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Yeast One-Hybrid Screens for Detection of Transcription Factor DNA Interactions

Part of the Methods in Molecular Biology book series (MIMB,volume 678)

Abstract

The yeast one-hybrid system is widely recognized as a valuable and straightforward technique to study interactions between transcription factors and DNA. By means of one-hybrid screens, transcription factors or other DNA-binding proteins, expressed from cDNA expression libraries, can be identified due to the interactions with a DNA sequence-of-interest that is linked to a reporter gene, such as the yeast HIS3 gene. Usually, the library is constructed in an E. coli-yeast shuttle vector designed for production of hybrid proteins consisting of a library protein and the trans-activating domain (AD) from the yeast GAL4 transcription factor. Here, we describe an optimized system of vectors for one-hybrid screenings together with detailed step-wise protocols, an elaborate trouble-shooting guide and many technical tips to conduct successful screenings. This system and other yeast genetic selection procedures derived from one-hybrid methodology proved highly useful to help understanding the regulatory networks controlling expression of the genome.

Key words

  • Yeast one-hybrid
  • Transcription factor
  • Regulatory sequence
  • pINT1 integration vector

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Acknowledgements

This work was supported by grants CerealGene Tags (European Commission FP5 project QLG2-CT-2001-01453) and NWO 901-07-206 (Netherlands Organization of Scientific Research) to P.B.F.O. and TF-STRESS (European Commission FP5 project QLK3-CT-2000-00328) and ZF-Models (European Commission FP6 project LSHG-CT-2003-503496) to A.H.M.

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Ouwerkerk, P.B.F., Meijer, A.H. (2011). Yeast One-Hybrid Screens for Detection of Transcription Factor DNA Interactions. In: Pereira, A. (eds) Plant Reverse Genetics. Methods in Molecular Biology, vol 678. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-682-5_16

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  • DOI: https://doi.org/10.1007/978-1-60761-682-5_16

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