Analytical and Bioanalytical Chemistry

, Volume 386, Issue 6, pp 1625–1632 | Cite as

Development of RNR3- and RAD54-GUS reporters for testing genotoxicity in Saccharomyces cerevisiae

Original Paper


S. cerevisiae RNR3 and RAD54 gene transcription becomes strongly activated upon DNA damage. This property was used to construct yeast strains in which DNA damage can be monitored by a very sensitive fluorogenic assay in a convenient 96-well microtiter plate format. These strains carried stably integrated fusions of RNR3 or RAD54 promoters to the E. coli β-glucuronidase GUS gene. GUS activity was measured by fluorogenic detection, a method that greatly increases the precision and sensitivity of the assay. Detection levels were similar to those of real-time quantitative PCR methods and close to the limits of biological response. The two reporters differed in terms of fold-induction, activation kinetics, sensitivity and specificity upon exposure to a variety of genotoxic compounds. While RNR3-GUS showed the fastest response, RAD54-GUS showed the highest sensitivity: similar to previous reported sensitivities for bacterial and eukaryotic genotoxic detection systems. These reporter strains may complement current genotoxicity tests, but they also have the advantages of higher flexibility, requirement for shorter incubation times, and the capability of being fully automated. In addition, the intrinsic features of the system facilitate its easy improvement by genetic manipulating the yeast strain or by introducing mammalian metabolizing enzymes.


DNA repair Mutagenesis Biosensors Fluorogenic reporters Cytotoxicity Yeast bioassays 



This work has been supported by the Spanish Ministry for Science and Technology (BIO2005-00840 and GEN2001-4707-C08-08). The contribution of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya is also acknowledged.


  1. 1.
    Ames BN, Lee FD, Durston WE (1973) Proc Natl Acad Sci USA 70:782–786CrossRefGoogle Scholar
  2. 2.
    Maron DM, Ames BN (1983) Mutat Res 113:173–215Google Scholar
  3. 3.
    Yasunaga K, Kiyonari A, Oikawa T, Abe N, Yoshikawa K (2004) Environ Mol Mutagen 44:329–345CrossRefGoogle Scholar
  4. 4.
    Quillardet P, Huisman O, D’Ari R, Hofnung M (1982) Proc Natl Acad Sci USA 79:5971–5975CrossRefGoogle Scholar
  5. 5.
    Jelinsky SA, Estep P, Church GM, Samson LD (2000) Mol Cell Biol 20:8157–8167CrossRefGoogle Scholar
  6. 6.
    Friedberg EC, Siede W, Cooper AJ (1991) In: Jones EW, Pringle JR, Broach JR (eds) The molecular and cellular biology of the yeast Saccharomyces cerevisiae. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 1, pp 147–192Google Scholar
  7. 7.
    Friedberg EC, Walker GC, Siede W (1995) DNA repair and mutagenesis. ASM, Washington, DCGoogle Scholar
  8. 8.
    Elledge SJ, Davis RW (1990) Genes Dev 4:740–751Google Scholar
  9. 9.
    Elledge SJ, Zhou Z, Allen, JB, Navas TA, Davis RW (1993) Bioessays 15:333–339CrossRefGoogle Scholar
  10. 10.
    Jia X, Zhu Y, Xiao W (2002) Mutat Res 519:83–92Google Scholar
  11. 11.
    Jia X, Xiao W (2003) Toxicol Sci 75:82–88CrossRefGoogle Scholar
  12. 12.
    Cole GM, Schild D, Lovett ST, Mortimer RK (1987) Mol Cell Biol 7:1078–1084Google Scholar
  13. 13.
    Gasch AP, Huang M, Metzner S, Botstein D, Elledge SJ, Brown PO (2001) Mol Biol Cell 12:2987–3003Google Scholar
  14. 14.
    Huang M, Zhou Z, Elledge SJ (1998) Cell 94:595–605CrossRefGoogle Scholar
  15. 15.
    Walsh L, Schmuckli-Maurer J, Billinton N, Barker MG, Heyer WD, Walmsley RM (2002) Curr Genet 41:232–240CrossRefGoogle Scholar
  16. 16.
    Afanassiev V, Sefton M, Anantachaiyong T, Barker G, Walmsley R, Wolfl S (2000) Mutat Res 464:297–308Google Scholar
  17. 17.
    Walmsley RM, Billinton N, Heyer WD (1997) Yeast 13:1535–1545CrossRefGoogle Scholar
  18. 18.
    Billinton N, Barker MG, Michel CE, Knight AW, Heyer WD, Goddard NJ, Fielden PR, Walmsley RM (1998) Biosens Bioelectron 13:831–838CrossRefGoogle Scholar
  19. 19.
    Knight AW, Goddard NJ, Billinton N, Cahill PA, Walmsley RM (2002) J Biochem Biophys Methods 51:165–177CrossRefGoogle Scholar
  20. 20.
    Lichtenberg-Frate H, Schmitt M, Gellert G, Ludwig J (2003) Toxicol In Vitro 17:709–716CrossRefGoogle Scholar
  21. 21.
    Cahill PA, Knight AW, Billinton N, Barker MG, Walsh L, Keenan PO, Williams, CV, Tweats DJ, Walmsley RM (2004) Mutagenesis 19:105–119CrossRefGoogle Scholar
  22. 22.
    Knight AW, Keenan PO, Goddard NJ, Fielden PR, Walmsley RM (2004) J Environ Monit 6:71–79CrossRefGoogle Scholar
  23. 23.
    Noguerol T, Boronat S, Jarque S, Barceló D, Piña B (2006) Talanta 69:358–359Google Scholar
  24. 24.
    Myung K, Kolodner RD (2003) DNA Repair (Amst) 2:243–258CrossRefGoogle Scholar
  25. 25.
    Drablos F, Feyzi E, Aas PA, Vaagbo CB, Kavli B, Bratlie MS, Pena-Diaz J, Otterlei M, Slupphaug G, Krokan HE (2004) DNA Repair (Amst) 3:1389–1407CrossRefGoogle Scholar
  26. 26.
    Thomas DC, Husain I, Chaney SG, Panigrahi GB, Walker IG (1991) Nucleic Acids Res 19:365–370CrossRefGoogle Scholar
  27. 27.
    Sebastian J, Kraus B, Sancar GB (1990) Mol Cell Biol 10:4630–4637Google Scholar
  28. 28.
    Koc A, Wheeler LJ, Mathews CK, Merrill GF (2004) J Biol Chem 279:223–230CrossRefGoogle Scholar
  29. 29.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) Embo J 6:3901–3907Google Scholar
  30. 30.
    Goldstein AL, McCusker JH (1999) Yeast 15:1541–1553CrossRefGoogle Scholar
  31. 31.
    Johnston M, Carlson M (1992) Gene expression. In: Jones EW, Pringle JR, Broach JR (eds) The molecular and cellular biology of the yeast Saccharomyces cerevisiae. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 193–282Google Scholar
  32. 32.
    Riu J, Marinez E, Barceló D, Ginebreda A, Tirapu LL (2001) Fresenius J Anal Chem 371:448–455CrossRefGoogle Scholar
  33. 33.
    Farré M, Gonçalves C, Lacorte S, Barceló D, Alpendurada MF (2002) Anal Bioanal Chem 373:696–703CrossRefGoogle Scholar
  34. 34.
    Routledge E, Sumpter J (1996) Environ Toxicol Chem 15:241–248CrossRefGoogle Scholar
  35. 35.
    Garcia-Reyero N, Grau E, Castillo M, López de Alda MJ, Barceló D, Piña B (2001) Environ Toxicol Chem 20:1152–1158CrossRefGoogle Scholar
  36. 36.
    Shetty RS, Deo SK, Liu Y, Daunert S (2004) Biotechnol Bioeng 88:664–670Google Scholar
  37. 37.
    Stocker J, Balluch D, Gsell M, Harms H, Feliciano J, Daunert S, Malik KA, Van der Meer JR (2003) Environ Sci Technol 37:4743–4750CrossRefGoogle Scholar
  38. 38.
    Ramanathan S, Shi WP, Rosen BP, Daunert S (1998) Anal Chim Acta 369:189–195CrossRefGoogle Scholar
  39. 39.
    Oda Y, Funasaka K, Kitano M, Nakama A, Yoshikura T (2004) Environ Mol Mutagen 43:10–19CrossRefGoogle Scholar
  40. 40.
    Carro D, Bartra E, Piña B (2003) Appl Environ Microbiol 69:2161–2165CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Institut de Biologia Molecular de BarcelonaConsejo Superior de Investigaciones CientíficasBarcelonaSpain

Personalised recommendations