Abstract
Yeast two-hybrid systems are artificial genetic systems that allow identification and characterization of protein-protein interactions. One common limit to the use of these techniques is when the intrinsic property of “bait” proteins of interest transcriptionally autoactivates reporters, eliminating the basis for interaction detection. To circumvent this problem, autoactivating baits can be alternatively used in bacteria wherein such activation does not occur. A single-vector system has been developed, which can be used either in yeast or in bacteria, streamlining and expanding capacity for protein-protein interaction screens. A concise proposal is provided for use of this system in bacteria; a companion article, chapter 15, describes use of the system in yeast.
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. (1989) A novel genetic system to detect protein-protein interaction. Nature 340, 245–246.
Fashena, S. J., Serebriiskii, I. G., and Golemis, E. A. (2000) The continued evolution of hybrid screening approaches in yeast: how to outwit different baits with different preys. Gene 250, 1–14.
Dove, S. L., Joung, J. K., and Hochschild, A. (1997) Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386, 627–630.
Dove, S. L. and Hochschild, A. (1998) Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target. Genes Dev. 12, 745–754.
Joung, J. K., Ramm, E. I., and Pabo, C. O. (2000) A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proc. Natl. Acad. Sci. USA 97, 7382–7387.
Vallet-Gely, I., Donovan, K. E., Fang, R., Joung, J. K., and Dove, S. L. (2005) Repression of phase-variable cup gene expression by H-NS-like proteins in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 102, 11,082–11,087.
Dove, S. L. and Hochschild, A. (2004) A bacterial two-hybrid system based on transcription activation. Methods Mol. Biol. 261, 231–246.
Serebriiskii, I. G., Fang, R., Latypova, E., et al. (2005) A combined yeast/bacteria two-hybrid system: development and evaluation. Mol. Cell Proteomics 4, 819–826.
Dove, S. L., Huang, F. W., and Hochschild, A. (2000) Mechanism for a transcriptional activator that works at the isomerization step. Proc. Natl. Acad. Sci. USA 97, 13,215–13,220.
Shaywitz, A. J., Dove, S. L., Kornhauser, J. M., Hochschild, A., and Greenberg, M. E. (2000) Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein. Mol. Cell Biol. 20, 9409–9422.
Struhl, K., Cameron, J. R., and Davis, R. W. (1976) Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 73, 1471–1475.
Struhl, K. and Davis, R. W. (1977) Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3). Proc. Natl. Acad. Sci. USA 74, 5255–5259.
Brennan, M. B. and Struhl, K. (1980) Mechanisms of increasing expression of a yeast gene in Escherichia coli. J. Mol. Biol. 136, 333–338.
Hollingshead, S. and Vapnek, D. (1985) Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase. Plasmid 13, 17–30.
Giesecke, A. V. and Joung, J. K. (2005) Bacterial Two-hybrid System for Studying Protein-Protein Interactions, in Protein-Protein Interactions: A Molecular Cloning Manual, (Golemis, E. A., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 195–216.
Miller, J. H. (1992) A short course in bacterial genetics, 1st ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sambrook, J. and Russell, D. (eds.), (2001) Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Thibodeau, S. A., Fang, R., and Joung, J. K. (2004) High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques 36, 410–415.
Serebriiskii, I. G. and Golemis, E. A. (2000) Uses of lacZ to study gene function: evaluation of beta-galactosidase assays used in the yeast two-hybrid system. Anal. Biochem. 285, 1–15.
Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795.
Walhout, A. J., Temple, G. F., Brasch, M. A., et al. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575–592.
Meng, X., Smith, R. M., Giesecke, A. V., Joung, J. K., and Wolfe, S. A. (2006) Counter-selectable marker for bacterial-based interaction trap systems. Biotechniques 40, 179–184.
Stanyon, C. A., Limjindaporn, T., and Finley, R. L., Jr. (2003) Simultaneous cloning of open reading frames into several different expression vectors. Biotechniques 35, 520–522, 524–526.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Serebriiskii, I.G., Milech, N., Golemis, E.A. (2007). A Bacterial/Yeast Merged Two-Hybrid System. In: Ochs, M.F. (eds) Gene Function Analysis. Methods in Molecular Biology™, vol 408. Humana Press. https://doi.org/10.1007/978-1-59745-547-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-59745-547-3_16
Publisher Name: Humana Press
Print ISBN: 978-1-58829-734-1
Online ISBN: 978-1-59745-547-3
eBook Packages: Springer Protocols