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Methods Mol Biol. 2017;1610:187-215. doi: 10.1007/978-1-4939-7003-2_13.

Identification of Protein-DNA Interactions Using Enhanced Yeast One-Hybrid Assays and a Semiautomated Approach.

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Department of Plant Biology and Genome Center, One Shields Ave., Davis, CA, 95616, USA.
Department of Plant Biology and Genome Center, One Shields Ave., Davis, CA, 95616, USA.


Yeast one-hybrid assays are an in vitro gene-centered approach to map transcription factor-DNA interactions. Here we describe this method and adaptations to screen for interactions between plant transcriptional regulators and their targets. Of particular note, the use of yeast one-hybrid assays fills in an important gap in available methodologies. When one is interested in a specific biological process of interest, the yeast one-hybrid assay is the only method that allows researchers to identify upstream regulators of the biological process of interest. This technique can be also used to further validate physical protein-DNA interactions or as a hypothesis-generating tool. In this method, promoters or DNA regions of interest are cloned and transformed into yeast and tested for interaction against a collection of transcription factors (TFs). Yeast one-hybrid screens are adaptable to the question the researcher is asking and the tools and components available. In this chapter we will describe large-scale and high-throughput Y1H screening; however, this can easily be scaled down for smaller studies.


High-throughput; Promoter; Transcription factor; Transcriptional regulation; Transcriptional regulatory network; Yeast one-hybrid

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