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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)

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.
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PMID:High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. 176 Dec 18

A family of plasmids containing short pieces of Escherichia coli lac promoter DNA has been constructed. DNA fragments from any source may be inserted directly into the unique EcoRI sites of some of these plasmids to achieve transcription under the control of the lacUV5 promoter. Alternatively, the plasmids serve as convenient sources of lac DNA fragments ('portable promoters') containing the 'up' promoter mutations UV5 or Ps (super promoter) as well as the wild-type promoter. pOP95-2, pOP95-5, pOP203-1, pOP203-2 and pOP203-3 are derivatives of pMB9 while pOP95-15 and pOP203-13 are derivatives of pBR322. The pOP95 plasmids contain the 95-bp AluI lac fragment. This fragment includes the UV5 promoter (minus the CAP binding site), the repressor binding site, and ends 2 bp before an ATG encoding the beta-Gal start codon. The pOP203 plasmids contain the 203-bp HaeIII lac fragment. This fragment contains the UV5 promoter (including the L8 mutation in the CAP binding site), the repressor binding site and sequences encoding the first 8 amino acids of beta-Gal. To shorten and introduce reading frame heterogeneity in the beta-Gal coding end of the pOP203 plasmids, the EcoRI site in pOP203-12 was moved upstream by digesting EcoRI cut plasmid DNA with T4 DNA polymerase and S1 nuclease followed by ligation in the presence of EcoRI linker. This produced the plasmids pOP203-24, pOP203-27, pOP203-28 and pOP203-29. pOP203-29 encodes essentially just that portion of the beta-Gal mRNA sequence which is protected from nuclease digestion by the bound ribosomal complex (Maizels, 1974).
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PMID:A family of cloning vectors containing the lacUV5 promoter. 629 48

DNA polymerase fingerprint analysis (DPFA) was employed for identifying DNA-carcinogen adduct formation in the human p53 and lac gene sequence. Two 'hot regions' at codons 223-250 and 257-283 of the p53 gene were easily attacked by nitroso-2-acetylaminofluorene or acetoxy-2-acetylaminofluorene. However, the promutational lesions in lac gene were rather randomly distributed. The chemical treated plasmid (pUC 19) which contains lac gene were transfected into Escherichia coli JM109 cells and the induced lac gene mutants were selected with X-Gal plate as indicated by the appearance of white colonies. No mutational hot regions were found in the lac gene.
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PMID:Preferential promutagenic lesions at exons 7-8 of human p53 genomic DNA induced by the direct-acting hepatocarcinogens N-nitroso-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene. 765 42

The Escherichia coli dnaE and dnaQ genes encode, respectively, the alpha (polymerase) and epsilon (proofreading) subunits of DNA polymerase III. Mutations in these genes resulting in mutator or antimutator phenotypes provide important tools to understand the mechanisms by which mutations occur. One way to isolate such strains is the use of papillation assays. We used one such assay based on the reversion of the galK2 allele in cells grown on MacConkey-Gal plates. Here, we describe the identification of the galK2 mutation and its possible reversion pathways, and the characterization of 7 mutators isolated using this system. 1 mutator resided in dnaE and 6 in dnaQ. Sequencing of the galK2 allele revealed a G.C-->T.A transversion at base pair 571 that changed a glu codon (GAA) to a stop codon (TAA). The analysis of 319 revertants showed that a Gal+ phenotype can be achieved by A.T-->G.C transition, A.T-->T.A transversion and A.T-->C.G transversion. We characterized the mutator phenotypes of the newly isolated mutators by determining (i) their mutation frequencies to resistance to rifampicin and nalidixic acid in both wild-type and mutL backgrounds, (ii) their temperature sensitivity and medium dependence and (iii) their mutational specificity (by analyzing the nature of galK revertants). Based on the genomic locations of their mutations, specificity of reversion pathways and magnitude of mutator effects, the mutators can be grouped into 3 classes. These classes may represent different mutational mechanisms that include defective base insertion, defective proofreading and interference with the postreplicative mismatch-repair system.
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PMID:The Escherichia coli galK2 papillation assay: its specificity and application to seven newly isolated mutator strains. 769 54

A system used for the determination of fidelity of DNA polymerase in PCR was developed in E.coli and was used to determine the fidelity of FD DNA polymerase in PCR amplication. Frame shift and base substitution mutations were created in vitro in the lacZ gene in pUC118 and pUC119. As a result, a set of six derived plasmids namely pFDFM118 and pFDFM119 (-1 frame shift), pFDFP118 and pFDFP119 (+1 frame shift), pFDFU118 and pFDFU119 (base substitution) were obtained. All of them failed to carry out lacZ alpha-complementation in E.coli MV1184 and the colonies appeared white on medium with X-Gal and IPTG consequently. PCR reaction was carried out using these derived plasmids as templates and the PCR products were ligated to specially constructed cloning vectors pFDFL118 or pFDFL119, and the ligated products were used to transform MV1184. If any back mutation happens to occur during PCR, the transformants would appear blue on medium with X-Gal and IPTG. By scoring the number of blue and white colonies, the fidelity of DNA polymerase can be calculated. With this system the error of replication of the FD DNA polymerase was found to be 10(-5)-10(-6).
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PMID:[Constuction of an improved system for the determination of fidelity of polymerase in PCR]. 925 77

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.
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PMID:Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence. 1241 54