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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fields S. and Song O. K. (1989) A novel genetic system to detect protein-protein Interactions. Nature 340, 245
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
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.
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
Van Aelst L., White M A., and Wigler M. H. (1994) Ras Partners. Symp Quant. Biol 59, 181–186
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
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
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.
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
Vojtek A., Hollenberg S M., and Cooper J A (1993) Mammalian Ras interacts directly with the serme/threonine kinase Raf. Cell 74, 205–214.
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.
Sambrook J., Frrtsch E F, and Mamatis T. (1989) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor, NY.
Ito H., Fukada Y., Murata K., and Kumura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacterlol 153, 163–168.
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.
Miller J. H. (1972) Experimentsin Molecular Genetics, vol. 50, Cold Spring Harbor Laboratory Cold Spring Harbor, NY.
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
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.
Harlow E. and Lane E (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
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
Katser P and Auer B. (1993) Raptd shuttle plasmid preparation from yeast cells by transfer to E coh Bio Techniques 14, 552
Hall M. N., Hereford L, and Herskowitz I (1984) Targeting of E coli betagalactosidase to the nucleus in yeast. Cell 36, 1057–1065.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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