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Yeast Two-Hybrid Screening for Proteins that Interact with Nuclear Hormone Receptors

  • Bertrand Le Douarin
  • David M. Heery
  • Claudine Gaudon
  • Elmar vom Baur
  • Régine Losson
Part of the Methods in Molecular Biology™ book series (MIMB, volume 176)

Abstract

The yeast two-hybrid system, originally developed by Fields and Song (1), is a sensitive genetic assay for the detection of protein-protein interactions. The system exploits the fact that eukaryotic transcriptional activators contain separable functional domains for DNA-binding (domain [DBD]) and transactivation (activation domain [AD]) (2)These domains cannot activate transcription when expressed as separate entities in yeast (either alone or together). However, they can function when joined noncovalently via protein-protein interactions. Thus, any pair of proteins that interact with each other may be used to bring separate DBDs and ADs together to reconstitute a functional transactivator. In a typical two-hybrid assay, one protein termed the “bait,” is expressed as a fusion with a specific DBD; the other is fused to an AD. If the two proteins interact in a yeast nucleus, transcription of reporter genes containing DBD sites will be enhanced. Using this approach, known proteins can be assayed for interaction, mutant proteins that are unable to interact with a given protein can be isolated, and libraries of AD fusion proteins can be screened for those that interact with a protein of interest. The authors laboratory has used domains from various steroid and nonsteroid nuclear receptors (NRs) as bait sequences to identify several interacting proteins that may mediate their transcriptional effects (8)

Keywords

Sodium Dodecyl Sulfate Reporter Strain Library Plasmid Bait Protein Bait Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Bertrand Le Douarin
    • 1
  • David M. Heery
    • 2
  • Claudine Gaudon
    • 1
  • Elmar vom Baur
    • 3
  • Régine Losson
    • 1
  1. 1.Institut de Génétique et de Biologie Moléculaire et CellulaireCNRS/INSERM/ULP/Collége de FranceStrasbourg
  2. 2.Department of BiochemistryUniversity of LeicesterLeicester
  3. 3.Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBoston

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