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One-Hybrid Systems f hor Detecting Protein-DNA Interactions

  • Protocol
Two-Hybrid Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 177))

Abstract

The yeast two-hybrid assay has proven useful for detecting protein-protein interactions. A variation on the theme can be used for finding proteins that interact with a particular DNA sequence. The one-hybrid assay, so far carried out only in Saccharomyces cerevisiae, in its simplest form (Fig. 1) consists of a DNA sequence of interest placed upstream of a reporter gene. The reporter gene can be either on a plasmid (1) or integrated into the chromosome (2). The protein or library being tested is cloned into a vector that expresses that protein fused to a transcription activation domain (TAD), the equivalent of the prey protein in a two-hybrid assay. This hybrid protein is expressed in the strain carrying the reporter gene. If the protein is able to interact with the sequence of interest, by either binding directly to the DNA or indirectly via interaction with a DNA-binding protein, transcription of the reporter gene is activated.

A generic one-hybrid assay. The DNA sequence of interest is placed upstream of a reporter gene with a minimal promoter, either on a plasmid or integrated into a chromosome. A hybrid protein consisting of a TAD fused to a protein that interacts with the target site is able to activate transcription of the reporter gene.

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Author information

Authors and Affiliations

  1. Lewis Thomas Lab, Princeton University, Princeton, NJ

    Mary Kate Alexander, Brenda D. Bourns & Virginia A. Zakian

Authors
  1. Mary Kate Alexander
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  2. Brenda D. Bourns
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  3. Virginia A. Zakian
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Editor information

Editors and Affiliations

  1. Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH

    Paul N. MacDonald

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© 2001 Humana Press Inc.

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Alexander, M.K., Bourns, B.D., Zakian, V.A. (2001). One-Hybrid Systems f hor Detecting Protein-DNA Interactions. In: MacDonald, P.N. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 177. Humana Press. https://doi.org/10.1385/1-59259-210-4:241

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  • DOI: https://doi.org/10.1385/1-59259-210-4:241

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-832-5

  • Online ISBN: 978-1-59259-210-4

  • eBook Packages: Springer Protocols

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