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A yeast two-hybrid system for discerning differential interactions using multiple baits

Nature Biotechnology volume 17, pages 1232–1233 (1999)Cite this article

The yeast two-hybrid system is a powerful genetic technique designed to identify novel protein–protein interactions that were previously limited to detection by biochemical studies. The original system1 has been manipulated and improved upon by interchanging DNA-binding domains2, by adding multiple reporter constructs2,3,4, and by using a reverse system to study disruption of protein interactions4,5.

All two-hybrid screening systems rely on the fact that transcriptional activation and DNA-binding domains of transcription factors are modular in nature6,7. In these systems, the coding sequence for the DNA-binding domain of a transcription factor such as Gal4 or LexA is fused to the cDNA of a protein of interest, termed the "bait". The fusion protein thus encoded tethers the bait to the promoter region of a reporter gene. A second fusion of a cDNA library with the coding sequence of a transcriptional activation domain is termed the "prey". Functional reconstitution of transcription factor activity occurs upon association of the bait and prey protein domains. This interaction is then detected by expression of reporter genes that are dependent upon the bait's DNA-binding domain.

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Figure 1: Differential yeast strain HW18.
Figure 2: Differential interactors.

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Acknowledgements

We thank Marc Vidal for the generous gifts of yeast strain MaV52, pMV252, oligos, and for his advice and critical reading of the manuscript. We thank Roger Brent for the kind gift of plasmids and Steve Buratowski for technical advice and insight. This work was funded by NIH grant GM55684 to P.H. M.G. was funded by US Army Breast Cancer Fellowship DAMD17-97-1-7167.

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  1. Martha J. Grossel

    Present address: department of zoology, Connecticut College, 270 Mohegan Avenue, New London, CT, 06320

Authors and Affiliations

  1. department of pathology, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, MA

    Martha J. Grossel, Hongmei Wang, Bedrick Gadea, Wilbert Yeung & Philip W. Hinds

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  1. Martha J. Grossel
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Correspondence to Philip W. Hinds.

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Grossel, M., Wang, H., Gadea, B. et al. A yeast two-hybrid system for discerning differential interactions using multiple baits. Nat Biotechnol 17, 1232–1233 (1999). https://doi.org/10.1038/70792

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