Chinese Science Bulletin

, Volume 46, Issue 5, pp 403–405 | Cite as

Clony color assay coupled with 5FOA negative selection greatly improves yeast three-hybrid library screening efficiency



The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactionsin vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.


colony color assay 5FOA negative selection yeast three-hybrid system RNA binding protein 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Sengupta, D. J., Zhang, B., Kraemer, B. et al., A three-hybrid system to detect RNA-protein interactions in vivo, Proc. Natl. Acad. Sci., 1996, 93: 8496.CrossRefGoogle Scholar
  2. 2.
    Bacharach, E., Goff, S. P., Binding of the human immunodeficiency virus type 1 Gag protein to the yeast three-hybrid system, J. Virol., 1998, 72: 6944.Google Scholar
  3. 3.
    Lee, E., Yeo-A, Kraemer, B. et al., The gag domains required for avian retroviral RNA encapsidation determined by using two independent assays, J. Virol., 1999, 73: 6282.Google Scholar
  4. 4.
    Wang, Z. F., Whitfield, M. L., Ingledue, III T. C. et al., The protein that binds the 3′ end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing, Genes Development, 1996, 10: 3028.CrossRefGoogle Scholar
  5. 5.
    Zhang, B., Gallegos, M., Puoti, A. et al., A conserved RNAbinding protein that regulates sexual fates in the C. elegans hermaphrodite germ line, Nature, 1997, 390: 477.CrossRefGoogle Scholar
  6. 6.
    Jan, E., Motzny, C. K., Graves, L. E. et al., The STAR protein, GLD1, is a translational regulator of sexual identity in Caenorhabditis elegans, EMBO J. 1999, 18: 258.CrossRefGoogle Scholar
  7. 7.
    Toh-e, A., Wickner, R. B., Curing of the 2 μ DNAplasmid from Saccharomyces Cerevisiae, J. Bacteriol., 1981, 145: 1421.Google Scholar
  8. 8.
    Park, Y. M., Tan, S. L., Katze, M. G., Differential sensitivity to 5-fluoro-orotic acid as a screen for bait RNA-independent false positives in a yeast three-hybrid system, Biotechniques, 1999, 26: 1102.Google Scholar
  9. 9.
    Boeke, J. D., Trueheart, J., Natsoulis, al., 5′-Fluoroorotic acid as a selective agent in yeast molecular genetics, Methos. Enzymol., 1987, 154: 164.CrossRefGoogle Scholar
  10. 10.
    Jemes, P., Halladay, J., Craig, E. A., Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast, Genetics, 1996, 144: 1425.Google Scholar
  11. 11.
    Liu, D. G., Akira, S., Kishimoto, T. et al., Overexpression of a reversion-related protein in the revertant RR cell, Science in China, Ser. C, 1996, 39(3): 300.Google Scholar
  12. 12.
    Gietz, D., St. Jean, A., Woods, R. A. et al., Improved method for high efficiency transformation of intact yeast cells, Nucleic Acids Res., 1992, 20: 1425.CrossRefGoogle Scholar
  13. 13.
    Bartel, P. L., Chien, C. T., Sternglanz, R. et al., Using the two-hybrid system to detect protein-protein interactions, in In Cellular Interactions in Development: A Practical Approach (ed. Hartley, D. A.), Oxford: Oxford University Press, 1993, 153.Google Scholar

Copyright information

© Science in China Press 2001

Authors and Affiliations

  1. 1.Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina

Personalised recommendations