독성물질 검출을 위한 Plasmid Vector 개발

  • 최연주 (부산대학교 자연과학대학 미생물학과) ;
  • 유진삼 (부산대학교 자연과학대학 미생물학과) ;
  • 하진목 (부산대학교 자연과학대학 미생물학과) ;
  • 백형석 (부산대학교 자연과학대학 미생물학과)
  • Published : 1997.04.01

Abstract

After DNA damage, umuDC is the only SOS operon that must be induced to promote SOS mutagenesis in Escherichia coli. The recombinant plasmid pBC401 and pBC402 were constructed to fuse the lac structural genes with promoter region of umuDC operon to induce the expression of lacZ gene by DNA damage. We transformed the plasmid pBC401 and pBC402 into E. coli MC1061, lacZ deleted strain and determined the activity of $\beta$-galactosidase for various mutagen; UV, mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroqunoline-1-oxide (NQO), ethyl methanesulfonate (EMS). The $\beta$-galactosidase activities of PBC401 and pBC402 for UV, MMC, and NQO were increased in proportion to expression time until 3 hours thereafter, the activities were constant or slightly decreased. The activities for MNNG and EMS were not so high as for UV, MMC, and NQO. When MNNG and EMS were treated, $\beta$-galactosidase activity of pBC402 was slightly lower than pBC401 but when UV, MMC, and NQO were treated in pBC402, $\beta$-galactosidase activity was slightly higher than in pBC401. Therefore, the pBC402 was better than the pBC401 in terms of sensitivity for frameshift mutagen. We suggest that the plasmid pBC401 and pBC402 are easy to detect mutagens which cause frameshift mutation rather than point mutation.

Keywords

References

  1. J. Mol. Biol. v.164 Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli Elledge, J. S.;G. C. Walker
  2. Gene v.23 Cloning and characterization of the umuoperon for inducible mutagenesis in Escherichia coli Shinagawa, H.;T. Kato, T.;Ise, K.;Makino;A. Nakata
  3. Proc. Natl. Acad. Sci. U.S.A. v.82 umuDC and mucAB operons whose products are required for UV light-and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology Perry, K. L;S. J. Elledge;B. B. Mitchell;L. Marsh;G. C. Walker
  4. Genet. v.239 Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli Sommer, S.;J. Knezevic;A. Bailone;R. Devoret
  5. Ann. Rev. Biochem. v.54 Induction DNA repair system Walker, G. C.
  6. Cell v.29 The SOS regulatory system of E. coli Little, J. W.;D. W. Mount
  7. Mol. Gen. Genet. v.229 Levels of chromosomally encoded Umu proteins and requirements for in vitro UmuD cleavage Woodgate, R.;D. G. Ennis
  8. Proc. Natl. Acad. Sci. U.S.A. v.77 Cleavage of LexA protein by RecA protease Little, J. W.;S. H. Edmistion;L. Z. Pacelli;D. W. Mount
  9. Proc. Natl. Acad. Sci. U.S.A. v.81 Autodigestion of LexA and phage λ repressors Little, J. W.
  10. Proc. Natl. Acad. Sci. U.S.A. v.85 UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA Burckhardt, S. E.;R. Woodgate, R. H.;Scheurmann;H. Echols
  11. Proc. Natl. Acad. Sci. U.S.A. v.85 RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis Shinagawa, H.;H. Iwasaki;T. Kato;A. Nakata
  12. Proc. Natl. Acad. Sci. U.S.A. v.85 RecA-meadiated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional dereperssion and posttranslational activation Nohmi, T.;J. R. Battista;L. A. Dodson;G. C. Walker
  13. Biocheimie v.73 A RecA protein mutant deficient in its interaction with the UmuDC complex Bailone, A.;S. Sommer;J. Knezevic;M. Dutreix;R. Devoret
  14. Proc. Natl. Acad. Sci. U.S.A. v.86 UmuC mutagenesis protein of Escherichia coli: Purification and interaction with UmuD and UmuC Woodgate, R.;M. Rajagopalan;C. Lu;H. Echols
  15. Proc. Natl. Acad. Sci. U.S.A. v.86 Activated-RecA protein affinity chromatography of LexA repressor and other SOS-rergulated proteins Freitag, N.;K. McEntee
  16. J. Bacteriol v.171 New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role of RecA protein in UV-mutagenesis Dutreix, M.;P. L. Moreau;A. Bailone;F. Galiberit;J. R. Battista;G. C. Walker;R. Devoret
  17. Mutat. Res. v.113 Revised methods for the Salmonella mutagenicity test Maron, D. M.;Ames, B. N.
  18. Mutat. Res. v.168 The Salmondella typhimurium/mammalian microsomal assay: a report of the U. S. Environmental Protection Agency Gene-Tox Program Kier, L. E.;D. J. Brusick;A. E. Auletta;E. S. VonHalle;M. M. Brown;V. F. Simmon;V. Dunkel;J. McCann;K. Mortelmans;M. Prival;T. K. Rao;V. Ray
  19. Casarett and Doull's Toxicology: the basic science of poisons(4th ed.) Amdur, M. O.;J. Doull;C. D. Klaassen
  20. J. Bacteriol. v.143 In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals Casadaban, M. J.;J. Chou;S. N. Cohen
  21. Molecular cloning(2nd ed.) Sambrook, J.;E. F. Fritsch;T Maniatis
  22. Proc. Natl. Acad. Sci. U.S.A. v.69 Non-chromosomal antibiotic resistance in bacteria; genetic transformation of E. coli by R. factor DNA Cohen, S. N.;A.C.Y. Chang;L. Hsu
  23. J. Bacteriol v.162 Cold sensitivity induced by overproduction of UmuDC in Escherichia coli Marsh. L.;G. C. Walker
  24. A short course in bacterial genetics Miller, J. H.
  25. Mutat. Res. v.314 Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: Ⅰ. Frameshift mutagenesis Watanabe, M.;T. Nohmi;T. Ohta
  26. Mutat. Res. v.314 Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: Ⅱ. Base substitution mutagenesis Watanabe, M.;T. Nohmi;T. Ohta