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

---

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

Investigations of mutants of human cytomegalovirus (CMV) that are resistant to foscarnet could shed light on mechanisms of selective drug action and features of drug resistance that may be clinically important. Preexisting foscarnet-resistant mutants could not be detected in a stock of wild-type strain AD169 at frequencies greater than 0.0025%. However, foscarnet-resistant mutants could be isolated by passage in increasing drug concentrations. Two independent mutants were shown by plaque reduction and dot-blot hybridization assays to be resistant to phosphonoacetic acid and acyclovir, sensitive to ganciclovir, vidarabine, 2'fluoro-5-iodoarabinosylcytosine, and (S)-1-(3-hydroxy-2- phosphonylmethoxypropyl)cytosine; and hypersensitive to aphidicolin and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. These results have implications for the mutation frequency of CMV, for the possibility that clinically important foscarnet- and acyclovir-resistant CMV infections could emerge, for possible therapies of drug-resistant virus infections, and for the role of viral DNA polymerase in mechanisms of selective action of anti-CMV drugs.
...
PMID:Isolation of foscarnet-resistant human cytomegalovirus patterns of resistance and sensitivity to other antiviral drugs. 165 62

Cyclosporin A (CSA)-induced gingival overgrowth was immunohistochemically compared with that phenytoin-induced and nonspecific inflammatory gingiva, and CSA concentration was determined for dental plaque. Leu-6+ epithelial dendric cells (EDC) were found to significantly decrease in number in CSA-induced gingival overgrowth, while the ratio of HLA-DR+ EDC to Leu-6+ EDC did not change significantly. The expression of class II major histocompatibility complex antigens, such as HLA-DR, -DP and -DQ on keratinocytes did not change by CSA-treatment. Leu-4+ mononuclear cells in CSA-induced gingival overgrowth were located primarily in the connective tissue far outside the epithelium. CSA concentration was much higher in dental plaque than in blood and other tissues. Immune response thus appears to be suppressed in the epithelial layer of CSA-induced gingival overgrowth through decrease in Leu-6+ HLA-DR+ EDC and T cell infiltration, both due to CSA in dental plaque. DNA polymerase alpha was detected in much fewer basal keratinocytes of CSA- and phenytoin-induced gingival overgrowth. Epithelial hyperplasia may thus be not due to increased keratinocyte proliferation, but rather to enhanced keratinocyte life span.
...
PMID:Immunohistochemical analysis of effects of cyclosporin A on gingival epithelium. 170 35

The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection.
...
PMID:Detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction. 170 93

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G----T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A----C substitutions, and two were G----T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G----T substitutions and misincorporation of oh8Gua as substrate causing A----C substitutions, both caused by oh8Gua.A mispairs.
...
PMID:8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. 173 May 83

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.
...
PMID:High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. 176 Dec 18

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.
...
PMID:Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction. 184 97

Cytomegalovirus (CMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in CMV infection is well documented. Traditionally the virus has been propagated in fibroblasts, however this process may alter CMV's characteristics, thereby limiting the fibroblast model's utility as a research tool. In our efforts to develop a more accurate in vitro model of CMV/endothelial cell interaction, we have propagated a recent isolate (CMV VHL) through multiple passages in human umbilical vein endothelial cells (HUVE) and, collaterally in neonatal human dermal fibroblasts (NHDF). Infection of HUVE inoculated with either sub-strain of the virus was confirmed by CMV-specific in situ hybridization and by immunocytochemical staining for CMV antigens. Whereas infection of HUVE by substrain VHL/E (endothelial-raised) was accompanied by dramatic cytopathology resembling that observed clinically, the endothelial cytopathic potential of VHL/F (fibroblast-raised) was lost by its 20th passage in NHDF. Similarly, the ability of VHL/F to initiate sustained productive infection in HUVE was severely attenuated; plaque assay of culture supernatants and infected cell fractions, as well as virus-specific DNA polymerase assay of cell lysates, demonstrated progressive viral reproductive activity in VHL/E-inoculated HUVE, whereas VHL/F reproduction was barely detectable. Since properties of VHL/F bear strong resemblance to those of the fibroblast-raised AD169, these studies suggest that while the fibroblast adaptation process commonly employed in the propagation of CMV restricts the host range of the virus and attenuates its spectrum of cytopathic potential, endothelial-based propagation preserves the natural endothelial cytopathogenicity of the original isolate.
...
PMID:Preservation of natural endothelial cytopathogenicity of cytomegalovirus by propagation in endothelial cells. 185 Feb 27

Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants. Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts. These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45. These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length. Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin. The DIPs could not be plaque-purified and needed standard (helper) virus to replicate; DIP-containing preparations interfered with standard virus replication in an interference assay, which explained the reduction in productivity of an AcNPV expression vector-insect cell system in continuous bioreactor operations. The origin of these DIPs and their possible generation mechanism are discussed.
...
PMID:Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. 185 72

Using electroporation with the phage PRD1 genome, we set up a high-frequency DNA transfer system for a linear dsDNA molecule with 5'-covalently linked terminal proteins. The transfer was saturated when more than 100 ng of PRD1 genome was used. Electroporation efficiency was about four orders of magnitude higher than that obtained with transfection. Removal of the terminal protein abolished plaque formation, which could not be rescued by supplying the terminal protein or phage DNA polymerase or both in trans.
...
PMID:High-frequency transfer of linear DNA containing 5'-covalently linked terminal proteins: electroporation of bacteriophage PRD1 genome into Escherichia coli. 188 19

We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.
...
PMID:Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity. 189 73


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---