Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Detection of Tissue-specific Expression of Porcine Cytochrome P450 Aromatase Genes by Use of Denaturing High Performance Liquid Chromatography(DHPLC) Technique

DHPLC 기술을 이용한 돼지 Cytochrome P450 Aromatase 유전자의 조직 - 특이적 발현양상 관찰

  • Chae, S.H. (School of Biological Resources, Yeungnam University) ;
  • Ghlmeray, A.K. (Institute of Biotechnology and Department of Biotechnology, Yeungnam University) ;
  • Hong, J.M. (Institute of Biotechnology and Department of Biotechnology, Yeungnam University) ;
  • Lee, E.J. (Institute of Biotechnology and Department of Biotechnology, Yeungnam University) ;
  • Chang, J.S. (National Livestock Research Institute (NLRI)) ;
  • Choi, I (School of Biological Resources and Institute of Biotechnology and Department of Biotechnology, Yeungnam University)
  • 채성화 (영남대학교 생물자원학부) ;
  • ;
  • 홍정민 (영남대학교 생명공학연구소 생명공학과) ;
  • 이은주 (영남대학교 생명공학연구소 생명공학과) ;
  • 장종수 (축산연구소) ;
  • 최인호 (영남대학교 생물자원학부, 생명공학연구소 생명공학과)
  • Published : 2004.06.30
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Abstract

Cytochrome P450 aromatase is the enzyme responsible for biosynthesis of female sex hormone(estrogen) and 19-nortestosterone(nandrolone), a unique steroid hormone endogenously synthesized in the pig. By use of RT-PCR coupled with DHPLC technique (WAVE analysis), expression pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. The result from the DHPLC demonstrated that PCR amplified DNA fragments of ovary and testis tissues. using unique PCR primers for all three types of aromatase genes, were different from those of type II and ill genes. Further nucleotide sequence analyses of the plasmid clones containing the PCR products revealed that nucleotide sequences of all clones were identical to type I aromatase gene(ovary type). Thus, the result from the present study indicates that the ovary and testis express the same type of aromatase gene. Therefore, the efficacy of DHPLC techniques used for this study helped us to analyze tissue-specific expression of isoform of genes containing the nucleotide sequences with high homology.

본 연구는 돼지에서 특이적으로 만들어지는 것으로 알려진 19-nortestosterone(nandrolone) 및 여성호르몬(estrogen)의 합성에 관여하는 효소인 Cytochrome P450 aromatase에 대한 유전자의 발현 양상을 밝혀내기 위해 실시되었다. RT-PCR과 최근에 개발된 DHPLC(Denature High Performance Liquid Chromatography 또는 WAVE라고 함) 분석 장치를 이용하여 정소와 난소에서 어떤 isoform의 aromatase 유전자가 발현되는지에 관해 조사하였다. 이러한 방법을 통해 같은 양의 RNA 중에 존재하는 정소내 aromatase mRNA가 난소보다 상대적으로 많이 존재한다는 것이 밝혀졌으며, 이는 돼지의 경우 수컷이 암컷 보다 혈중 여성호르몬이 더 높게 나타난다는 이전의 연구 발표가 돼지에서 여성호르몬을 만드는 aromatase 유전자가 난소에 비해 정소에서 더 많이 만들어지기 때문이라는 사실을 입증하였다. 또한, 정소와 난소에서 발현되는 aromatse 유전자를 PCR를 이용하여 증폭한 후 DHPLC를 이용하여 분석한 결과 type II, III와 다르다는 것을 확인하였다. RT-PCR에 의해 증폭된 aromatase DNA 단편을 plasmid vector에 cloning한 후에 그 염기 서열을 분석한 결과, 정소 및 난소에서 발현되는 aromatse는 모두 type I(난소형)으로 밝혀졌다. 이는 어떻게 정소와 난소의 두 다른 조직에서 같은 aromatase 효소로부터 다른 steroid가 만들어 질 수 있는지에 대한 새로운 의문을 제시하는 연구결과이며, 현재 추가적인 연구가 진행 중이다. 또한, DHPLC 기술을 활용하여 염기서열이 매우 유사한 isoform 유전자들의 발현을 관찰할 수 있다는 사실이 증명되었다.

Keywords

References

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