초고온성 아케아 파이로코코스 NA2로부터 열안정 핵산 중합쇼소 유전자 클로닝 및 특성 분석

Abstract

An extremely thermophilic archaeal strains, NA2 was isolated from a deep-sea hydrothermal vent area at the PACMANUS field (3° 14' S, 151° 42' E) in the East Manus Basin. On the basis of 16S rRNA gene sequences, the NA2 belonged to genus Pyrococcus. The α-like DNA polymerase gene was screened from a genomic library of Pyrococcus sp. NA2 by colony and southern hybridization using the partial DNA polymerase gene segment as a probe. We have obtained six positive clones from 196 clones and one clone (#2-D4) has been chosen for further analysis. The nucleotide sequence of the insert DNA (30kb) of the clone was determined and the coding region of the α-like DNA polymerase gene was found by using ORF finder. The DNA polymerase gene was 2,325 nucleotides in length and the translated protein had two conserved regions. One was the 3'-5' exonuclease motif consisting of ExoⅠ, Ⅱ, and Ⅲ. The other region was the DNA polymerase domain consisting of six regions (region Ⅰ to Ⅵ) conserved among α-like DNA polymerases. In a pairwise alignment, the amino acid sequence from NA2 showed highest identities to the following family B DNA polymerases of Pyrococcus sp. ST700 Pol B (GeneBank accession no. AJ250332) (94% identity), of P. abyssi ST855 Pol B (GeneBank accession no. Z54174) (89% identity), of T. kodakaransis KOD1 Pol B (GeneBank accession no. D29671) (78% identity), and of Crenarchaeota Pyrodictium occulum Pol B (GeneBank accession no. E10436) (33% identity).