Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Intramolecular DNA Triplexes in Escherichia. coli

Methylase를 사용한 Escherichia coli에서 Triplex 존재에 관한 연구

  • Rhim, Hyangshuk (Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea) ;
  • Kim, Sungjo (Graduate School of Biotechnology, Korea University) ;
  • Kang, Seongman (Graduate School of Biotechnology, Korea University)
  • 임향숙 (가톨릭대학교 의과학원 분자유전학연구소) ;
  • 김성조 (고려대학교 생명공학원) ;
  • 강성만 (고려대학교 생명공학원)
  • Received : 1998.09.28
  • Accepted : 1998.11.01
  • Published : 1998.12.01
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Abstract

We have introduced a genetic assay to study the existence of intramolecular triplexes in Escherichia coli. A plasmid containing the gene that encodes a temperature-sensitive EcoRI methylase was cotransformed with different plasmids containing inserts, $(G)_9AATTC(G)_9$ and $(GAA)_9TTC(GAA)_8$, that are able to form intramolecular triplexes in vitro. Inhibition of methylation in vivo was found for $(G)_9AATTC(G)_9$ and $(GAA)_9TTC(GAA)_8$, suggesting that the pur pyr sequences adopt unusual strucures in E. coli. In addition, experiments using two dimensional gel electrophoresis confirmed that intramolecular triplexes are formed for the pur pyr sequences under negative supercoiling. These results demonstrate the existence of intramolecular triplexes in E. coli.

Escherichia coli 세포에서 intramolecular triplex가 형성될 수 있는지를 조사하기 위하여 genetic assay를 도입하였다. Two dimensional gel electrophoresis 방법을 사용하여 $(G)_9AATTC(G)_9$ 혹은 $(GAA)_9TTC(GAA)_8$ sequences가 in vitro에서 intramolecular triplex 구조를 형성하는 것을 확인하였다. Genetic assay를 위하여 temperature-sensitive EcoRI methylase 유전자를 포함한 플라스미드와 triplex를 형성할 수 있는 $(G)_9AATTC(G)_9$ 혹은 $(GAA)_9TTC(GAA)_8$ sequences를 포함한 플라스미드로 E. coli를 cotransform 하였다. EcoRI methylase가 발현되는 permissive temperature에서 pur pyr sequences를 포함하는 플라스미드는 EcoRI methylase 작용에 kinetically 저항성을 보여주었다. 이러한 결과는 pur pyr sequence들이 EcoRI methylase가 작용하기 어려운 triplex 구조를 E. coli 세포에서 형성한다는 것을 증거한다.

Keywords

Acknowledgement

Supported by : 과학기술부