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Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus.

ABSTRACT: 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.

SUBMITTER: Uemori T

PROVIDER: S-EPMC309101 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus.

Uemori T T Ishino Y Y Toh H H Asada K K Kato I I

Nucleic acids research 19930101 2

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 un ...[more]

PMID: 8441634

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Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100Â°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 â 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs. Pyrococcus furiosus (DSM 3638) was grown at 95Â°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 Â´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4Â°C. Total RNA was extracted using acid-phenol and stored at -80Â°C until needed. A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.

Dataset Information

Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus.

Publications

Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus.

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