생명과학회지 (Journal of Life Science)

  • 제13권4호
  • /
  • Pages.522-528
  • /
  • 2003
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

균류 Coprinus cinereus에서 DNA 회복에 관여하는 RAD4 유사유전자의 분리와 특성

Characterization of RAD4 Homologous Gene from Coprinus cinereus

  • Choi, In-Soon (Marine-Biotechnology Center for Bio-Functional Material Industries Department of Life Science, Silla University)
  • 발행 : 2003.08.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 출아형 효모 Saccharomyces cerevisiae에서 자외선의 상해 시 이를 정상으로 회복시키는 절제회복 (excision repair) 유전자로 알려진 RADA4의 특성 규명을 위하여 균류 Coprinus cinereus에서 이와 유사한 유전자를 분리하였다. RAD4 유사 유전자를 분리하기 위하여 균류 C. cinereus의 염색체 DNA를 전기영동하여 분리한 다음 효모 RAD4 DNA를 probe로하여 이와 hybridization하였다. 이 결과 RAD4 유사 유전자는 3.2 kb의 insert DNA를 갖고 있었다. 또한 Southern hybridization으로 이 유사 유전자는 fungus C. cinereus의 염색체에 존재함을 확인하였다. 분리한 RAD4 유사 유전자의 전사체 크기는 2.5 kb 였으며, 자외선의 상해 시 전혀 'inducibility가 없음을 Northern hybridization으로 확인하였다. 또한 유사유전자 부분을 삭제하였을 때 이 부분이 없는 세포는 전혀 생존을 못하였다. 이 결과 분리한 RAD4 유사유전자는 세포의 생존에 관여함을 알 수 있었다.

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 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 RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the RAD4 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. In order to investigation whether the increase of transcripts by DNA damaging agent, transcripts levels were examined after treating the cells. The level of transcript did not increase by untraviolet light (UV). This result indicated that the RAD4 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the RAD4 homologous gene is essential for cell viability.

키워드

참고문헌

  1. Baker, S. M., Margison, G. P., and Striker, P., (1992): Inducible alkytransferase DNA repair proteins in the filamentous fungus nidulans. Nucleic Acids. Res. 20 (4), 645-651. https://doi.org/10.1093/nar/20.4.645
  2. Brody, H. Griffith, J. Cuticchia, A. J. Arnold, J. and W. E. Timberlake. 1991. Chromosome specific recombinant DNA libraries from the fungus Aspergillus nidulans. Nucleic Acids. Res. 19(11), 3105-3109. https://doi.org/10.1093/nar/19.11.3105
  3. Caldecott, K. W., McKeown, C. K., Tucker, J. D., Ljungquist, S., and L. H. Thompson. 1994. An interation between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol. 14, 68-76.
  4. Carr, A. M., H. Schmidt, S. Kirchhoff, W. J. Muriel. K. S. Sheldrick, D.J. Griffiths, C. N. Basmacioglu, S. Subramani, M. Clegg, A. Nasim and A. Lehmann. 1994. The rad16 gene of Schzosaccharomyces pombe: A Homolog of the RAD1 gene of Saccharomyces cerevisiae. Mol. Cell. Biol. 14, 2029-2040.
  5. Choi, I. S., Kim, J. B. and S. D. Park. 1990. Nuc1eotide sequence of RAD4 gene of Saccharomyces cerevisiae that can be propagated in Escherichia coli without inactivation. Nucl.eic Acids Res. 18, 7137. https://doi.org/10.1093/nar/18.23.7137
  6. Choi, I. S., Kim, J. B., Jeon, S. H. and S. D. Park. 1993. Expression of RAD4 gene of Saccharomyces cerevisiae that can be propagated in Escherichia coli without inactivation. Biochem. Biophy. Res. Commu. 193(1), 91-197.
  7. Fasullo, M, T. Bennett, P. Ahching and J. Koudelik. 1998. The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocation. Mol. Cell Biol. 18-3, 1190-1200.
  8. Fenech, M., Carr, A. M., Murray, J., Watts, F. Z. and A. R. Lehmann. 1991. Cloning and characterization of the RAD4 gene of Schizosaccharomyces pombe; a gene showing short regions of sequence similarity to the human XRCC1 gene. Nucleic Acids Res. 19-24, 6737-6741.
  9. Friedberg, E. C. 1988. Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae. Microbiol. Rev. 52, 70-102.
  10. Guha, S. and W. Guschlbauer. 1992. Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyadenine is removed by two repair pathways. Nucleic Acids Res. 20(14), 3607-3615. https://doi.org/10.1093/nar/20.14.3607
  11. Hoeijmakers, J. H. J. and D. Bootsma. 1990. Molecular genetics of eukaryotic DNA excision repair. Cancer Cells 2, 311-320.
  12. Ito, H. Fukuda, Y., Murata, K. and A. Kimmura, 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168.
  13. Jang, Y.K, Jin, Y. H., Kim, M. J., Seong, R. H., Hong, S. H. and S. D. Park. 1995. Identification of the DNA damage-responsive elements of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizocaccharomyces pombe. Biochem. Mol. Biol. Int. 37, 337-344.
  14. Kim, J. B., Jeon, S. H., Choi, I. S. and S. D. Park. 1994. Overexpressed RAD4 protein required for excision repair of Saccharomyces cerevisiae is toxic to the host Escherichia coli. In Vitro Toxicology 7(3), 269-275.
  15. McCready, S. J., Burkill, H., Evans, S. and B. S. Cox. 1989. The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation. Curr. Genet. 15, 27-30. https://doi.org/10.1007/BF00445748
  16. Murray, J. M., Carr, A. M., Lehmann, A. R. and F. Z. Watts. 1991. Cloning and characterization of the rad15 gene, a homologue to the S. cerevisiae RAD3 and human ERCC2 gene. Nucleic Acids Res. 19, 3525-3531. https://doi.org/10.1093/nar/19.13.3525
  17. Reynolds, R. J. and E. C. Friedgerg. 1981. Molecular mechanisms of pyrimidine dimer excision of ultraviolet-irradiated deoxyribonucleic acid. J. Bacteriol. 146, 692-704.
  18. Reynolds, P. R., Biggar, S., Prakash, L. and S. Prakash. 1992. The Shizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae. Nucleic Acids Res. 20(9), 2327-2334. https://doi.org/10.1093/nar/20.9.2327
  19. Thompson, L. H., Mitchell, D. L., Regan, J. D., Bouffler, S. D., Stewart, S. A., Carrier, W. L., Nairn, R. S. and R. T. Johnson. 1988. CHO mutant UV61 removes photoproducts but not cyclobutane dimers. Mutagenesis 4, 140-146. https://doi.org/10.1093/mutage/4.2.140
  20. Van Duin, M., De Wit, J., Odijk, H., Westerveld, A., Yasui, A., Koken, M. H. M., Hoeijmakers, J. H. J. and D. Bootsma. 1986. Molecular characterization of the human excision repair gene ERCC1: cDNA cloning and amino acid homology with the yeast DNA repair gene RAD10. Cell 44, 913-923. https://doi.org/10.1016/0092-8674(86)90014-0
  21. Weber, C. A., Salazar, E. P., Stewart, S. A. and L. H. Hampton. 1990. ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3. EMBO J. 9, 1437-1447.
  22. Weeda, G., Van Ham, R. C. A., Vermeulen, W., Bootsma, D., Van Der Eb, A. J. and J. H. J. Hoeijmakers. 1990. Molecular cloning and biological characterization of the human excision repair gene ERCC3. Cell 62, 6160-6171.
  23. Zolan, M. E., Crittenden, J., Heyler, N. K. and L. C. Seitz. 1992. Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome specific libraries. Nucleic Acids Res. 20(15), 3993-3999. https://doi.org/10.1093/nar/20.15.3993