Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Characterization of RAD3 Homologous Gene from Coprinus cinereus

균류 Coprinus cinereus에서 DNA 회복에 관여하는 RAD3 유사유전자의 분리와 특성

  • Published : 2004.12.01
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Abstract

The RAD3 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. An yeast RAD3 gene has been previously isolated by functional complementation. In order to identify the RAD3 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD3 DNA, and then isolated RAD3 homologous DNA from C. cinereus chromosome. The RAD3 homolog DNA was contained in 3.2 kb DNA fragment. Here, we report the results of characterization of a fungus C. cinereus homolog to the yeast RAD3 gene. Southern blot analysis confirmed that the C. cinereus chromosome contains the RAD3 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from the C. cinereus cells were hybridized with the 3.4 kb PvuII DNA fragment of the S. cerevisiae RAD3 gene, transcripts size of 2.8 kb were detected. In order to investigate whether the increase of the amount of transcripts by DNA damaging agent, transcript levels were examined after treating agents to the cells. The level of transcripts were not increased by untraviolet light (UV). This result indicated that the RAD3 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the HRD3 gene is essential for viability of the cells and DNA repair function. These observations suggest an evolutionary conservation of other protein components with which HRD3 interacts in mediating its DNA repair and viability functions.

본 연구는 출아형 효모 Saccharomyces cerevisiae에서 자외선의 상해 시 이를 정상으로 회복시키는 절제회복(excision repair) 유전자로 알려진 RADS의 특성 규명을 위하여 균류 Coprinus cinereus에서 이와 유사한 유전자를 분리하였다. RADS 유사 유전자를 분리하기 위하여 균류 C. cinereus의 염색체 DNA를 전기영동하여 분리한 다음 효모 RADS DNA를 probe로 하여 이와 hybridization하였다. 이 결과 RADS유사 유전자는 3.4kb의 insert DNA를 갖고 있었다. 또한 Southern hybridization으로 이 유사 유전자는 fungus C. cinereus의 염색체에 존재함을 확인하였다. 분리한 RADS 유사 유전자의 전사체 크기는 2.8kb 였으며, 자외선의 상해시 전혀 자외선에 대한 유도성이 없음을 Northern hybridization으로 확인하였다. 또한 유사유전자 부분을 삭제하였을 때 이 부분이 없는 세포는 전혀 생존을 못하였다. 이 결과 분리한 RADS 유사유전자는 세포의 생존에 필수적인 유전자임을 알 수 있었다.

Keywords

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