Two‐Hybrid Dual Bait System

Elena Kotova1, Thomas Coleman1, Ilya Serebriiskii1

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.7
DOI:  10.1002/0471142727.mb2007s86
Online Posting Date:  April, 2009
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Abstract

The yeast two‐hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein‐protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA‐binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA‐binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain‐fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also expressed to allow dual hybrid‐mediated transcriptional activation. Curr. Protoc. Mol. Biol. 86:20.7.1‐20.7.32. © 2009 by John Wiley & Sons, Inc.

Keywords: interactions; yeast two‐hybrid; interaction trap; interaction mating; increased specificity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Baits and Library: Characterizing Bait Proteins
  • Basic Protocol 2: Preparation of Baits and Library: Transforming and Characterizing the Library
  • Basic Protocol 3: Selecting an Interactor
  • Basic Protocol 4: Analysis of Primary Interactors
  • Alternate Protocol 1: Screening for Interaction Trap Positives by Yeast Plasmid Recovery
  • Support Protocol 1: Colorimetric Assay of β‐Galactosidase and β‐Glucuronidase Activity by Agarose Overlay
  • Support Protocol 2: Detection of Bait Protein Expression
  • Support Protocol 3: Preparation of High‐Quality Digested Library Plasmid
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Baits and Library: Characterizing Bait Proteins

  Materials
  • DNA encoding the proteins of interest
  • LexA‐fusion plasmids and controls (Table 20.7.1): pMW103 (Figs. and ), pEG202‐Krev1 (control), and pEG202‐Krit (control)
  • cI‐fusion plasmids and controls (Table 20.7.2): pGLS23 (Figs. and ), pGLS22‐EE 12345L (control), and pGLS22‐Ras (control)
  • Yeast strains (Table 20.7.3): PRT50
  • YPD plates and liquid media (unit 13.1)
  • pLacGus (Fig. )
  • 90‐ or 100‐mm complete minimal (CM) medium dropout plates (unit 13.1) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal) and 1% raffinose (Raff):
    • Glu/CM −Lys
    • Glu/CM −Ura −His
    • Glu/CM −Trp (also in 24 × 24–cm plates)
    • Gal‐Raff/CM −Lys
    • Gal‐Raff/CM −Ura −His
    • Gal‐Raff/CM −Ura −His −Leu
    • Gal‐Raff/CM −Ura −His −Lys
  • H 2O, sterile
  • 96‐well microtiter plate
  • Insert grid from a rack of 200‐µl micropipet tips
  • Tape
  • 200‐µl micropipet tips, sterile
  • Metal frogger (e.g., Dankar Scientific) or plastic replicator (Bel‐Blotter; Bel‐Art Products or Fisher)
  • Additional reagents and equipment for subcloning DNA fragments (unit 3.16) or alternative cloning strategies (e.g., in vivo recombination; Ma et al., ), lithium acetate transformation of yeast (unit 13.7), Xgal overlay assay (see protocol 6), and immunoblotting and immunodetection (see protocol 7)
    Table 0.7.1   MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

    Plasmid name Yeast selection E. coli selection Comment/description Contact information
    LexA Fusion plasmids
    pMW103 HIS3 KmR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. RB a
    pEG202 HIS3 ApR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. Origene b, MoBiTecc
    pJK202 HIS3 ApR pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus) Origene b
    pNLexA HIS3 ApR Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked Origene b
    pGilda HIS3 ApR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast Origene b
    pDD HIS3 KmR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast R. Hopkins a
    Control LexA‐fused baits
    pEG202‐Krev1 HIS3 ApR A negative control for activation and positive control for interaction with Krit1 and RalGDS I. Serebriiskii a
    pEG202‐Krit1 HIS3 ApR A moderate positive control for activation I. Serebriiskii a
    pRFHM1 (control) HIS3 ApR The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay. Origene b
    pSH17‐4 (control) HIS3 ApR GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation. Origene b
    Plasmid name (reading frames) Yeast selection E. coli selection Comment/description c
    cI‐Fusion plasmid
    pGLS23 d (A, B) HIS5 CmR ADH1 promoter expresses cI followed by polylinker
    Control cI‐fused baits
    pGKS3‐Krev HIS5 CmR Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
    pGLS22‐Ras HIS5 CmR Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d.
    pGLS22‐EE 12345L HIS5 CmR Expresses cI‐EE 12345L fusion protein. Use as positive control for activation assay.
    Name Relevant genotype Number of operators Comment/description Contact information
    PRT50 MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2 6 lexA, 3 cI Provides the most sensitive LexA‐responsive LEU2 reporter I. Serebriiskii e
    PRT475 MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2 4 lexA, 3 cI Mating partner for PRT50 strain; can be also used as a reporter strain itself. I. Serebriiskii e

     aContact R. Brent at ; R. Hopkins at ; and Ilya Serebriiskii at .
     bSee http://www.origene.com or http://www.mobitec‐germany.com.
    Table 0.7.2   MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

    Plasmid name Yeast selection E. coli selection Comment/description Contact information
    LexA Fusion plasmids
    pMW103 HIS3 KmR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. RB a
    pEG202 HIS3 ApR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. Origene b, MoBiTecc
    pJK202 HIS3 ApR pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus) Origene b
    pNLexA HIS3 ApR Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked Origene b
    pGilda HIS3 ApR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast Origene b
    pDD HIS3 KmR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast R. Hopkins a
    Control LexA‐fused baits
    pEG202‐Krev1 HIS3 ApR A negative control for activation and positive control for interaction with Krit1 and RalGDS I. Serebriiskii a
    pEG202‐Krit1 HIS3 ApR A moderate positive control for activation I. Serebriiskii a
    pRFHM1 (control) HIS3 ApR The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay. Origene b
    pSH17‐4 (control) HIS3 ApR GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation. Origene b
    Plasmid name (reading frames) Yeast selection E. coli selection Comment/description c
    cI‐Fusion plasmid
    pGLS23 d (A, B) HIS5 CmR ADH1 promoter expresses cI followed by polylinker
    Control cI‐fused baits
    pGKS3‐Krev HIS5 CmR Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
    pGLS22‐Ras HIS5 CmR Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d.
    pGLS22‐EE 12345L HIS5 CmR Expresses cI‐EE 12345L fusion protein. Use as positive control for activation assay.
    Name Relevant genotype Number of operators Comment/description Contact information
    PRT50 MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2 6 lexA, 3 cI Provides the most sensitive LexA‐responsive LEU2 reporter I. Serebriiskii e
    PRT475 MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2 4 lexA, 3 cI Mating partner for PRT50 strain; can be also used as a reporter strain itself. I. Serebriiskii e

     cContact Ilya Serebriiskii at
     dControl plasmids are cloned in a pGLS22 background; pGLS22 is identical to pGLS23 except for an extra EcoRI site outside the polylinker.
    Table 0.7.3   MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

    Plasmid name Yeast selection E. coli selection Comment/description Contact information
    LexA Fusion plasmids
    pMW103 HIS3 KmR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. RB a
    pEG202 HIS3 ApR Basic plasmids to clone bait as fusion with LexA. Expression is driven by the ADH1 promoter. Origene b, MoBiTecc
    pJK202 HIS3 ApR pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus) Origene b
    pNLexA HIS3 ApR Polylinker is upstream of LexA, which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked Origene b
    pGilda HIS3 ApR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast Origene b
    pDD HIS3 KmR GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast R. Hopkins a
    Control LexA‐fused baits
    pEG202‐Krev1 HIS3 ApR A negative control for activation and positive control for interaction with Krit1 and RalGDS I. Serebriiskii a
    pEG202‐Krit1 HIS3 ApR A moderate positive control for activation I. Serebriiskii a
    pRFHM1 (control) HIS3 ApR The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay. Origene b
    pSH17‐4 (control) HIS3 ApR GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation. Origene b
    Plasmid name (reading frames) Yeast selection E. coli selection Comment/description c
    cI‐Fusion plasmid
    pGLS23 d (A, B) HIS5 CmR ADH1 promoter expresses cI followed by polylinker
    Control cI‐fused baits
    pGKS3‐Krev HIS5 CmR Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
    pGLS22‐Ras HIS5 CmR Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d.
    pGLS22‐EE 12345L HIS5 CmR Expresses cI‐EE 12345L fusion protein. Use as positive control for activation assay.
    Name Relevant genotype Number of operators Comment/description Contact information
    PRT50 MATα, trp1, his3, his 4, ura3, lexAop‐LEU2, cIop‐Lys2 6 lexA, 3 cI Provides the most sensitive LexA‐responsive LEU2 reporter I. Serebriiskii e
    PRT475 MATa, his3, HIS5, leu2, trp1, ura3, lexAop‐LEU2, cIop‐LYS2 4 lexA, 3 cI Mating partner for PRT50 strain; can be also used as a reporter strain itself. I. Serebriiskii e

     eContact at .

Basic Protocol 2: Preparation of Baits and Library: Transforming and Characterizing the Library

  Materials
  • Yeast strains (Table 20.7.3), fresh: PRT475
  • YPD liquid media (unit 13.1) without G418 ( appendix 1K)
  • H 2O, sterile
  • TE buffer ( appendix 22)/0.1 M lithium acetate
  • Library DNA in pJG4‐5 (Fig. ) or pYesTrp (Table 20.7.4)
  • Carrier DNA, freshly denatured
  • Negative control plasmids (Table 20.7.4; optional): pJG4‐5 or pYesTrp2, pJG4‐5‐Raf1, pJG4‐5‐Krit, and pYesTrp2‐RalGDS
  • 40% (w/v) PEG 4000/0.1 M lithium acetate/TE buffer, pH 7.5
  • Dimethyl sulfoxide (DMSO)
  • 90‐mm and 24 × 24–cm Glu/CM −Trp plates (unit 13.1)
  • TE buffer ( appendix 22), sterile (optional)
  • Glycerol solution: 65% (w/v) sterile glycerol/0.1 M MgSO 4/25 mM Tris·Cl, pH 8.0 ( appendix 22)
  • Orbital shaker, 30°C
  • 50‐ml conical tubes, sterile
  • 42°C heat block
  • 3‐ to 4‐mm glass balls, sterile (Thomas Scientific or Fisher)
  • Additional reagents and equipment for lithium acetate transformation of yeast (unit 13.7)
    Table 0.7.4   MaterialsMost Commonly Used Activation Domain Fusion Plasmids and Controls

    Plasmid Yeast selection E. coli selection Comment/description Contact information
    pJG4‐5 TRP1 ApR Library construction plasmid; GAL1 promoter provides efficient expression of a gene fused to a cassette consisting of nuclear localization sequence, transcriptional activation domain, and HA epitope tag Origene, MoBiTec f
    pYesTrp TRP1 ApR GAL1 promoter expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site. Used to express cDNA libraries Invitrogen f
    Control activation domain–fusions
    pJG4‐5‐Raf TRP1 ApR A positive control for interaction with Ras I. Serebriiskii g
    pYesTrp‐RalGDS TRP1 ApR A positive control for interaction with Ras and Krev I. Serebriiskii g
    pJG4‐5‐Krit TRP1 ApR A positive control for interaction with Krev I. Serebriiskii g

     fSee http://www.origene.com, http://www.mobitec‐germany.com, or http://www.invitrogen.com.
     gContact at .

Basic Protocol 3: Selecting an Interactor

  Materials
  • Complete minimal (CM) medium dropout plates (or medium; unit 13.1) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal) and 1% raffinose (Raff):
    • Glu/CM −Ura −His
    • Glu/CM −Ura −His −Trp
    • Glu/CM −Ura −His −Trp −Leu
    • Glu/CM −Ura −His −Trp −Lys
    • Glu/CM −Leu (optional)
    • Glu /CM −Lys (optional)
    • Gal‐Raff/CM −Ura −His −Trp
    • Gal‐Raff/CM −Ura −His −Trp −Leu
    • Gal‐Raff/CM −Ura −His −Trp −Lys
    • Gal‐Raff/CM −Lys
    • Gal‐Raff/CM −Leu (optional)
    • Gal‐Raff/CM −Ura −His −Trp −Lys −Leu (optional)
  • Bait strains containing appropriate combinations of plasmids (i.e., pMW103‐Bait1 + pLacGus + pGLS23‐Bait2) on Glu/CM −Ura −His (see protocol 1)
  • Pretransformed library strain (see protocol 2)
  • H 2O, sterile
  • 90‐ or 100‐mm‐diameter YPD plates (unit 3.1)
  • Glycerol solution: 65% (w/v) sterile glycerol/0.1 M MgSO 4/25 mM Tris·Cl, pH 8.0
  • 30°C incubator with and without shaker
  • 1.5‐ml microcentrifuge tube, sterile
  • 3‐ to 4‐mm sterile glass balls (Thomas Scientific or Fisher)
  • Markers or wax pencils of different colors
  • Insert grid from a rack of 200‐µl pipet tips
  • Metal frogger (e.g., Dankar Scientific) or plastic replicator (i.e., Bel‐Blotter; Bel‐Art Products or Fisher)
  • 96‐well microtiter plate
  • Additional reagents and equipment for lithium acetate transformation of yeast (unit 13.7) and protocol 6

Basic Protocol 4: Analysis of Primary Interactors

  Materials
  • Glu/CM −Ura −His −Trp master plate (see protocol 3)
  • β‐Glucuronidase solution: dilute crude β‐glucuronidase type HP‐2 from H. pomatia (Sigma) 1:50 in 50 mM Tris·Cl, pH 7.5 ( appendix 22)/10 mM EDTA; prepare fresh
  • Library plasmid specific primers—e.g., for JG4‐5:
  • Forward primer (FP1): 5′‐CTG AGT GGA GAT GCC TCC
  • Reverse primer (FP2): 5′ CTG GCA AGG TAG ACA AGC CG
  • HaeIII (unit 3.1)
  • PRT50 containing pMW103‐Bait1 + pLacGus + pGLS23‐Bait2 (see protocol 1)
  • PRT50 containing pEG202‐Krev1 + pLacGus + pGLS22‐Ras (see protocol 1)
  • Raf1 and Krit1 PCR fragment (see protocol 8; optional)
  • Glu/CM −Ura −His −Trp dropout plates (unit 13.1)
  • Metal frogger (e.g., Dankar Scientific) or plastic replicator (i.e., Bel‐Blotter; Bel‐Art Products or Fisher)
  • 96‐well microtiter plate
  • Horizontal shaker, 37°C
  • Tape
  • 150‐ to 212‐µm glass beads (e.g., Sigma G‐1145)
  • Vortex with flat‐top surface
  • Additional reagents and equipment for PCR (unit 15.1), restriction endonuclease digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), purification of DNA fragments (unit 2.6), lithium acetate transformation of yeast (unit 3.17), sequencing (Chapter 7), rapid miniprep isolation of yeast DNA (unit 3.6), and miniprep isolation of bacterial DNA (unit 1.6).

Alternate Protocol 1: Screening for Interaction Trap Positives by Yeast Plasmid Recovery

  Materials
  • Plates containing spotted yeast colonies to be assayed
  • Chloroform
  • Xgal agarose: prepare 1% low‐melting agarose in 100 mM KHPO 4, pH 7.0, and heat to boil; cool to ∼60°C and add Xgal to 0.25 mg/ml; prepare fresh
  • Xgluc agarose: prepare as described for Xgal agarose, except replace Xgal with Xgluc

Support Protocol 1: Colorimetric Assay of β‐Galactosidase and β‐Glucuronidase Activity by Agarose Overlay

  Materials
  • Master plates containing primary transformants of each bait (see protocol 1) and positive control
  • Selective medium (unit 13.1)
  • 2× Laemmli sample buffer (unit 20.1)
  • Antibodies to LexA and cI (Invitrogen)
  • Additional reagents and equipment for minigel electrophoresis (unit 2.5)

Support Protocol 2: Detection of Bait Protein Expression

  • Empty library plasmid: pJG4‐5
  • Restriction enzymes (e.g., EcoRI and XhoI; unit 3.1)
  • pJG4‐5‐Raf1 (Table 20.7.4)
  • pJG4‐5‐Krit1 (Table 20.7.4)
  • PRT475 (Table 20.7.3)
  • Glu/CM −Trp dropout plates (unit 13.1)
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Figures

Videos

Literature Cited

Literature Cited
   Estojak, J., Brent, R., and Golemis, E.A. 1995. Correlation of two‐hybrid affinity data with in vitro measurements. Mol. Cell. Biol. 15:5820‐5829.
   Fields, S. and Song, O. 1989. A novel genetic system to detect protein‐protein interaction. Nature 340:245‐246.
   Gyuris, J., Golemis, E.A., Chertkov, H., and Brent, R. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791‐803.
   Inouye, C., Dhillon, N., Durfee, T., Zambryski, P.C., and Thorner, J. 1997. Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: Application of a differential interaction trap assay for examining protein‐protein interactions. Genetics 147:479‐492.
   Jiang, R. and Carlson, M. 1996. Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10:3105‐3115.
   Ma, H., Kunes, S., Schatz, P.J., and Botstein, D. 1987. Plasmid construction by homologous recombination in yeast. Gene 58:201‐216.
   Petermann, R., Mossier, B.M., Aryee, D.N., and Kovar, H. 1998. A recombination‐based method to rapidly assess specificity of two‐hybrid clones in yeast. Nucl. Acids Res. 26:2252‐2253.
   Reeder, M.K., Serebriiskii, I.G., Golemis, E.A., and Chernoff, J. 2001. Analysis of small GTPase signaling pathways using Pak1 mutants that selectively couple to Cdc42. J. Biol. Chem. 276:40606‐40613.
   Serebriiskii, I., Khazak, V., and Golemis, E.A. 1999. A two‐hybrid dual bait system to discriminate specificity of protein interactions. J. Biol. Chem. 274:17080‐17087.
   Serebriiskii, I.G., Khazak, V., and Golemis, E.A. 2001. Redefinition of the yeast two‐hybrid system in dialogue with changing priorities in biological research. BioTechniques 30:634‐655.
   Serebriiskii, I.G., Mitina, O., Pugacheva, E., Benevolenskaya, E., Kotova, E., Toby, G.G., Khazak, V., Kaelin, W.G., Chernoff, J., and Golemis, E.A. 2002. Detection of peptides, proteins, and drugs that selectively interact with protein targets. Genome Res. 12:1785‐1791.
   Serebriiskii, I.G., Fang, R., Latypova, E., Hopkins, R., Vinson, C., Joung, J.K., and Golemis, E.A. 2005. A combined yeast/bacteria two‐hybrid system: Development and evaluation. Mol Cell Proteomics 4:819‐826.
   Xu, C.W., Mendelsohn, A.R., and Brent, R. 1997. Cells that register logical relationships among proteins. Proc. Natl. Acad. Sci. U.S.A. 94:12473‐12478.
Internet Resource
   http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html
  Analysis of the two‐hybrid usage; database of false positives detected in two‐hybrid experiments; database of available libraries, strains, and specialized vectors (maps, sequences) for Interaction Trap/Dual Bait system; protocols related to the two‐hybrid screening and β‐galactosidase assays in yeast
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