Skip to main content

Two-Hybrid Analysis of Ras–Raf Interactions

  • Protocol
  • 626 Accesses

Part of the Methods In Molecular Biology™ book series (MIMB,volume 84)

Abstract

The yeast two-hybrid system (1) is a genetic method that enables the experimentor to determine whether two proteins can form complexes within yeast cells. The method comprises expressing the proteins of interest as “hybrid” proteins, one fused to a DNA-binding domain and the other protein fused to a transcription-activating domain. If the fusion proteins interact, a reporter gene IS transcribed (see Fig. 1).

The two-hybrid system. (A) The hybrid of the DNA-binding domain (BD) and protein X does not activate transcription if protein X does not contain an activation domain. (B) The hybrid of the activation domain (AD) and protein Y does not activate transcription because it does not localize to the DNA-binding site. (C) Interaction between proteins X and Y brings the activation domain into close proximity to the DNA-binding site and results in transcription of the reporter genes: LacZ and HIS3.

Keywords

  • Yeast Cell
  • Control Plasmid
  • Frame Fusion
  • Altered Phenotype
  • Mutant Target

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1385/0-89603-488-7:201
  • Chapter length: 22 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   79.99
Price excludes VAT (USA)
  • ISBN: 978-1-59259-568-6
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Fig. 2.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Fields S. and Song O. K. (1989) A novel genetic system to detect protein-protein Interactions. Nature 340, 245

    PubMed  CrossRef  CAS  Google Scholar 

  2. Hollenberg S. M, Sternglanz R., Cheng P. F, and Weintraub H. (1995) Identifrcatron of a new family of trssue-specific basic helix-loop-helix proteins with a two-hybrid system Mol Cell Biol l53813–3822

    Google Scholar 

  3. Van Aelst L., Barr M., Marcus S., Polvermo A, and Wigler M H. (1993) Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sct., USA 90, 6213–6217.

    CrossRef  Google Scholar 

  4. White M. A., Nicolette C, Mmden A, Polvermo A., Van Aelst L., Karin M, and Wigler M. H. (1995) Multiple Ras functions can contribute to mammalian cell transformation Cell 80, 533–541

    PubMed  CrossRef  CAS  Google Scholar 

  5. Van Aelst L., White M A., and Wigler M. H. (1994) Ras Partners. Symp Quant. Biol 59, 181–186

    Google Scholar 

  6. Khosravr-Far R., White M A, Westwick J. K, Solskr P A., ChrzanowskaWodmcka M., Van Aelst L, Wrgler M. H., and Der C. J. (1996) Oncogemc Ras actrvatron of Raf/MAP kinase-independent pathways is sufficient to cause tumongenrc transformatron. Mol Cell Biol 16, 3923–3933

    Google Scholar 

  7. Bartel P L, Chien C T, Sternglanz R., and Fields S (1993) Using the twohybrid system to detect protein-protein interactions, in Cellular Interactions In Development A Practical Approach (Hariley D. A., ed), Oxford University Press, Oxford, UK, p. 153

    Google Scholar 

  8. Hannon G. J., Demetrick D, and Beach D. (1993) Isolation of the Rb-related p130 through Its Interaction with CDK2 and cyclins. Genes and Deveopment 7, 2378–2391.

    CrossRef  CAS  Google Scholar 

  9. Chang E. C., Barr M., Wang Y., Jung V, Xu H-P., and Wigler M H. (1994) Cooperatrve interaction of S. pombe proteins requrred for mating and morphogemsrs. Cell 79, 131–141

    PubMed  CrossRef  CAS  Google Scholar 

  10. Vojtek A., Hollenberg S M., and Cooper J A (1993) Mammalian Ras interacts directly with the serme/threonine kinase Raf. Cell 74, 205–214.

    PubMed  CrossRef  CAS  Google Scholar 

  11. Feilotter H J, Hannon G J, Ruddell C. J, and Beach D. (1994) Construction of an improved host strain for two hybrid screening Nucleic Acids Res 22, 1502–1503.

    PubMed  CrossRef  CAS  Google Scholar 

  12. Sambrook J., Frrtsch E F, and Mamatis T. (1989) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor, NY.

    Google Scholar 

  13. Ito H., Fukada Y., Murata K., and Kumura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacterlol 153, 163–168.

    CAS  Google Scholar 

  14. Breeden L and Nasmyth K (1985) Regulation of the yeast HO gene, in Cold Spring Symposia on Quantiitatve Biology, vol 50, Cold Spring Harbor Laboratory Cold Spring Harbor, NY, pp 643–650.

    Google Scholar 

  15. Miller J. H. (1972) Experimentsin Molecular Genetics, vol. 50, Cold Spring Harbor Laboratory Cold Spring Harbor, NY.

    Google Scholar 

  16. Estojak J., Brent R, and Golemis E. A (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell Biol 15, 5820–5829

    PubMed  CAS  Google Scholar 

  17. Harper J W., Adamt G. R., Wei N., Keyomarsi K., and Elledge S. J. (1993) The p21 Cdk-interacting protein Cipl is a potent inhrbrtor of Gl cyclin-dependent kinases Cell 75, 805–816.

    PubMed  CrossRef  CAS  Google Scholar 

  18. Harlow E. and Lane E (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  19. Hoffman C S. and Wmston F (1987) A ten mmute DNA preparation from yeast efficiently releases autionomous plasmids for transformation of Eschemliza colz Gene 57, 267–272

    CAS  Google Scholar 

  20. Katser P and Auer B. (1993) Raptd shuttle plasmid preparation from yeast cells by transfer to E coh Bio Techniques 14, 552

    Google Scholar 

  21. Hall M. N., Hereford L, and Herskowitz I (1984) Targeting of E coli betagalactosidase to the nucleus in yeast. Cell 36, 1057–1065.

    PubMed  CrossRef  CAS  Google Scholar 

  22. Mosteller R D, Han J, and Broek D (1994) Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors. Mol Cell Biol 14, 1104–1112.

    PubMed  CAS  Google Scholar 

  23. Zhou Y., Zhang X, and Ebrtght R. H (1991) Random mutagenesis of genesized DNA molecules by use of PCR with TAQ DNA polymerase. Nucl Acids Res 19, 6052.

    PubMed  CrossRef  CAS  Google Scholar 

  24. Joneson T, White M A., Wigler M. H, and Bar-Sagi D. (1996) Stimulation of membrane ruffling and MAP kinase activation by distinct effecters of Ras Science 271, 810–812.

    PubMed  CrossRef  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 1998 Humana Press Inc.

About this protocol

Cite this protocol

Van Aelst, L. (1998). Two-Hybrid Analysis of Ras–Raf Interactions. In: Bar-Sagi, D. (eds) Transmembrane Signaling Protocols. Methods In Molecular Biology™, vol 84. Humana Press. https://doi.org/10.1385/0-89603-488-7:201

Download citation

  • DOI: https://doi.org/10.1385/0-89603-488-7:201

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-488-4

  • Online ISBN: 978-1-59259-568-6

  • eBook Packages: Springer Protocols