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Yoshizumi Ishino - One of the best experts on this subject based on the ideXlab platform.
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Comprehensive search for DNA Polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus.
Nucleosides Nucleotides and Nucleic Acids, 2006Co-Authors: Sonoko Ishino, Yoshizumi IshinoAbstract:DNA Polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA Polymerases in this organism. Three main fractions containing DNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA Polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA Polymerases, did not bind tightly to any of the three DNA Polymerases. A primer usage preference was also shown for each purified DNA Polymerase. Considering their biochemical properties, the roles of the three DNA Polymerases during DNA replication in the cells are discussed.
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organization and nucleotide sequence of the dna Polymerase gene from the archaeon pyrococcus furiosus
Nucleic Acids Research, 1993Co-Authors: Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin KatoAbstract:We cloned the gene encoding the thermostable DNA Polymerase from the archaeon Pyrococcus furiosus. The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA Polymerase was sequenced. DNA Polymerase (Pfu Polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA Polymerases (family B) represented by human DNA Polymerase alpha and Escherichia coli DNA Polymerase II. An unrooted phylogenetic tree of the alpha-like DNA Polymerases based on the amino acid sequence alignment was constructed. Pfu Polymerase, with two other archaeon Polymerases, constitutes a group with some animal viruses. The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P. furiosus and E. coli, and the promoters were assigned upstream of the pol coding region. A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P. furiosus.
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Escherichia coli DNA Polymerase II is homologous to alpha-like DNA Polymerases.
Molecular & general genetics : MGG, 1991Co-Authors: Hiroshi Iwasaki, Yoshizumi Ishino, Atsuo Nakata, Hideo ShinagawaAbstract:The Escherichia coli polB gene encodes DNA Polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA Polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89,917, and is structurally homologous to alpha-like DNA Polymerases, which include eukaryotic replicative DNA Polymerases. Comparison of the sequences of the alpha-like DNA Polymerases including E. coli DNA Polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA Polymerases based on the differences in these conserved regions. The DNA Polymerases of herpes groups viruses and the DNA Polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA Polymerases, including E. coli DNA Polymerase II, human DNA Polymerase alpha, and yeast DNA Polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the -35 sequence of the polB promoter.
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SOS-inducible DNA Polymerase II of E coli is homologous to replicative DNA Polymerase of eukaryotes.
Biochimie, 1991Co-Authors: Hideo Shinagawa, Yoshizumi Ishino, Hiroshi Iwasaki, Atsuo NakataAbstract:Abstract The polB gene of Escherichia coli encodes DNA Polymerase II whose role in vivo is not defined. the polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor. Nucleotide sequencing of polB reveals that E coli DNA Polymerase II as highly homologous to replicative DNA Polymerases of eukaryotes which include human DNA Polymerase α and Saccharomyces cerevisiae DNA Polymerases I, II and III. the polB gene is not required for growth, UV-repair and UV-mutagenesis.
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Escherichia coli DNA Polymerase II is homologous to α-like DNA Polymerases
Molecular Genetics and Genomics, 1991Co-Authors: Hiroshi Iwasaki, Yoshizumi Ishino, Atsuo Nakata, Hideo ShinagawaAbstract:The Escherichia coli polB gene encodes DNA Polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA Polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89917, and is structurally homologous to α-like DNA Polymerases, which include eukaryotic replicative DNA Polymerases. Comparison of the sequences of the α-like DNA Polymerases including E. coli DNA Polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA Polymerases based on the differences in these conserved regions. The DNA Polymerases of herpes groups viruses and the DNA Polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA Polymerases, including E. coli DNA Polymerase II, human DNA Polymerase α, and yeast DNA Polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the −35 sequence of the polB promoter.
Yannick Gueguen - One of the best experts on this subject based on the ideXlab platform.
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PCR performance of the highly thermostable proof‐reading B‐type DNA Polymerase from Pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA Polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA Polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA Polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA Polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA Polymerases described, exhibiting very high accuracy compared to other available commercial DNA Polymerases and robust thermostable activity. This new DNA Polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
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pcr performance of the highly thermostable proof reading b type dna Polymerase from pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA Polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA Polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA Polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA Polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA Polymerases described, exhibiting very high accuracy compared to other available commercial DNA Polymerases and robust thermostable activity. This new DNA Polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
J H Rivera - One of the best experts on this subject based on the ideXlab platform.
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The DNA Polymerases of Leishmania mexicana.
FEMS microbiology letters, 1992Co-Authors: L L Nolan, J H RiveraAbstract:Two previously isolated DNA Polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA Polymerases. DNA Polymerase A, a high molecular mass enzyme, and DNA Polymerase B, a beta-like DNA Polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA Polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
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The DNA Polymerases of Leishmania mexicana.
Fems Microbiology Letters, 1992Co-Authors: L L Nolan, J H RiveraAbstract:Two previously isolated DNA Polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA Polymerases. DNA Polymerase A, a high molecular mass enzyme, and DNA Polymerase B, a β-like DNA Polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA Polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
Ikunoshin Kato - One of the best experts on this subject based on the ideXlab platform.
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organization and nucleotide sequence of the dna Polymerase gene from the archaeon pyrococcus furiosus
Nucleic Acids Research, 1993Co-Authors: Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin KatoAbstract:We cloned the gene encoding the thermostable DNA Polymerase from the archaeon Pyrococcus furiosus. The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA Polymerase was sequenced. DNA Polymerase (Pfu Polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA Polymerases (family B) represented by human DNA Polymerase alpha and Escherichia coli DNA Polymerase II. An unrooted phylogenetic tree of the alpha-like DNA Polymerases based on the amino acid sequence alignment was constructed. Pfu Polymerase, with two other archaeon Polymerases, constitutes a group with some animal viruses. The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P. furiosus and E. coli, and the promoters were assigned upstream of the pol coding region. A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P. furiosus.
Jacques Dietrich - One of the best experts on this subject based on the ideXlab platform.
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PCR performance of the highly thermostable proof‐reading B‐type DNA Polymerase from Pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA Polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA Polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA Polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA Polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA Polymerases described, exhibiting very high accuracy compared to other available commercial DNA Polymerases and robust thermostable activity. This new DNA Polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
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pcr performance of the highly thermostable proof reading b type dna Polymerase from pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA Polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA Polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA Polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA Polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA Polymerases described, exhibiting very high accuracy compared to other available commercial DNA Polymerases and robust thermostable activity. This new DNA Polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
