Genetic Strategies in Saccharomyces cerevisiae to Study Human Tumor Suppressor Genes

  • Takahiko Kobayashi
  • Ting Wang
  • Hua Qian
  • Rainer K. Brachmann
Part of the Methods in Molecular Biology™ book series (MIMB, volume 223)


Baker’s yeast or Saccharomyces cerevisiae is the most intensely studied eukaryotic microorganism for very good reasons. Many fundamental processes such as cell cycle control and DNA repair show evolutionary conservation from yeast to human, thus making studies in yeast highly relevant to our understanding of mammalian cells. Yeast also allows for the efficient pursuit of questions that are often almost impossible to answer in mammalian assay systems.


Tumor Suppressor Protein Auxotrophic Marker Yeast Assay Tumor Suppressor Gene Mutation Reporter Gene HIS3 


  1. 1.
    Kolonin, M. G., Zhong, J., and Finley, R. L. (2000) Interaction mating methods in two-hybrid systems. Meth. Enzymol. 328, 26–46.PubMedCrossRefGoogle Scholar
  2. 2.
    Cagney, G., Uetz, P., and Fields, S. (2000) High-throughput screening for protein-protein interactions using two-hybrid assay. Meth. Enzymol. 328, 3–14.PubMedCrossRefGoogle Scholar
  3. 3.
    Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329–3330.PubMedCrossRefGoogle Scholar
  4. 4.
    Lorenz, M. C. Muir, R. S., Lim, E., McElver, J., Weber, S. C., and Heitman, J. (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158, 113–117.PubMedCrossRefGoogle Scholar
  5. 5.
    Ma, H., Kunes, S., Schatz, P. J., and Botstein, D. (1987) Plasmid construction by homologous recombination in yeast. Gene 58, 201–216.PubMedCrossRefGoogle Scholar
  6. 6.
    Muhlrad, D., Hunter, R., and Parker, R. (1992) A rapid method for localized mutagenesis of yeast genes. Yeast 8, 79–82.PubMedCrossRefGoogle Scholar
  7. 7.
    Burke, D., Dawson, D., and Stearns, T. (2000) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Plainview, NY, 205.Google Scholar
  8. 8.
    Lundblad, V. (1998) Saccharomyces cerevisiae, in Current Protocols in Molecular Biology, Vol. 2 (Ausubel, F. M., ed.) Wiley, New York, 13.10.11–13.14.17.Google Scholar
  9. 9.
    Fields, S. and Jang, S. K. (1990) Presence of a potent transcription activating sequence in the p53 protein. Science 249, 1046–1049.PubMedCrossRefGoogle Scholar
  10. 10.
    Kern, S. E., Kinzler, K. W., Bruskin, A., et al. (1991) Identification of p53 as a sequence-specific DNA-binding protein. Science 252, 1708–1711.PubMedCrossRefGoogle Scholar
  11. 11.
    Scharer, E. and Iggo, R. (1992) Mammalian p53 can function as a transcription factor in yeast. Nucleic Acids Res. 20, 1539–1545.PubMedCrossRefGoogle Scholar
  12. 12.
    Ishioka, C., Frebourg, T., Yan, Y. X., et al. (1993) Screening patients for heterozygous p53 mutations using a functional assay in yeast. Nat. Genet. 5, 124–129.PubMedCrossRefGoogle Scholar
  13. 13.
    Flaman, J. M., Frebourg, T., Moreau, V., et al. (1995) A simple p53 functional assay for screening cell lines, blood, and tumors. Proc. Natl. Acad. Sci. USA 92, 3963–3967.PubMedCrossRefGoogle Scholar
  14. 14.
    Robert, V., Michel, P., Flaman, J. M., et al. (2000) High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis. Carcinogenesis 21, 563–565.PubMedCrossRefGoogle Scholar
  15. 15.
    Di Como, C. J. and Prives, C. (1998) Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay. Oncogene 16, 2527–2539.PubMedCrossRefGoogle Scholar
  16. 16.
    Brachmann, R. K., Vidal, M., and Boeke, J. D. (1996) Dominant-negative p53 mutations selected in yeast hit cancer hot spots. Proc. Natl. Acad. Sci. USA 93, 4091–4095.PubMedCrossRefGoogle Scholar
  17. 17.
    Inga, A., Cresta, S., Monti, P., et al. (1997) Simple identification of dominant p53 mutants by a yeast functional assay. Carcinogenesis 18, 2019–2021.PubMedCrossRefGoogle Scholar
  18. 18.
    Marutani, M., Tonoki, H., Toda, M., et al. (1999) Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme. Cancer Res. 59, 4765–4769.PubMedGoogle Scholar
  19. 19.
    Monteiro, A. N., August, A., and Hanafusa, H. (1996) Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc. Natl. Acad. Sci. USA 93, 13595–13599.PubMedCrossRefGoogle Scholar
  20. 20.
    Humphrey, J. S., Salim, A., Erdos, M. R., Collins, F. S., Brody, L. C., and Klausner, R. D. (1997) Human BRCA1 inhibits growth in yeast: potential use in diagnostic testing. Proc. Natl. Acad. Sci. USA 94, 5820–5825.PubMedCrossRefGoogle Scholar
  21. 21.
    Xu, G. F., Lin, B., Tanaka, K., et al. (1990) The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell 63, 835–841.PubMedCrossRefGoogle Scholar
  22. 22.
    Martin, G. A., Viskochil, D., Bollag, G., et al. (1990) The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 63, 843–849.PubMedCrossRefGoogle Scholar
  23. 23.
    Ballester, R., Marchuk, D., Boguski, M., et al. (1990) The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 63, 851–859.PubMedCrossRefGoogle Scholar
  24. 24.
    Ishioka, C., Ballester, R., Engelstein, M., et al. (1995) A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene. Oncogene 10, 841–847.PubMedGoogle Scholar
  25. 25.
    Ishioka, C., Suzuki, T., FitzGerald, M., et al. (1997) Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast. Proc. Natl. Acad. Sci. USA 94, 2449–2453.PubMedCrossRefGoogle Scholar
  26. 26.
    Furuuchi, K., Tada, M., Yamada, H., et al. (2000) Somatic mutations of the APC gene in primary breast cancers. Am. J. Pathol. 156, 1997–2005.PubMedCrossRefGoogle Scholar
  27. 27.
    Zhang, C. L., Tada, M., Kobayashi, H., Nozaki, M., Moriuchi, T., and Abe, H. (2000) Detection of PTEN nonsense mutation and psiPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay. Oncogene 19, 4346–4353.PubMedCrossRefGoogle Scholar
  28. 28.
    Beroud, C. and Soussi, T. (1998) p53 gene mutation: software and database. Nucleic Acids Res 26, 200–204.PubMedCrossRefGoogle Scholar
  29. 29.
    Hernandez-Boussard, T., Rodriguez-Tome, P., Montesano, R., and Hainaut, P. (1999) IARC p53 mutation database: a relational database to compile and analyze p53 mutations in human tumors and cell lines. International Agency for Research on Cancer. Hum. Mutat. 14, 1–8.PubMedCrossRefGoogle Scholar
  30. 30.
    Hussain, S. P. and Harris, C. C. (1998) Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Res. 58, 4023–4037.PubMedGoogle Scholar
  31. 31.
    Fronza, G., Inga, A., Monti, P., et al. (2000) The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts. Mutat. Res. 462, 293–301.PubMedCrossRefGoogle Scholar
  32. 32.
    Inga, A., Iannone, R., Monti, P., et al. (1997) Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology. Oncogene 14, 1307–1313.PubMedCrossRefGoogle Scholar
  33. 33.
    Moshinsky, D. J. and Wogan, G. N. (1997) UV-induced mutagenesis of human p53 in a vector replicated in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94, 2266–2271.PubMedCrossRefGoogle Scholar
  34. 34.
    Murata, J., Tada, M., Iggo, R. D., Sawamura, Y., Shinohe, Y., and Abe, H. (1997) Nitric oxide as a carcinogen: analysis by yeast functional assay of inactivating p53 mutations induced by nitric oxide. Mutat. Res. 379, 211–218.PubMedGoogle Scholar
  35. 35.
    Epstein, C. B., Attiyeh, E. F., Hobson, D. A., Silver, A. L., Broach, J. R., and Levine, A. J. (1998) p53 mutations isolated in yeast based on loss of transcription factor activity: similarities and differences from p53 mutations detected in human tumors. Oncogene 16, 2115–2122.PubMedCrossRefGoogle Scholar
  36. 36.
    Sengstag, C., Morbe, J. L., and Weibel, B. (1999) Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background. Mutat. Res. 430, 131–144.PubMedGoogle Scholar
  37. 37.
    Lowe, S. W. (1995) Cancer therapy and p53. Curr. Opin. Oncol. 7, 547–553.PubMedCrossRefGoogle Scholar
  38. 38.
    Donehower, L. A. and Bradley, A. (1993) The tumor suppressor p53. Biochim. Biophys. Acta 1155, 181–205.PubMedGoogle Scholar
  39. 39.
    Lane, D. P. and Hall, P. A. (1997) MDM2—arbiter of p53’s destruction. Trends Biochem. Sci. 22, 372–374.PubMedCrossRefGoogle Scholar
  40. 40.
    Gibbs, J. B. and Oliff, A. (1994) Pharmaceutical research in molecular oncology. Cell 79, 193–198.PubMedCrossRefGoogle Scholar
  41. 41.
    Foster, B. A., Coffey, H. A., Morin, M. J., and Rastinejad, F. (1999) Pharmacological rescue of mutant p53 conformation and function. Science 286, 2507–2510.PubMedCrossRefGoogle Scholar
  42. 42.
    Brachmann, R. K., Yu, K., Eby, Y., Pavletich, N. P., and Boeke, J. D. (1998) Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations. EMBO J. 17, 1847–1859.PubMedCrossRefGoogle Scholar
  43. 43.
    Freeman, J., Schmidt, S., Scharer, E., and Iggo, R. (1994) Mutation of conserved domain II alters the sequence specificity of DNA binding by the p53 protein. EMBO J. 13, 5393–5400.PubMedGoogle Scholar
  44. 44.
    Saller, E., Tom, E., Brunori, M., et al. (1999) Increased apoptosis induction by 121F mutant p53. EMBO J. 18, 4424–4437.PubMedCrossRefGoogle Scholar
  45. 45.
    Inga, A., Monti, P., Fronza, G., Darden, T., and Resnick, M. A. (2001) p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay. Oncogene 20, 501–513.PubMedCrossRefGoogle Scholar
  46. 46.
    Thiagalingam, S., Kinzler, K. W., and Vogelstein, B. (1995) PAK1, a gene that can regulate p53 activity in yeast. Proc. Natl. Acad. Sci. USA 92, 6062–6066.PubMedCrossRefGoogle Scholar
  47. 47.
    Merrill, G. F., Dowell, P., and Pearson, G. D. (1999) The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase. Cancer Res. 59, 3175–3179.PubMedGoogle Scholar
  48. 48.
    Pearson, G. D. and Merrill, G. F. (1998) Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression. J. Biol. Chem. 273, 5431–5434.PubMedCrossRefGoogle Scholar
  49. 49.
    Tokino, T., Thiagalingam, S., El-Deiry, W. S., Waldman, T., Kinzler, K. W., and Vogelstein, B. (1994). p53 tagged sites from human genomic DNA. Hum. Mol. Genet. 3, 1537–1542.PubMedCrossRefGoogle Scholar
  50. 50.
    Oda, K., Arakawa, H., Tanaka, T., et al. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 102, 849–862.PubMedCrossRefGoogle Scholar
  51. 51.
    Gutmann, D. H., Boguski, M., Marchuk, D., Wigler, M., Collins, F. S., and Ballester, R. (1993) Analysis of the neurofibromatosis type 1 (NF1) GAP-related domain by site-directed mutagenesis. Oncogene 8, 761–769.PubMedGoogle Scholar
  52. 52.
    Mori, S., Satoh, T., Koide, H., Nakafuku, M., Villafranca, E., and Kaziro, Y. (1995) Inhibition of Ras/Raf interaction by anti-oncogenic mutants of neurofibromin, the neurofibromatosis type 1 (NF1) gene product, in cell-free systems. J. Biol. Chem. 270, 28834–28838.PubMedCrossRefGoogle Scholar
  53. 53.
    Gil, R. and Seeling, J. M. (1999) Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1. Mol. Cell Biochem. 202, 109–118.PubMedCrossRefGoogle Scholar
  54. 54.
    Golubic, M., Tanaka, K., Dobrowolski, S., et al. (1991) The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid. EMBO J. 10, 2897–2903.PubMedGoogle Scholar
  55. 55.
    Nigro, J. M., Sikorski, R., Reed, S. I., and Vogelstein, B. (1992) Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 1357–1365.PubMedGoogle Scholar
  56. 56.
    Moorthamer, M., Panchal, M., Greenhalf, W., and Chaudhuri, B. (1998) The p16(INK4A) protein and flavopiridol restore yeast cell growth inhibited by Cdk4. Biochem. Biophys. Res. Commun. 250, 791–797.PubMedCrossRefGoogle Scholar
  57. 57.
    Koerte, A., Chong, T., Li, X., Wahane, K., and Cai, M. (1995) Suppression of the yeast mutation rft1-1 by human p53. J. Biol. Chem. 270, 22556–22564.PubMedCrossRefGoogle Scholar
  58. 58.
    Zheng, L., Chen, Y., Riley, D. J., Chen, P. L., and Lee, W. H. (2000) Retinoblastoma protein enhances the fidelity of chromosome segregation mediated by hsHec1p. Mol. Cell. Biol. 20, 3529–3537.PubMedCrossRefGoogle Scholar
  59. 59.
    Macleod, K. (2000) Tumor suppressor genes. Curr. Opin. Genet. Dev. 10, 81–93.PubMedCrossRefGoogle Scholar
  60. 60.
    Haber, D. and Harlow, E. (1997) Tumour-suppressor genes: evolving definitions in the genomic age. Nat. Genet. 16, 320–322.PubMedCrossRefGoogle Scholar
  61. 61.
    Sellers, W. R. and Kaelin, W. G., Jr. (1997) Role of the retinoblastoma protein in the pathogenesis of human cancer. J. Clin. Oncol. 15, 3301–3312.PubMedGoogle Scholar
  62. 62.
    Vogelstein, B., Lane, D., and Levine, A. J. (2000) Surfing the p53 network. Nature 408, 307–310.PubMedCrossRefGoogle Scholar
  63. 63.
    Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Moll, U. M., Hope, T. J., and Wahl, G. M. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672.PubMedCrossRefGoogle Scholar
  64. 64.
    Lutzker, S. G. and Levine, A. J. (1996) A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat. Med. 2, 804–810.PubMedCrossRefGoogle Scholar
  65. 65.
    Sikorski, R. S. and Boeke, J. D. (1991) In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Meth. Enzymol. 194, 302–318.PubMedCrossRefGoogle Scholar
  66. 66.
    White, M. A. (1996) The yeast two-hybrid system: forward and reverse. Proc. Natl. Acad. Sci. USA 93, 10001–10003.PubMedCrossRefGoogle Scholar
  67. 67.
    Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., and Boeke, J. D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc. Natl. Acad. Sci. USA 93, 10315–10320.PubMedCrossRefGoogle Scholar
  68. 68.
    Shih, H. M., Goldman, P. S., De Maggio, A. J., Hollenberg, S. M., Goodman, R. H., and Hoekstra, M. F. (1996) A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc. Natl. Acad. Sci. USA 93, 13896–13901.PubMedCrossRefGoogle Scholar
  69. 69.
    Leanna, C. A. and Hannink, M. (1996) The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions. Nucleic Acids Res. 24, 3341–3347.PubMedCrossRefGoogle Scholar
  70. 70.
    Wang, T., Kobayashi, T., Takimoto, R., et al. (2001) hADA3 is required for p53 activity. EMBO J. 20, 6404–6413.PubMedCrossRefGoogle Scholar
  71. 71.
    Boeke, J. D., LaCroute, F., and Fink, G. R. (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197, 345–346.PubMedCrossRefGoogle Scholar
  72. 72.
    Iwabuchi, K., Li, B., Massa, H. F., Trask, B. J., Date, T., and Fields, S. (1998) Stimulation of p53-mediated transcriptional activation by the p53-binding proteins, 53BP1 and 53BP2. J. Biol. Chem. 273, 26061–26068.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Takahiko Kobayashi
    • 1
  • Ting Wang
    • 2
  • Hua Qian
    • 3
  • Rainer K. Brachmann
    • 4
    • 5
  1. 1.Hokkaido University Medical HospitalSapporoJapan
  2. 2.Department of GeneticsWashington University School of MedicineSt. Louis
  3. 3.Department of SurgeryShanghai Second Medical UniversityShangaiP. R. China
  4. 4.Department of MedicineUniversity of California at IrvineIrvine
  5. 5.Department of Biological ChemistryUniversity of California at IrvineIrvine

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