substitution in nonstructural protein (nsP) three at amino acid ...
Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies. Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 pro...
Differences between individuals in high-molecular weight glycoproteins from mammary epithelia of several species. Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human mi...
