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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.
Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin. Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrat...
Use of repetitive sequence-based polymerase chain reaction for molecular epidemiologic analysis of Streptococcus equi subspecies equi. To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease. Methods: An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease. Methods: An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was perfor...
Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes. To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. Methods: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. Methods: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related ge...
Molten globule structure of equine beta-lactoglobulin probed by hydrogen exchange. The molten globule structure of equine beta-lactoglobulin has been inferred from the hydrogen exchange protection of the backbone amide protons. In order to make it possible to measure the hydrogen exchange kinetics of the individual backbone amide protons, the uniformly (15)N-labeled recombinant protein was expressed in Escherichia coli and the NMR peak assignment was obtained for most of the backbone protons. The chemical shift and NOE results obtained under the condition where the protein assumes the native structure are fully consistent with the known secondary structure of bovine beta-lac...
Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR. A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evalua...
Horse conceptuses secrete insulin-like growth factor-binding protein 3. Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radi...
Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine. Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-administration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which isoxsuprine is recovered has never been established, although it has long been assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Using ESI/MS/MS positive mode as an analytical tool, urine samples collected 4-8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions...
Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-li...
The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis. Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429...
Identification and phylogenetic comparison of Salem virus, a novel paramyxovirus of horses. A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemabsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramy...
Sequence analysis of trinucleotide repeat microsatellites from an enrichment library of the equine genome. Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsatellites have been elucidated. Thus, we constructed the equine genomic library enriched for DNA fragments containing (CAG)n repeats. The enriched method includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedures are useful for the cloning of less abundant trinucleotide microsatellites. ...
Primary nucleotide structure of predominant and alternate splice forms of equine insulin-like growth factor I and their gene expression patterns in tissues. To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression in tissues and cartilage from horses. Methods: Horses of various ages. Methods: Total RNA was isolated from tissues and purified. Complementary DNA (cDNA) was derived by reverse transcription and polymerase chain reaction (PCR) amplification and subcloned to plasmid vectors for sequencing and comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube...
Resistance of Rhodococcus equi to acid pH. Rhodococcus equi is an important gram-positive intracellular facultative pathogen in foals of less than 3 months of age, that causes suppurative bronchopneumonia, lymphadenitis and/or enteritis. The disease in young foals mainly occurs in spring and summer when weather conditions are favorable for survival and multiplication of the bacteria in the environment. R. equi is widespread in the environment of horsebreeding farms: it has been isolated from the soil of paddocks and from the feces of adult horses and foals. Aerosol infection via dust of paddocks seems to be the major route of foal infe...
Tissue-specific localization of cytochrome P450 aromatase in the equine embryo by in situ hybridization and immunocytochemistry. Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C(18) estrogens from C(19) androgens requires cytochrome P450 aromatase (P450(arom)) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450(arom) in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12-15 of p...
Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs. In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 sp...
Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope. Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open readi...
Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses. The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/...
Protein analysis of Babesia caballi merozoites by two-dimensional polyacrylamide gel electrophoresis and western blotting. Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among...
A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide. The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)...
Molecular cloning of equine transforming growth factor-beta1 reveals equine-specific amino acid substitutions in the mature peptide sequence. This study cloned and sequenced equine transforming growth factor (TGF)-beta1, yielding a unique nucleotide structure which predicted amino acid substitutions not seen in other mammalian species. The nucleotide sequence homology was 89% to bovine, 91% to man, 90% to ovine, and 86% to rat. Derived amino acid sequence comparison showed that the equine protein was unique, differing by two residues from man, cow, sheep, pig, and dog, and by three residues in the rat. Subsequent use of the cDNA clones to examine the expression of the TGF-beta1 gene in various tissues indicated predominant expressio...
Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11. Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the ...
Generation of an equine oviductal epithelial cell line for the study of sperm-oviduct interactions. Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 T-ag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortaliz...
Heme orientation affects holo-myoglobin folding and unfolding kinetics. Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C).
