Molecular Cloning and Nucleotide Sequence Analysis of pyrB Gene Encoding Aspartate Transcarbamylase from Psychrophilic Sporosarcina psychrophilia

저온성균 Sporosarcina psychrophilia로부터 Aspartate Transcarbamylase 유전자의 클로닝 및 염기서열 분석

  • 성혜리 (경북대학교 미생물학과) ;
  • 안원근 (경북대학교 생물학과) ;
  • 김사열 (경북대학교 미생물학과)
  • Published : 2002.12.01

Abstract

The Sporosarcina psychrophilia pyrB gene, which encodes aspartate transcarbamylase (ATcase), was cloned on Sau3AI restriction endonuclease fragment inserted into pUC19 plasmid vector, S. psychrophilia pyrB gene was expressed in Escherichia coli pyrB mutant for the complementation test. The sequence of 2,606 nucleotides including putative pyrB gene was determined. The region contained one full open reading frame (ORf) and two partial ORFs. The deduced amino acid sequence of the second ORF showed 59% identity with that of Bacillus caldolyticus ATCase. The first and third partial ORFs were closely related to the uracil permease (pyrP) and dihydroorotase (pyrC), respectively. Besides, potential terminator, antiterminator, and anti-antiterminator structures were found in the intergenic region between pyrP and pyrB. These results suggested that S. psychrophilia pyrimidine nucleotide biosynthesis genes are clustered as well as other Bacillus sp. Over-expressed product of pyrB encoding ATCase was purified and analyzed by the SDS-PAGE. The purified PyrB protein turned out to be molecular mass of 27 kDa and showed ATCase activity.

Keywords

Sporosarcina psychrophilia;pyrB;aspartate transcarbamylase;nucleotide sequence;expression

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