Yeast One‐Hybrid Screening for DNA‐Protein Interactions

Pieter B.F. Ouwerkerk1, Annemarie H. Meijer1

1 Leiden University, Leiden, The Netherlands
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.12
DOI:  10.1002/0471142727.mb1212s55
Online Posting Date:  August, 2001
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Abstract

One‐hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence). In this technique, the interaction between two proteins (bait and prey) is detected via in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene. Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter‐gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans‐activating domain. The advantage of cloning transcription factors or other DNA‐binding proteins via one‐hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.

     
 
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Table of Contents

  • Basic Protocol 1: Construction and Characterization of Yeast Reporter Strains
  • Basic Protocol 2: Library Screening
  • Basic Protocol 3: Assessment of His+ Colonies: Identification of True Positives
  • Support Protocol 1: Automatic Subcloning of λ Phages into Plasmid Libraries
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction and Characterization of Yeast Reporter Strains

  Materials
  • DNA fragment or oligonucleotides, containing the bait sequence
  • Plasmids pHIS3NB or pHIS3NX and pINT1 (Table 12.12.1, Fig. ; available from P.B.F. Ouwerkerk or A.H. Meijer; and )
  • Yeast strain Y187 (Table 12.12.2)
    Table 2.2.2   MaterialsYeast Strain Genotypes

    Strain Genotype Reference
    Y187 d MATαura3‐52his3‐Δ200ade2‐101trp1‐901leu2‐3,112 Harper et al., ; Clontech
    metgal4Δgal80ΔURA3::GAL1 UAS‐GAL1 TATA‐lacZMEL1
    YPH500 d MATαura3‐52lys2‐801ade2‐101trp1‐Δ63his3‐Δ200leu2‐Δ1 Sikorski and Hieter,
    YJO d MATa gal4Δgal80Δura3‐52his3ade2‐101trp1leu2‐3,112MEL1 Leuther and Johnston, e
    YM954 d MATaura3‐52his3‐Δ200ade2‐101lys2‐801trp1‐901 Wilson et al.,
    leu2‐3, 112gal4‐Δ512gal80‐Δ538tyr1‐501
    YM4271 d MATaura3‐52his3‐Δ200ade2‐101lys2‐801trp1‐901 Liu et al.,
    leu2‐3, 112gal4‐Δ512gal80‐Δ538tyr1‐501ade5::hisG
    YPO101 f Y187 PDC6::pINT1‐HIS3 P.B.F. Ouwerkerk (unpub. observ.)

     dThese strains are suitable hosts for one‐hybrid screenings and are mentioned in the text. Use of Y187 is recommended for the procedure described in this unit.
     eThis reference refers to YJO‐Z which is a derivative of YJO, containing a GAL1lacZ fusion chromosomally integrated.
     fIn strain YPO101 a HIS3 reporter lacking an upstream bait sequence was integrated via the pHIS3/pINT1 system. This strain can serve as a control strain for the analysis of library clones.
  • Liquid YAPD medium: YPD medium (unit 13.1) supplemented with 20 mg/liter adenine hemisulfate
  • recipe10× TE buffer, pH 7.5 (see recipe)
  • recipe10× lithium acetate stock (see recipe)
  • recipe50% (w/v) polyethylene glycol (PEG) stock (see recipe)
  • 10 mg/ml YEASTMAKER carrier DNA (Clontech; also see unit 13.7 or unit 20.1)
  • YAPD‐G418 plates; YPD plates (unit 13.1) supplemented with 20 mg/liter adenine hemisulfate and G418 (150 mg/liter; add from 1000‐fold stock solution)
  • 87% (v/v) glycerol (sterilize by autoclaving for 20 min; store indefinitely at room temperature)
  • Complete minimal medium (CM) plates (unit 13.1) prepared with Difco Bacto‐Agar or Duchefa Micro‐Agar
  • CM −His plates (unit 13.1) with a concentration series of recipe3‐amino‐1,2,4‐triazole (3‐AT) (see recipe for 3‐AT)
  • 30°C incubator, with and without shaker
  • 50‐ml centrifuge tubes
  • Low‐speed tabletop centrifuge (preferably swing‐out) for 50‐ml tubes
  • 1.5‐ml microcentrifuge tubes (e.g., Eppendorf or equivalent)
  • 42°C water bath
  • 10‐ to 15‐ml tubes or flasks
  • Additional reagents and equipment for miniprep DNA isolation procedures (unit 1.6), transformation of E. coli (unit 1.8), gel electrophoresis (unit 2.5), gel extraction procedures (unit 2.6), and restriction analyses (unit 3.1)

Basic Protocol 2: Library Screening

  Materials
  • pINT1‐HIS3 reporter yeast strain(s) (see protocol 1 for construction)
  • Liquid YAPD medium: YPD medium (unit 13.1) supplemented with 20 mg/liter adenine hemisulfate
  • cDNA library plasmid
  • recipe10× TE buffer, pH 7.5 (see recipe)
  • Complete minimal medium (CM) standard‐size plates, minus leucine ( CM −Leu plates; unit 13.1) prepared with Difco Bacto‐Agar or Duchefa Micro‐Agar
  • CM −Leu −His standard and large (15‐cm wide) plates (unit 13.1), with optimal concentration of 3‐AT (see protocol 1, step ; see recipe for recipe3‐AT)
  • 30°C incubator, with and without shaker

Basic Protocol 3: Assessment of His+ Colonies: Identification of True Positives

  Materials
  • His+ positive yeast colonies (see protocol 2)
  • Liquid complete minimal medium (CM; unit 13.1) minus leucine (CM −Leu)
  • 0.9 M sorbitol/50 mM EDTA, pH 8.0; sterilize by autoclaving
  • 750 U/mg lyticase from Arthrobacter luteus (750 U/mg from Sigma, L‐4025)
  • recipe1× TE buffer, pH 7.5 (see recipe)
  • pINT1‐HIS3 reporter yeast strain(s) (see protocol 1)
  • Empty cDNA library vector
  • CM −Leu plates (unit 13.1) prepared with Difco Bacto‐Agar or Duchefa Micro‐Agar
  • CM −Leu −His, plates with optimal concentration of recipe3‐AT (see protocol 1, step ; see recipe for 3‐AT)
  • 50‐ml centrifuge tubes
  • 30°C incubator, with and without shaking
  • Low‐speed tabletop centrifuge for 50‐ml tubes (preferably swing‐out)
  • 1.5‐ml microcentrifuge (e.g., Eppendorf tubes or equivalent)
  • Additional reagents and equipment for β‐galactosidase filter lift assay (unit 13.6), miniprep DNA isolation procedures (unit 1.6), transformation of E. coli (unit 1.8), restriction analyses (unit 3.1), gel electrophoresis (unit 2.5), PCR amplification (unit 15.1), Southern blotting (unit 2.9), hybridizations (unit 2.10), sequence analysis (unit 15.2), and in vitro binding assays (units 12.2, 12.3 & 12.4)

Support Protocol 1: Automatic Subcloning of λ Phages into Plasmid Libraries

  Materials
  • cDNA synthesis kit (e.g., ZAP‐cDNA Synthesis Kit, Stratagene)
  • λACTII‐cDNA or equivalent expression library
  • Cre protein‐expressing E. coli (e.g., BNN132)
  • Liquid LB medium (unit 1.1), supplemented with 0.2% maltose, 10 mM MgSO 4, and 50 mg/liter kanamycin (add from 1000‐fold kanamycin stock)
  • recipeLB‐Cb plates (see recipe)
  • Liquid LB medium (unit 1.1) containing 200 mg/liter carbenicillin
  • Commercial maxiprep kits (optional; e.g., Qiagen or Promega)
  • 30°C and 37°C incubators, with and without shaker
  • 1.5‐ml microcentrifuge tubes (e.g., Eppendorf or equivalent)
  • Sterile cell scrapers
  • Centrifuge with large buckets (e.g., Sorvall GSA or GS‐3 or Beckman JA‐10 rotor, or equivalent)
  • Additional reagents and equipment for handling λ phages (unit 1.11), miniprep (unit 1.6) and maxiprep (unit 1.7), DNA isolation procedures, gel electrophoresis (unit 2.5), restriction analyses (unit 3.1), and quantitation of DNA via UV spectrophotometry ( appendix 3D)
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Figures

Videos

Literature Cited

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Key References
   Meijer et al., 1998. See above.
  First description of the pHIS3/pINT1 vector system for yeast reporter strain construction.
Internet Resources
  http://www.atcc.org/
  American Type Culture Collection(ATCC): source for yeast vectors and strains.
  http://www.ncbi.nlm.nih.gov/
  National Center for Biotechnology Information (NCBI, Genbank): public database of genes and vectors sequences, computational tools for sequence analysis
  http://www.clontech.com/
  Commercial sources for vectors, AD‐hybrid libraries, yeast strains, and supplies.
  http://www.stratagene.com/
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