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 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...
Nonequivalence of the two subunits of horse erythrocyte glutathione transferase in their reaction with sulfhydryl reagents. Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activit...
Repetitive DNA probes for the detection of Babesia equi. This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigemina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively.
Synthesis and aromatization of 19-norandrogens in the stallion testis. The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost ...
Surface antigens on equine sarcoid cells and normal dermal fibroblasts as assessed by xenogeneic antisera. To characterise the expression of surface antigens on equine sarcoid cells compared to normal equine fibroblasts, immune sera were produced in rabbits against transformed cells of a virus-containing sarcoid cell line (Mc-1) and normal dermal fibroblasts, respectively. The specificities of the sera were analysed by antibody-dependent cellular cytotoxicity against 51Cr-labelled target cells using human lymphocytes as effector cells. Anti-Mc-1 antiserum induced strong cytotoxicity against transformed cells of two sarcoid cell lines (Mc-1 and Bay Mc-1), whereas the cytotoxicity against transformed...
Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen. The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)
Purification of chicken liver ferritin by two novel methods and structural comparison with horse spleen ferritin. Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results ...
Comparison of partial amino acid sequences of two protamine 2 variants from stallion sperm. Structural evidence that the variants are products of different genes. Protamine 1 and two protamine 2 variants were isolated from stallion sperm and separated by acetic acid-urea gel electrophoresis. After electroblotting onto polyvinyldifluoride filters, their amino-terminal amino acid sequences were determined by pulse-liquid peptide sequencing. The sequences of the two protamine 2 variants are homologous but slightly different in length and amino acid composition and indicate for the first time the existence of two different genes for this protamine species.
A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane. Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited p...
A human minisatellite sequence reveals DNA polymorphism in the equine species. Southern blot analysis of equine DNA's digested with the restriction endonuclease Hinfl hybridized with a 32 PdCTP labeled human VNTR probe revealed a highly polymorphic pattern of restriction fragments upon autoradiography. The horses were unrelated individuals of the quarter horse breed. This heterologous probe can thus be used in the equine species for individual identification and pedigree analysis.
Identification and nucleotide sequence of a gene in equine herpesvirus 1 analogous to the herpes simplex virus gene encoding the major envelope glycoprotein gB. A gene in equine herpesvirus 1 (EHV-1; equine abortion virus) equivalent to the gB glycoprotein gene of herpes simplex virus (HSV) has been identified by DNA hybridization and nucleotide sequencing. A 4.3 kbp EHV-1 PstI-ClaI sequence (0.40 to 0.43 map units) contained an open reading frame flanked by appropriate control elements and was capable of encoding a polypeptide of 980 amino acids. This had 50 to 60% identity over a 617 amino acid conserved region with the gB gene products of HSV and three other alphaherpesviruses, and 20 to 30% identity with those of human cytomegalovirus and Epstein-...
Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy. It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to lo...
Crystallization and preliminary X-ray study of horse pancreatic lipase. Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content...
The structure and properties of horse muscle acylphosphatase in solution. Mobility of antigenic and active site regions. The solution structure of acylphosphatase determined by proton nuclear magnetic resonance spectroscopy is described. The results allow us to discuss the fold of the protein (101 amino acids), to correlate the exposure and the mobility of the backbone with the antigenicity, and to locate the active site.
Physical mapping of the genomic heterogeneity of isolates of equine herpesvirus 2 (equine cytomegalovirus). The BamHI, EcoRI, and HindIII physical maps of the genomes of 14 isolates of equine herpesvirus 2 (EHV 2) were determined by Southern blot analysis using DNA fragments of a previously mapped EHV 2 strain 86/67. No two isolates had identical maps for all 3 enzymes, the number of differing cleavage sites between pairs of isolates varying from 3 to 21. Overall 75 cleavage sites were mapped, of which 40 were variable. Cleavage sites occurred throughout the genome, including within the terminal repeat regions. Additionally, fragment length polymorphisms, independent of cleavage site loss or gain, w...
Methylation at the CpG doublet in equine adenovirus genome. Viral DNA obtained from the equine adenovirus propagated in equine transitional cell carcinoma (ETCC) cells and in equine fetal dermis cells were compared by cleaving with isoschizomeric restriction enzymes, HpaII and MspI, and then electrophoresed in 1.4 per cent agarose gels. Differences between the HpaII and MspI cleavage patterns were evident in viral DNA obtained only from the equine adenovirus propagated in ETCC cells, suggesting site specific methylation at CpG sequences.
Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins. An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two-dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second-dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ch...
Purification of F(ab’)2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immu...
The continuum of events leading to maternal recognition of pregnancy in mares. Endometria from pregnant mares are able to produce PGF in vitro, but when co-incubated with conceptus membranes the amount and rate of PGF production is considerably reduced. To estimate the molecular weight of conceptus factors that inhibited PGF production, Day-14 conceptus membranes were placed inside bags constructed of dialysis tubing and co-incubated with endometria from Day-14 pregnant mares. PGF production was significantly reduced when membranes were in bags with molecular weight exclusion limits of 12,000, 6000, and 3500, but not of 1000, suggesting that conceptus PGF-inhibitory fact...
Enzymatic deacylations of esterified saccharides–III. Comparison of de-esterifications by serum esterases from different sources. 1. 14C-labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a substrate for esterases from rabbit, guinea pig, mouse, donkey, pig, horse, sheep and human sera. 2. Stepwise de-esterification of the diester substrate 1 occurred with rabbit, guinea pig and mouse serum. Data on time-course experiments and kinetic data are reported. 3. The use of donkey, pig, horse, sheep and human serum led to the migration of the 2-O-pivaloyl group in substrate 1 to the position 4- in the sugar molecule, followed by stepwise de-esterifications of both 1 and the newly formed methyl 4,6-di-O-pi...