Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5'-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: (formula; see text) where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.
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PMID:Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization. 298 30

Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of EHV-1 and developed PCR/sequencing procedures enabling differentiation of EHV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of EHV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role.
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PMID:Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. 1657 21

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.
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PMID:A point mutation in a herpesvirus polymerase determines neuropathogenicity. 1799

Equine herpesvirus 1 (EHV-1) causes rhinopneumonitis, abortion, and rarely, myeloencephalopathy. The neurovirulence of this virus is due to a point mutation in the DNA polymerase gene. Diagnosis by virus isolation has been replaced by real-time polymerase chain reaction (RT-PCR) assays that can detect strains, viral loads, and states; this may aid in control and management of the disease.
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PMID:Equine herpesvirus-1, non-neurogenic pathotype, in a 9-year-old American Saddlebred with neurological signs. 1943 83

Infection with Equid Herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism (A(2254)/G(2254)) in the genome region of the open reading frame 30 (ORF30), which results in an amino acid variation (N(752)/D(752)) of the EHV-1 DNA polymerase, is significantly associated with the neuropathogenic potential of naturally occurring strains. In order to estimate the prevalence of the EHV-1 neuropathogenic genotype in our country, we analyzed the ORF30 genome region of Argentinean EHV-1 isolates. The study was carried out by real time allelic discrimination PCR in 90 equine EHV-1-positive samples, being 89 from 54 cases of abortion outbreaks (two of which were in association with neurological disease) and one from the respiratory tract of a healthy horse in training. Our results indicate that 7% (4/54) of the abortion outbreaks studied were induced by the neuropathogenic (G(2254)) genotype of EHV-1 and 50% (2/4) of them were associated with simultaneous neurological disease. This information emphasizes the necessity to extreme the hygienic and preventive measures to diminish EHV-1 infections and consequently reduce the risk of epizootic neurological disease as has been recently observed in other countries.
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PMID:Neuropathogenic and non-neuropathogenic genotypes of Equid Herpesvirus type 1 in Argentina. 1958 51

A single nucleotide polymorphism in the equine herpesvirus 1 (EHV-1) DNA polymerase gene (ORF30 A(2254) to G) has been associated with clinical signs of equine herpes myeloencephalopathy (EHM). The purpose of our study was to determine the odds ratio for this genetic marker and EHM using a panel of field isolates from North America collected over the past twenty-three years. EHV-1 isolates cultured at the Cornell University Animal Health Diagnostic Laboratory from 1984 to 2007 were retrieved along with their clinical histories. DNA was extracted from these EHV-1 cultures and allelic discrimination was performed using real-time PCR. The results were confirmed by sequencing of the target region in ORF30. PCR and sequencing were in 100% agreement and showed that 19 out of the 176 isolates had the ORF30 G(2254) allele (11%), of which 16 were EHM cases and 3 respiratory or abortion cases. The odds of having neurologic disease with the ORF30 G(2254) genotype were computed as 162 times greater than those with the opposite allele ORF30 A(2254) (95% confidence interval: 35-742). Despite this strong statistical significance, 24% (5/21) of horses with neurologic disease in our study population harbored the "non-neurologic" form of the allele (ORF30 A(2254)), suggesting that other factors may also contribute to the onset of EHM.
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PMID:Investigation of the prevalence of neurologic equine herpes virus type 1 (EHV-1) in a 23-year retrospective analysis (1984-2007). 1961 31

We studied the pathology, epidemiology, and clinical significance of genital herpesvirus infection in a zoo collection of bottlenose dolphins (Tursiops truncatus). Samples from the genital mucosa of male (n=21) and female (n=15) dolphins were tested by nested polymerase chain reaction (PCR) targeting the DNA polymerase of herpesvirus. Herpesvirus infection was significantly associated with the occurrence of mucosal plaques on penis (n=3) or vulva (n=4). Biopsies from a penile plaque showed epithelial hyperplasia by histology, contained herpesvirus-like particles by electron microscopy, and tested positive for herpesvirus by PCR. Herpesvirus was successfully cultivated from penile plaque samples and identified as a member of the Gammaherpesvirinae by DNA sequencing and phylogenetic analysis. We used the newly cultivated bottlenose dolphin herpesvirus (TTHV) to develop a direct enzyme-linked immunosorbent assay for anti-TTHV antibodies in banked sera of these dolphins. The percentage of positive samples was higher in adults (20/21, 95%) than in juveniles (7/15, 47%). Seroconversion occurred around the age of onset of sexual behavior. Although herpesvirus infection has been associated with abortion, perinatal mortality, and urogenital neoplasia in other species, we found no evidence of herpesvirus infection by PCR in tissues from six cases of abortion and perinatal mortality, and no diagnoses of urogenital tumors in 24 bottlenose dolphins from this zoo collection that died since 1990. Together, we here report the first successful cultivation from bottlenose dolphins of a herpesvirus that probably causes benign genital plaques, is endemic in this group of dolphins, and is likely transmitted by sexual contact.
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PMID:Herpesvirus in bottlenose dolphins (Tursiops truncatus): cultivation, epidemiology, and associated pathology. 1990 66

Equine herpesvirus 1 (EHV-1) is a common pathogen of the horse which may induce mild respiratory distress, abortion, neonatal death and neurological disease. A single nucleotide polymorphism in the EHV-1 DNA polymerase (ORF30 A(2254) to G(2254)) has been associated with clinical signs of Equine herpes myeloencephalopathy (EHM). The aim of this work was to analyze the ORF30 genomic region among a panel of EHV-1 DNA extract in order to estimate the prevalence of the EHV-1 neuropathogenic genotype in France. Samples coming from cases associated with EHM, horses with respiratory symptoms and aborted mares, each obtained between 2002 and 2009, were investigated. DNA was directly extracted from biological samples and allelic discrimination was performed using real-time PCR. Thirty of the 125 analysed horses (24%) presented the G(2254) genotype of ORF 30. Among them, 7/16 were provided by EHM cases, 1/24 by respiratory cases and 22/85 by abortion cases. Concerning EHM, the 7 G(2254) genotype of ORF30 were all isolated in 2009 during two outbreaks where mortality was observed. Regarding the 22 G(2254) genotype of ORF 30, 17 were identified in foetuses on which EHV-1 was detected by PCR, without any certainty of viral implication in the abortion. These findings clearly suggest that other factors need to be considered for a better understanding of the impact of DNA polymerase genotype upon EHV-1 neuropathogenic phenotype.
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PMID:Neuropathogenic and non-neuropathogenic variants of equine herpesvirus 1 in France. 2042 33

A single nucleotide polymorphism within EHV-1 gene ORF 30, which encodes for the viral DNA polymerase, allows the differentiation of the neuropathogenic (G2254) from non-neuropathogenic genotype (A2254). The aim of our study was to investigate the distribution of the neuropathogenic and non-neuropathogenic genotype of EHV-1 isolates associated with abortions in Germany. To determine the nucleotide sequence at the polymorphic site the amplification product of ORF 30 gene specific nested PCR was digested with restriction enzyme SalI and sequenced. Thirty-two EHV-1 isolates from six abortion outbreaks and 34 archived isolates from individual cases were obtained between 1987 and 2009 from stud farms in different regions of Germany. 89.4% (59/66) of the EHV-1 abortion isolates was of non-neuropathogenic genotype (N752/A2254) and 10.6% (7/66) contained the neuropathogenic marker (D752/G2254). Two out of seven EHV-1 abortion isolates with the mutation (G2254) came from the same outbreak and were derived from mares with neurological signs. Interestingly, the semen of a stallion from the same stud was tested positive for the neuropathogenic genotype (G2254) too. The other five EHV-1 strains came from individual abortion cases with no neurological signs. In addition to the A2254 to G2254 substitution, two EHV-1 reference strains (Ab4 and RacH) and one field isolate from an individual abortion case showed an exchange of adenine to cytosine at position 2258. In sum, we confirmed coherence between the occurrence of abortions and the non-neuropathogenic EHV-1 (A2254), but 10.6% of the abortion strains carried the mutation (G2254). The relevance of this finding as well as the role of the additional mutation and of stallions as carriers should be further investigated.
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PMID:Detection of neuropathogenic strains of Equid Herpesvirus 1 (EHV-1) associated with abortions in Germany. 2061 72

The alphaherpesvirus, equine herpesvirus type 1 (EHV-1), is a highly prevalent cause of equine infectious abortion and encephalomyelopathy. These syndromes have been attributed to ischemic necrosis from thrombosis in placental and neural vessels, although the mechanisms underlying thrombosis are unknown. After inhalation, EHV-1 establishes a peripheral blood mononuclear cell-associated viremia, with monocytes being a target of infection. Monocytes are also the main source of tissue factor (TF) in diseased states. Since TF is the primary activator of coagulation, increased monocyte TF expression could be involved in EHV-1-associated thrombosis. We hypothesized that EHV-1 infection would induce TF-dependent procoagulant activity in equine monocytes. Monocyte-enriched fractions of blood were infected with abortigenic (RacL11, NY03) and neuropathogenic (Ab4) EHV-1 strains. All strains induced procoagulant activity, to variable degrees, within 1 to 4 h, with maximal activity at 24 h, after infection. Virus-induced procoagulant activity was similar to that seen with lipopolysaccharide, a known stimulant of TF-mediated procoagulant responses. Virus-induced procoagulant activity was factor VIIa-dependent and temporally associated with TF gene transcription, implicating TF as the main driver of the activity. Procoagulant activity was mildly decreased (30-40%) when virus was inactivated by ultraviolet light or when infected cells were treated with aphidicolin, a virus DNA polymerase inhibitor, suggesting early events of virus infection (attachment, entry or intracellular trafficking) are the primary stimulus of procoagulant activity. Our results indicate that EHV-1 rapidly stimulates procoagulant activity in equine monocytes in vitro. The EHV-1-induced procoagulant activity in monocytes may contribute to clinical thrombosis in horses with EHV-1 infection.
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PMID:Equine herpesvirus type 1 infection induces procoagulant activity in equine monocytes. 2349 76


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