초고온성 해양 아케아로부터 열안정한 핵산 중합효소의 클로닝

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 genomic library of Pyrococcus sp. NA002 by colony and southern blot analysis using the partial DNA polymerase gene segment. We have obtained six positive clones from 196 clones and one clone (#2-D4) has been chosen for further analysis. The inserted DNA (30 kb) of the #2-D4 clone was fully sequenced and we found the coding region of the α-like DNA polymerase gene. The open reading frame was 2,328 nucleotides and predicted amino acid sequence from the ORF had two conserved regions. One was the 3'-5' exonuclease motif consisting of ExoⅠ, Ⅱ, and Ⅲ, which is responsible for proofreading ability (repair system). The other region was the DNA polymerase domain consisting of six regions (region Ⅰ to Ⅵ) that are conserved among α-like DNA polymerases. The DNA polymerase gene is under cloning and expression by using a pET vector system.