The Yeast Two-Hybrid SystemWhat is the yeast two-hybrid system used for?How does it work?The ModelSteps to Screen a LibrarySequence analysisReporter GenesPlasmid ConstructsSample PlasmidFalse PositivesElimination of False PositivesAdvantagesExamples of Uses of the Yeast Two-Hybrid SystemReferencesReferences continuedThe Yeast Two-Hybrid SystemThe Yeast Two-Hybrid SystemAnne C. LuebkeAnne C. LuebkeWhat is the yeast two-hybrid system used for?What is the yeast two-hybrid system used for?Identifies novel protein-protein interactionsIdentifies novel protein-protein interactionsCan identify protein cascades Can identify protein cascades Identifies mutations that affect protein-Identifies mutations that affect protein-protein bindingprotein bindingCan identify interfering proteins in known Can identify interfering proteins in known interactions (Reverse Two-Hybrid System)interactions (Reverse Two-Hybrid System)How does it work?How does it work?Uses yeast as a model for eukaryotic protein Uses yeast as a model for eukaryotic protein interactionsinteractionsA library is screened or a protein is A library is screened or a protein is characterized using a bait constructcharacterized using a bait constructInteractions are identified by the transcription Interactions are identified by the transcription of reporter genesof reporter genesPositives are selected using differential mediaPositives are selected using differential mediaThe ModelThe ModelDNA-BindingDomainBait Protein Prey ProteinTranscriptionActivatingRegionReporter GeneDNA-Binding SiteSteps to Screen a LibrarySteps to Screen a LibraryCreate the Bait Plasmid Construct from the gene of Create the Bait Plasmid Construct from the gene of interest and the DNA binding domain of Gal4 or interest and the DNA binding domain of Gal4 or LexA or other suitable domainLexA or other suitable domainTransform with the bait construct a yeast strain Transform with the bait construct a yeast strain lacking the promoter for the reporter genes and lacking the promoter for the reporter genes and select for transformed yeastselect for transformed yeastTransform the yeast again with the library plasmidsTransform the yeast again with the library plasmidsSelect for interactionSelect for interactionSequence analysisSequence analysisIsolate plasmid from yeast and transform E. Isolate plasmid from yeast and transform E. colicoliPurify plasmid from E. coli and sequencePurify plasmid from E. coli and sequenceBlast sequence against database for known Blast sequence against database for known proteins or construct a possible protein proteins or construct a possible protein sequence from the DNA sequence and sequence from the DNA sequence and compare to other proteinscompare to other proteinsReporter GenesReporter GenesLacZ reporter - Blue/White ScreeningLacZ reporter - Blue/White ScreeningHIS3 reporter - Screen on His+ media (usually HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity)need to add 3AT to increase selectivity)LEU2 reporter - Screen on Leu+ mediaLEU2 reporter - Screen on Leu+ mediaADE2 reporter - Screen on Ade+ mediaADE2 reporter - Screen on Ade+ mediaURA3 reporter - Screen on Ura+ media (can URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)do negative selection by adding FOA)Plasmid ConstructsPlasmid ConstructsPlasmids are constructed with the Gal4 Plasmids are constructed with the Gal4 DNA binding domain (or other suitable DNA binding domain (or other suitable domain) in front of a Multiple Cloning Site domain) in front of a Multiple Cloning Site (MCS)(MCS)The plasmid contains genes that can be used The plasmid contains genes that can be used for selection such as Amp, Leu2, Ura3, or for selection such as Amp, Leu2, Ura3, or Trp1Trp1Sample PlasmidSample PlasmidFrom Golemis Lab HomepageFalse PositivesFalse PositivesFalse positives are the largest problem with False positives are the largest problem with the yeast two-hybrid systemthe yeast two-hybrid systemCan be caused by:Can be caused by:Non-specific binding of the preyNon-specific binding of the preyAbility to induce transcription without Ability to induce transcription without interaction with the bait (Majority of false interaction with the bait (Majority of false positives)positives)Elimination of False PositivesElimination of False PositivesSequence AnalysisSequence AnalysisPlasmid Loss AssaysPlasmid Loss AssaysRetransformation of both strain with bait Retransformation of both strain with bait plasmid and strain without bait plasmidplasmid and strain without bait plasmidTest for interaction with an unrelated Test for interaction with an unrelated protein as baitprotein as baitTwo (or more) step selectionsTwo (or more) step selectionsAdvantagesAdvantagesImmediate availability of the cloned gene of Immediate availability of the cloned gene of the interacting proteinthe interacting proteinOnly a single plasmid construction is Only a single plasmid construction is requiredrequiredInteractions are detected Interactions are detected in vivoin vivoWeak, transient interactions can be detectedWeak, transient interactions can be detectedCan accumulate a weak signal over timeCan accumulate a weak signal over timeExamples of Uses of the Yeast Two-Hybrid SystemExamples of Uses of the Yeast Two-Hybrid SystemIdentification of caspase substratesIdentification of caspase substratesInteraction of Calmodulin and L-Isoaspartyl Interaction of Calmodulin and L-Isoaspartyl MethyltransferaseMethyltransferaseGenetic characterization of mutations in Genetic characterization of mutations in E2F1 E2F1 Peptide hormone-receptor interactionsPeptide hormone-receptor interactionsPha-4 interactions in C. elegansPha-4 interactions in C. elegansReferencesReferencesBartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924.System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924.Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and clone genes for Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and