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)

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.
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PMID:An efficient site-directed mutagenesis using polymerase chain reaction. 188 38

Twenty-nine HBV carriers were randomised to receive ARA-AMP 10 mg/kg/day given as intramuscular injections 12 h apart for 5 days followed by 5 mg/kg/day for 23 days (15 patients) or no treatment (14 patients). Serum HBV-DNA polymerase and DNA decreased in all patients during therapy and 6 patients lost HBeAg. Five developed anti-HBe and 1 lost HBsAg and developed anti-HBs. No such changes occurred in the untreated control group over the 18-month period of observation. A 1-month course of ARA-AMP inhibits productive HBV replication and in some patients this is associated with a later loss of HBeAg and development of anti-HBe. However, intercurrent infection occurred in 3 of the treated patients with other hepatotrophic viruses known to inhibit HBV replication.
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PMID:A randomised controlled trial of adenine arabinoside 5'-monophosphate (ARA-AMP) in chronic hepatitis B virus infection. 243 68

A 1.4 kb region downstream of the DNA polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected RNA and RNA synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an ATAAG sequence and a GTAAG sequence 11 and 87 bases upstream of the ATG codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of DNA replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the beta-galactosidase gene suggest that the former gene is essential for viral replication.
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PMID:Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide. 267 27

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.
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PMID:Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme. 627 Mar 49

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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PMID:Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. 759 Mar 20

A 1,632-bp fragment, flanking the original 483-bp region of the DNA polymerase gene of a novel herpesvirus found in tissues of green turtles (Chelonia mydas) with fibropapilloma, was amplified from the circularized EcoRI-cut DNA extracted from tumor tissues by inverse PCR. The resultant 2,019-bp partial sequence of the DNA polymerase gene of the newfound herpesvirus, including the original 483-bp region, showed a high degree of homology at both the nucleotide and amino acid levels with that of other human and animal herpesviruses. Phylogenetic analysis confirmed that this novel herpesvirus belonged to the Alphaherpesvirinae subfamily.
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PMID:Amplification and analysis of DNA flanking known sequences of a novel herpesvirus from green turtles with fibropapilloma Brief report. 1120 12

A 1.7-kb cDNA clone, pGEM-cDP, was isolated from a cDNA library of IUdR-induced p3HR1 cells. It contains the upstream nucleotide sequence of the Epstein-Barr virus (EBV) DNA polymerase gene from 156,859 to 155,088, and was subcloned into expression vector pET3cp* by the polymerase chain reaction, giving the plasmid pDP1. Using a T7 RNA polymerase expression system, a 77-kD polypeptide was produced from pDP1 in Escherichia coli and specific hyperimmune serum was generated in mice. The truncated EBV DNA polymerase was shown to possess the authentic antigenicity by an indirect immunofluorescence assay and by immunoblotting using EBV-containing cells as antigens. Serum from nasopharyngeal carcinoma (NPC) patients and healthy donors was examined for antibodies against the 77-kD polypeptide by Western blot analyses and ELISAs. About 70% NPC patients were positive, while less than 15% of healthy persons showed weak reactivities in ELISAs. Copyright 1994 S. Karger AG, Basel
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PMID:Serological Responses of Patients with Nasopharyngeal Carcinoma to an N-Terminal Epstein-Barr Virus DNA Polymerase Protein Expressed in Prokaryotic Cells. 1172 14

The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase gamma (pol gamma) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol gamma was a possible target of ROS with H2O2 and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 microM H2O2 significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H2O2 treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol gamma resulting from H2O2 treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol gamma was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol gamma as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.
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PMID:The mitochondrial DNA polymerase as a target of oxidative damage. 1208 65

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oil-filled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94 degrees C-96 degrees C, which is identical to commercial machines. The optimal extension temperature of 62 degrees C-66 degrees C is lower than reported for Taq DNA polymerase (70 degrees C-80 degrees C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 minfor 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.
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PMID:Thirty-cycle temperature optimization of a closed-cycle capillary PCR machine. 1223 66

A 1-year-old male chinchilla with a 2-week history of conjunctivitis suffered subsequently from neurological signs comprising seizures, disorientation, recumbency and apathy. After 3 weeks of progressive central nervous disease the animal was killed in view of the poor prognosis. A non-suppurative meningitis and polioencephalitis with neuronal necrosis and intranuclear inclusion bodies were observed at necropsy and by light microscopy. The brain stem and cerebral cortices were most severely affected. Both eyes displayed ulcerative keratitis, uveitis, retinitis and retinal degeneration, and optical neuritis. Additionally, a purulent rhinitis with focal erosions, epithelial degeneration and intranuclear inclusion bodies was present. Ultrastructurally, herpes virus particles were detected in neurons of the brain. Immunohistochemistry with antisera specific for human herpes virus types 1 and 2 resulted in viral antigen labeling in neurons, glial cells and in neuronal processes. Viral antigen was found in the rhinencephalon, cerebral cortices, hippocampus, numerous nuclei of the brain stem, single foci in the cerebellum, and in a solitary erosive lesion of the right nasal vestibulum. Viral antigen was not detected in the eyes. The virus was isolated from the CNS, and nucleic acid sequence analysis of the glycoprotein B and the DNA polymerase revealed a sequence homology with human herpes virus type 1 of 99% and 100%, respectively. The clinical signs, the distribution of the lesions and the viral antigen suggest a primary ocular infection with subsequent spread to the CNS. Chinchillas are susceptible to human herpes virus 1 and may play a role as a temporary reservoir for human infections.
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PMID:Spontaneous human herpes virus type 1 infection in a chinchilla (Chinchilla lanigera f. dom.). 1241 Mar 90


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