Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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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

  • Published : 2002.12.01
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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.

저온성 균인 Sporosarcina psychrophilia의 염색체 DNA를 추출하여 Sau3AI으로 부분 절단하고 pUC19 vector에 ligation 시킨 후, Escherichia coli pyrB mutant 균주에 형질전환하여 uracil이 없는 AB배지에서 생존하는 균주를 선택한 후, 그 plasmid를 분리하여 pSMI과 pSM2라고 명명하였다. 두 plasmid의 염기배열을 결정한 결과 pSM2 insert DNA는 pSMl insert DNA 부분을 포함하는 2,606 nucleotide 단편이었다. 염기서열을 분석하였을 때 이것은 1개의 완전한 open reading frame(ORF)과 2개의 부분 ORFs를 포함하고 있었다. 두 번째 위치한 완전한 ORF는 Bacillus caldolyticus aspartate transcarbamylase(pyrB)와 아미노산 서열 수준에서 59% 상동성을 보였고, 첫 번째와 세 번째 위치한 부분적 ORFs는 각각 Bacillus속의 uracil permease(pyrP)와 dihydoorotase(pyrC)와 높은 상동성을 보였다. 그리고 pyrB와 pyrP사이에 intergenic 부분에는 잠재적인 terminator, antiterminator, anti-antiterminator 구조를 포함하고 있었다. 이러한 결과는 S. psychrophilia pyrimidine 생합성에 관련된 유전자들은 다른 Bacillus속에서 알려진 바와 같이 유전자군을 형성하고 있을 것으로 추정했다. S. psychrophilia pyrB 유전자의 생성물을 과다발현 시키고 정제해서 그 단백질을 SDS-PAGE로 확인한 결과 27 kDa 부근에서 band를 확인할 수 있었으며, 정제한 단백질도 ATCase 효소활성을 지니고 있었다.

Keywords

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