Advertisement

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

  • Yuanzheng He
  • Lei Bao
  • Dinggan Liu
Notes
  • 55 Downloads

Abstract

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.

Keywords

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

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  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, G.et 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