Abstract
The yeast two-hybrid system is a powerful technique for studying protein-protein interactions. Two proteins are separately fused to the independent DNA-binding and transcriptional activation domains of the Gal4p transcription factor. If the proteins interact, they reconstitute a functional Gal4p that activates expression of reporter gene(s). In this way, two individual proteins may be tested for their ability to interact, and a transcriptional readout can be measured to detect this interaction. Furthermore, novel interacting partners can be found by screening a single protein or domain against a library of other proteins using this system. It is this latter feature—the ability to search for interacting proteins without any prior knowledge of the identity of such proteins—that is the most powerful application of the two-hybrid technique.
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References
Keegan, L., Gill, G., and Ptashne, M. (1986) Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science 231, 699–704.
Ma, J. and Ptashne, M. (1987) Deletion analysis of GAL4 defines two transcriptional activating segments. Cell 48, 847–853.
Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803.
Ma, J. and Ptashne, M. (1987) A new class of yeast transcriptional activators. Cell 51, 113–119.
Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S. (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582.
Hudson, J.R. Jr., Dawson, E.P., Rushing, K.L., et al. (1997) The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7, 1169–1173.
James, P., Halladay, J., and Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.
Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.
Bartel, P. L., Roecklein, J. A., SenGupta, D., and Fields, S. (1996) A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet. 12, 72–77.
Soni, R., Carmichael, J. P., and Murray, J.A. (1993) Parameters affecting lithium acetate-mediated transformation of Saccharomyces cerevisiae and development of a rapid and simplified procedure. Curr. Genet. 24, 455–459.
Pierce Chemical Co., P.O. Box 117, Rockford IL 61105. http://www.piercenet.com.
Flajolet, M., Rotondo, G., Daviet, L., et al. (2000) A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242, 369–379.
BD Biosciences Clontech, 1020 E Meadow Circle, Palo Alto, CA 94303. MATCHMAKER Library User Manual (PT1020-1) version PR2562. http:// www.clontech.com.
Zhu, L., Gunn, D., and Kuchibhatla, S. (1997) The Yeast Two-Hybrid System (Bartel, P. L. and Fields, S., eds.) Oxford University Press, New York, NY, pp. 73–96.
Bai, C. and Elledge, S.J. in ref. 15, pp. 11–28.
Sambrook, J. and Russell, D.W. (2001) Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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© 2004 Humana Press Inc., Totowa, NJ
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Miller, J., Stagljar, I. (2004). Using the Yeast Two-Hybrid System to Identify Interacting Proteins. In: Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 261. Humana Press. https://doi.org/10.1385/1-59259-762-9:247
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DOI: https://doi.org/10.1385/1-59259-762-9:247
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