Abstract
The yeast one-hybrid (Y1H) system has been among the methods of choice to detect protein–DNA interactions. However, conventional Y1H systems with a single auxotrophic reporter gene often suffer from high incidence of false positives to demonstrate a limited power in large-scale screenings. Here we describe a refined Y1H system that uses two independent bait sequences, each controlling a distinct reporter gene integrated in the host genome. With these modifications and a method of targeted DNA methylation, we succeeded in efficient isolation of clones for methylated DNA-binding proteins from mammalian cDNA libraries.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hess J, Angel P, Schorpp-Kistner M (2004) AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117(Pt 25):5965–5973
Black AR, Black JD, Azizkhan-Clifford J (2001) Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188(2):143–160
O'Dea E, Hoffmann A (2009) NF-κB signaling. Wiley Interdiscip Rev Syst Biol Med 1(1):107–115
Dey B, Thukral S, Krishnan S, Chakrobarty M, Gupta S, Manghani C et al (2012) DNA-protein interactions: methods for detection and analysis. Mol Cell Biochem 365(1–2):279–299
Reece-Hoyes JS, Walhout AJ (2012) Gene-centered yeast one-hybrid assays. Methods Mol Biol 812:189–208
Furey TS (2012) ChIP-seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nat Rev Genet 13(12):840–852
Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340(6230):245–246
Hannon GJ, Demetrick D, Beach D (1993) Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev 7(12A):2378–2391
James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144(4):1425–1436
Lopato S, Bazanova N, Morran S, Milligan AS, Shirley N, Langridge P (2006) Isolation of plant transcription factors using a modified yeast one-hybrid system. Plant Methods 2:3
Feng SY, Ota K, Ito T (2010) A yeast one-hybrid system to screen for methylated DNA-binding proteins. Nucleic Acids Res 38(20):e189. doi:10.1093/nar/gkq757
Lõoke M, Kristjuhan K, Kristjuhan A (2011) Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50(5):325–328. doi:10.2144/000113672
Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34
Sitterlin D, Tiollais P, Transy C (1997) The RAR alpha-PLZF chimera associated with acute promyelocytic leukemia has retained a sequence-specific DNA-binding domain. Oncogene 14(9):1067–1074
Acknowledgment
We thank Kazuyuki Mizushima for his contribution to vector construction.
This work was supported by Genome Network Project and Cell Innovation Project from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T.I.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ota, K., Feng, SY., Ito, T. (2014). Detecting Protein–DNA Interactions Using a Modified Yeast One-Hybrid System. In: Miyamoto-Sato, E., Ohashi, H., Sasaki, H., Nishikawa, Ji., Yanagawa, H. (eds) Transcription Factor Regulatory Networks. Methods in Molecular Biology, vol 1164. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0805-9_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0805-9_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0804-2
Online ISBN: 978-1-4939-0805-9
eBook Packages: Springer Protocols