matchmaker library construction & screening kits user...

Matchmaker™ Library Construction & Screening Kits User Manual

PT3529-1 (PR6Z2169)Published 22 December 2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 2 Version No. PR6Z2169


I. Introduction 4

II. ListofComponents 7

III. AdditionalMaterialsRequired 9

IV. YeastStrains 11

V. YeastVectors 14

VI. ProtocolOverview:One-HybridLibraryConstruction&Screening 16

VI. ProtocolOverview:One-HybridLibraryConstruction&Screening 17

VII. ConstructingaReporterVectorforOne-HybridAnalysis 18

VIII. ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysis 19

IX. GeneratingacDNALibrary 21

X. Constructing&ScreeningOne-HybridandTwo-HybridLibraries(overview) 27

XI. Constructing&ScreeningaOne-HybridLibrary 28

XII. Constructing&ScreeningaTwo-HybridLibrary 30

ProtocolA:ScreenbyYeastMating 30

ProtocolB:ScreenbyCotransformation 35

XIII. AnalyzingPositiveInteractions 38

XIV. TroubleshootingGuide 44

XV. References 47

XVI. RelatedProducts 50

AppendixA:TypicalResultsofdscDNASynthesis 52

AppendixB:PreparationofCompetentYeastCells—LiAcMethod 53

AppendixC:One-HybridControlVectorInformation 54

AppendixD:Two-HybridControlVectorInformation 55

ListofTables

TableI. MatchmakerYeastStrainGenotypes 11TableII. MatchmakerYeastStrainPhenotypes 11TableIII. One-HybridSystemVectors 14TableIV. Two-HybridSystemVectors 15TableV. ComparisonofMatchmakerDNA-BDVectors 19TableVI. RelationshipBetweenAmountofRNAandOptimalNumberofThermalCycles 24TableVII. Set-UpforOne-HybridControlCotransformations 29TableVIII. ControlOne-HybridCotransformations:ExpectedResults 29TableIX. Set-UpforControlTwo-HybridTransformations 33TableX. Set-UpforControlTwo-HybridMatings 34TableXI. Set-UpforControlTwo-HybridCotransformations 36TableXII. ControlTwo-HybridCotransformations:ExpectedResults 37TableXIII. AssemblingMasterMixesforPCRColonyScreening 40

TableofContents

Protocol No. PT3529-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2169 3


TableofContentscontinued

ListofFigures

Figure1. Screeningforprotein-DNAinteractionswiththeMatchmaker One-HybridSystem. 4Figure2. Screeningforprotein-proteininteractionswiththeMatchmaker Two-HybridSystem. 4Figure3. Generalstepsofyeastone-hybridandtwo-hybridscreening. 5Figure4. ConstructingandscreeningMatchmakerOne-HybridandTwo-Hybridlibraries. 6Figure5. ReportergeneconstructsinyeaststrainsAH109andY187. 12Figure6. MatchmakerOne-HybridLibraryConstruction&Screening. 16Figure7. MatchmakerTwo-HybridLibraryConstruction&Screening. 17Figure8. Synthesisofhigh-qualitydscDNAusingSMART™technology. 21Figure9. CHROMASPINColumnandCollectionTubes. 26Figure10. ConstructingADfusionlibrariesbyrecombination-mediatedcloninginyeast. 27Figure11. ScreeninganADfusionlibraryfortwo-hybridinteractions. 32Figure12. Strategiesforanalyzingandverifyingputativepositive one-hybridandtwo-hybridinteractions. 39Figure13. Yeastmatingtoverifyprotein-protein(two-hybrid)interactions. 42Figure14. Double-strandedcDNAsynthesizedfromControlHumanPlacentaPolyA+RNA. 52Figure15. Mapofp53HIS2ControlVector. 54Figure16. MapofpGAD-Rec2-53ADControlVector. 54Figure17. MapofpGADT7-RecTADControlVector. 55Figure18. MapofpGBKT7-53DNA-BDControlVector. 55Figure19. MapofpGBKT7-LamDNA-BDControlVector. 56

NoticetoPurchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs, in vitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.

Practiceofthetwo-hybridsystemiscoveredbyU.S.PatentNos.5,283,173,5,468,614,and5,667,973assignedtotheResearchFoundationoftheStateUniversityofNewYork.PurchaseofanyClontechtwo-hybridreagentdoesnotimplyorconveyalicensetopracticethetwo-hybridsystemcoveredbythesepatents.CommercialentitiespurchasingthesereagentsmustobtainalicensefromtheResearchFoundationoftheStateUniversityofNewYorkbeforeusingthem.Clontechisrequiredbyitslicensingagreementtosubmitareportofallpurchasersoftwo-hybridreagentstoSUNYStonyBrook.PleasecontacttheOfficeofTechnologyLicensing&IndustryRelationsatSUNYStonyBrookforlicenseinformation(Tel:631.632.9009;Fax:631.632.1505).

SMART™TechnologyiscoveredbyU.S.PatentNos.5,962,271and5,962,272.For-ProfitandNot-For-ProfitpurchasersofSMART™Productsareentitledtousethereagentsforinternalresearch.However,thefollowingusesareexpresslyprohibited:(1)performingservicesforthirdparties;(2)identifyingnucleicacidsequencestobeincludedonnucleicacidarrays,blots,orinlibrariesorothercDNAcollectionswhicharethensoldtothirdparties.Reproduction,modification,reformulation,orresaleofthereagentsprovidedinSMART™Productsisnotpermitted.ForinformationonlicensingSMART™Technologyforcommercialpurposes,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

ThepBridgeVectoristhepropertyoftheInstitutNationaldelaSantéetdelaRechercheMédicale(INSERM).InquiriesregardingthecommercialuseorresaleofthisvectormustbedirectedtoINSERM,France.

NucleoSpin®andNucleoBond®arearegisteredtrademarksofMacherey-NagelGmbH&Co.

Parafilm®isaregisteredtrademarkoftheAmericanNationalCanCo.

Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc. ClontechisaTakaraBioCompany.©2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 � Version No. PR6Z2169


Matchmaker™LibraryConstruction&ScreeningKitsprovideasimplemethodforconstructingcDNAlibrariesforyeasttwo-hybridandone-hybridscreening.ThesekitscombineMatchmakerSystemswithSMARTcDNASynthesistechnology,whichallowsyoutoconstructcDNAlibrariesfromanytissuesourcestartingwithaslittleas1µgofpolyA+RNAortotalRNA.Followingaroutinein vivo cloningstep,youcanthenscreentheselibrariesforone-hybridandtwo-hybridinteractionsusingasensitivetranscriptionalassayprovidedbyourMatchmakerSystems.

Principleoftheone-hybridassay—aprotein-DNAinteractionassay

One-hybridassaysenableyoutoidentifyandcharacterizeproteinsthatbindtoatarget,cis-actingDNAsequence—anupstreamelementthatenhancestranscriptionfromadownstreamminimalpromoter.TheassaymayalsobeusedtomaptheDNA-bindingdomainofpreviouslyknown,ornewlyidentified,DNA-bindingproteins.WiththeMatchmakerOne-HybridSystem,youcanreadilyobtainthegenesencodingthecorrespondingDNA-bindingprotein.

InaMatchmakerone-hybridassay,potentialDNA-bindingproteinsareexpressedasfusionstotheGAL4activationdomain(AD)bycloningthecorrespondingcDNAintopGADT7-Rec2,alow-copyvectordesignedforone-hybridscreening.OneormoretandemcopiesofthetargetDNAsequenceareclonedintopHIS2,areportervectorthatcontainsthenutritionalreportergeneHIS3.InteractionbetweenaDNA-bindingproteinandthetargetsequencestimulatestranscriptionofHIS3 (Figure1),enablingtheyeasthoststrain,Y187,aHisauxotroph,togrowonminimalmedialackinghistidine.

Principleofthetwo-hybridassay—aprotein-proteininteractionassay

Two-hybridassayscanbeusedtoidentifynovelprotein-proteininteractions,confirmsuspectedinteractions,anddefineinteractingdomains.InaMatchmakerTwo-Hybridassay,abaitgeneisexpressedasafusiontotheGAL4DNA-bindingdomain(DNA-BD),whileanothergeneorcDNAisexpressedasafusiontotheGAL4activationdomain(AD;Fields&Song,1989;Chienet al.,1991).WhenbaitandlibraryfusionproteinsinteractinayeastreporterstrainsuchasAH109,theDNA-BDandADarebroughtintoproximityandactivatetranscriptionofthereportergenes:ADE2, HIS3, lacZ, andMEL1 (Figure2).

DNA-BDandADfusionsarecreatedbycloningcDNAsintotheyeastexpressionvectorspGBKT7andpGADT7-Rec.pGBKT7expressesproteinsasfusionstotheGAL4DNA-BD,whilepGADT7-RecexpressesproteinsasfusionstotheGAL4AD.Inyeast,bothfusionsareexpressedathighlevelsfromtheconstitutiveADH1promoter(PADH1).OtherGAL4-basedDNA-BDcloningvectorssuchaspGBT9,pAS2-1,andpBridgearealsocompatiblewiththiskit.pBridgeVectorcanbeusedtoperformthree-hybridassaystoidentifyternaryproteincomplexes.

Biologicalbasisforone-hybridandtwo-hybridassays

One-hybrid(andtwo-hybrid)methodsarebasedonthefindingthatmanyeukaryotictranscription factors are modular; theirtranscriptionactivatingandDNA-bindingdomainsarestructurallyandfunctionallydistinct.This allows researchers to con-struct various gene fusions that, whenexpressed as fusion proteins in yeast,cansimultaneouslybindtoatargetDNAsequenceandactivate transcriptionofadownstream reporter (Figures 1 and 2).Matchmakersystemsusethetranscriptionactivating and DNA-binding domains ofGAL4,awell-characterizedyeasttranscrip-tion factor.To learn more about GAL4-basedyeasthybridtechnology,seeZhu,L.&Hannon,G.J.,2000.

Figure1.Screeningforprotein-DNAinteractionswiththeMatchmakerOne-HybridSystem.Inthisconstruct,threecopiesoftheDNAtarget(T)havebeeninsertedintothepHIS2reportervector.

GAL UAS minimal promoter ADE2, HIS3, lacZ, and MEL1

transcriptionLibrary protein

GAL4 AD

Bait protein

GAL4 DNA-BD

Figure2.Screeningforprotein-proteininteractionswiththeMatchmakerTwo-HybridSystem.

GAL� AD

Libraryprotein

HIS3T TT minimal promoter

Transcription activator

transcription

I. Introduction



I. Introductioncontinued

ConstructingandscreeningMatchmakerone-hybridandtwo-hybridlibraries

ConstructingandscreeningMatchmakerone-hybridandtwo-hybridlibrariesconsistsoffourmainsteps(Figure3).Noticethatbothproceduresfollowthesamegeneralpath.

Ifyouintendtoscreenfortwo-hybridinteractions,thefirststep(Step1)istoconstructaDNA-BDfusionvector.If,ontheotherhand,youintendtoscreenforone-hybridinteractions,yourfirststepistoconstructaDNAtarget-HIS3 reportervector.Next(Step2),usetheSMARTreagentsweprovidetogenerateacDNAlibraryfromthepolyA+ortotalRNAthatyouprovide.

Inthecaseofyeasttwo-hybridscreening,youmayskipRNAisolation,cDNAsynthesis,andADfusionlibraryconstruction(Step3)if,insteadofpreparingyourownlibrary,youintendtoscreenoneofourmanypremadeMatchmakercDNAlibraries.Representingabroadrangeoftissues,theselibrariesareavailableasglycerolstocksorpretransformedinyeaststrainY187.WealsoofferaMatchmakerCustomLibraryService.Tousethisservice,sendusthetissueorcellsyouwishtoscreen,andwewillmaketheADfusionlibraryforyou.Pleasenote,however,thatmanyofourpremadeandpretransformedMatchmakercDNALibrariesareconstructedinhigh-copyyeastexpressionvectors,idealfortwo-hybridwork,butlesssuitableforone-hybridanalysis.WerecommendusinglowcopyvectorssuchaspGADT7-Rec2andpHIS2forone-hybridscreeningbecausetheygeneratefewerfalsepositives.

FollowingcDNAsynthesis,constructaGAL4ADfusionlibrarybycloningthecDNAintooneofourMatchmakerADCloningVectors:pGADT7-Rec2ifyouareconstructingaone-hybridlibrary;pGADT7-Rec if you are constructing a two-hybrid library.The cloning takes place in vivo viahomologousrecombination(Figure4).ThissteptakesadvantageofthehighlyefficientrecombinationsysteminyeasttofusedscDNAwiththeappropriateGAL4ADplasmid.Withrecombination-mediatedcloning,libraryconstructionandscreening(Steps3and4)canbecompletedinquicksuccession without the need for any bacterial transformation or amplification steps. SimplytransformyeastwiththecDNAlibraryandtheappropriateMatchmakervectors;thenspreadthecellsondropoutmediumtoselectforone-hybridortwo-hybridinteractions.

1. Construct a DNA target-reporter vector by cloning your DNA target sequence into pHIS2. Section VII

1. Construct a DNA-BD fusion vector by cloning your bait gene into pGBKT7 or other GAL4 DNA-BD vector. Section VIII

2. Generate a cDNA library from any tissue or cell source using SMART™ cDNA Synthesis technology. Section IX

One-Hybrid Library Construction & ScreeningOverview: Section VI

Two-Hybrid Library Construction & ScreeningOverview: Section VI

3. Construct a GAL� AD fusion library using homologous recombination. Sections XI & XII

�. Screen for one-hybrid or two-hybrid interactions by yeast mating or transformation. Sections XI & XII

Figure3.Generalstepsofyeastone-hybridandtwo-hybridscreening.Formoredetailedflowchartsoftheone-hybridandtwo-hybridprocedures,pleaserefertoFigures6and7.



SMART™ cDNA synthesis and amplification by LD-PCR

ds cDNA with SMART™ III & CDS III anchors

Plate on selective mediumto screen for one-hybrid or two-hybrid interactions

Homologous recombination in yeast mediates GAL� AD Vector assembly

Steps 2 & 3:Library construction(in vivocloning)~ 2 hours

Step �:Screening3 days

Prepare competent yeast cells and

transform

GAL4 AD LEU2

pGADT�-Rec[2]AD Vector

Sma I-linearized

pGBKT�Two-Hybrid

DNA-BD VectorGAL4 DNA-BD

protein bait

Amp

GAL4 AD LEU2

Step 1:cDNAsynthesis~ 5 hours

TRP1

Kanr

Ampr

pHIS2One-Hybrid

Reporter Vector

DNA target

TRP1

Kanr

MCS

HIS3

pGBKT7

or

pHIS2

pGADT�-Rec[2]AD Vector

Sma I-linearized

I. Introductioncontinued

Figure 4. Constructing and screening Matchmaker One-Hybrid andTwo-Hybrid libraries. As this diagram shows,recombination-mediatedcloningmakeslibraryconstructionandscreeningquickandefficient.Thoughnotshownhere,two-hybridlibrariescanalsobescreenedbyyeastmating(SeeSectionXIIfordetails).FordetailsaboutSMARTcDNAsynthesis and amplification, please refer to Section IX. pGADT7-Rec is used for two-hybrid library construction andscreening,whilepGADT7-Rec2isusedforone-hybridlibraryconstructionandscreening.Thoughrelated,thetwovectors,denotedaspGADT7-Rec[2]inthefigure,havedifferentreplicationelements.SeeSectionXandthecorrespondingVectorInformationPacketsformoreinformation.

Protocol No. PT3529-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2169 �


This kit contains sufficient reagents to make 5 one-hybrid (Cat. No. 630304) or 5 two-hybrid(Cat.No.630445)libraries.

StoreDeionizedH2O,CHROMASPINColumns,NaClSolution,Dropout(DO)Supplements,NaOAc,LiAc,PEG,TEBuffer,andYPDPlusMediumatroomtemperature.Storeyeaststrains,ControlPolyA+RNA,andtheSMARTIIIOligoat–70°C.Storeallotherreagentsat–20°C.

First-strandcDNAsynthesis• 10µl SMARTIIIOligo(10µM;5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3')• 10µl CDSIIIPrimer(10µM;5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN-3')*• 10µl CDSIII/6Primer(10µM;5'-ATTCTAGAGGCCGAGGCGGCCGACATG-NNNNNN-3')* *N=A,G,C,orT;V=A,G,orC

• 20µl MMLV(MoloneyMurineLeukemiaVirus)ReverseTranscriptase• 7µl RNaseH• 100µl 5XFirst-StrandBuffer(250mMTris(pH8.3);30mMMgCl2;375mMKCl)• 100µl DTT(dithiothreitol;20mM)• 5µl ControlPolyA+RNA(HumanPlacenta;1µg/µl)• 50µl dNTPMix(dATP,dCTP,dGTP,dTTP,10mMeach)• 500µl DeionizedH2O(Cat.No.630445only)

cDNAamplification• 50µl 5'PCRPrimer(10µM;5'-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3')

• 50µl 3'PCRPrimer(10µM;5'-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3')

• 500µl 10XGC-MeltSolution

cDNApurification• 10 CHROMASPIN+TE-400Columns• 300µl SodiumAcetate(3M;pH4.8)

One-HybridLibraryConstruction(Cat.No.630304)• 20µg pHIS2ReporterVector(500ng/µl)• 20µg pGADT7-Rec2ADCloningVector(Sma I-linearized;500ng/µl)• 20µg pGAD-Rec2-53ControlVector(500ng/µl)• 20µg p53HIS2ControlVector(500ng/µl)• 0.5ml S. cerevisiae strainY187• 50ml NaClSolution(0.9%)• 10g –LeuDOSupplement• 10g –TrpDOSupplement• 10g –Leu/–TrpDOSupplement• 10g –His/–Leu/–TrpDOSupplement

Two-HybridLibraryConstruction(Cat.No.630445)• 20µg pGBKT7DNA-BDCloningVector(500ng/µl)• 25µg pGADT7-RecADCloningVector(Sma I-linearized;500ng/µl)• 20µg pGBKT7-53ControlVector(500ng/µl)• 20µg pGBKT7-LamControlVector(500ng/µl)• 20µl SV40Large-TPCRFragment(25ng/µl)• 0.5ml S. cerevisiae strainAH109

II. ListofComponents

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 � Version No. PR6Z2169


II. ListofComponentscontinued

• 0.5ml S. cerevisiae strainY187• 50ml NaClSolution(0.9%)• 10g –LeuDOSupplement• 10g –Leu/–TrpDOSupplement• 10g –Ade/–His/–Leu/–TrpDOSupplement

YeastmakerYeastTransformationSystem2(Cat.No.630439)includesthefollowing:• 50ml 1MLiAc(10X)• 50ml 10XTEBuffer• 50ml YPDPlusLiquidMedium• 20µl pGBT9(0.1µg/µl;controlplasmid)• 2x1ml HerringTestesCarrierDNA,denatured(10mg/ml)• 2x50ml 50%PEG3350



Thefollowingreagentsarerequiredbutnotsupplied.Storeallreagentsandsolutionsatroomtemperature(20–22°C)unlessspecifiedotherwise.

First-strandcDNAsynthesisandSMART™PCRcDNAamplification• Advantage®2PCRKit(Cat.Nos.639206&639207)• Sterile,0.5-mlmicrocentrifugetubes• PolyA+ortotalRNA• Mineraloil• ThermalCycler Note:Thecyclingparametersinthisprotocolweresetusingahot-lidthermalcyclerandmaynotbeoptimalfor

nonhot-lidcyclers.

• DNAsizemarkers(1-kbDNAladder)

• 1.2%Agarose/EtBrgel

cDNAsizefractionation• 1.5-mlsterilemicrocentrifugetubes• Ring-standwithsmallclampforholdingCHROMASPINcolumns• 95%ethanol(–20°C)• 1%xylenecyanol

Constructingbaitplasmids• CompetentE. colicells.UseageneralpurposestrainsuchasDH5αorFusion-Blue

CompetentCells(Cat.No.636700) Fusion-BlueCompetentCellsareanE. coli K-12strainthatprovideshightransformation

efficiency paired with blue-white screening capability when used with the appropriateplasmids.ThestraincarriesrecAandendAmutationsthatmakeitagoodhostforobtaininghighyieldsofplasmidDNA.

• T4DNAligase• 10XT4ligationbuffer(Sambrooket al.,1989;orthebufferprovidedwiththeenzyme)• LB/ampplates• 50mMNaCl• MaterialsforpurifyingplasmidfromE. colitransformants

Yeasttransformation(Preparereagentsinsterilecontainers)• PEG/LiAcSolution(polyethyleneglycol3350/lithiumacetate) Prepareafresh10-mlsolutionjustpriortotransformationusingthestocksolutionsprovided:

Mix8mlof50%PEG3350with1mlof10XTEBufferand1mlof1MLiAc(10X).• 1.1XTE/LiAcSolution Shouldbefreshlypreparedbeforeeachtransformationusingthestocksolutionsprovided:

Combine1.1mlof10XTEwith1.1mlof1MLiAc(10X).Bringthetotalvolumeto10mlusingsterile,deionizedH2O.

• DimethylSulfoxide(DMSO;SigmaCat.No.D8418)

III. AdditionalMaterialsRequired



Yeastculture&mating• YPDMediumCat.No.630409;orprepareyourown;seetheYeastProtocolsHandbook

(PT3024-1)• YPDAmedium(YPDmediumsupplementedwith30mg/Ladeninehemisulfate;seethe

YeastProtocolsHandbook)• TEbufferorsterile,distilledH2O• Appropriatesteriletubesorflasksfortransformations• 100-and150-mmcultureplates• Sterileglassrod,bentpasteurpipette,or5-mmglassbeadsforspreadingcellsonplates• X-α-Gal(Cat.No.630407)forblue/whitescreeningofyeasttwo-hybridsexpressingMEL1 (α-galactosidase)• MinimalSDBasewithandwithoutagar(Cat.Nos.630412and630411)• 3-amino-1,2,4-triazole(3-AT;SigmaCat.No.A8056;forsuppressingbackgroundgrowth

onSDminimalmedialackingHis)• Kanamycinstocksolution(50mg/mlinH2O;1000X);Storeat–20°C.

• Ampicillinstocksolution(50mg/mlinH2O;1000X);Storeat–20°C.

Long-termlibrarystorage• 100%Glycerol• FreezingMedium:YPDmediumwith25%(v/v)glycerol

Two-HybridLibraryConstruction&ScreeningThe following dropout (DO) supplements are not supplied with the Matchmaker LibraryConstruction&ScreeningKit(Cat.No.630445).YoumustobtainthesesupplementsseparatelyfromacommercialsupplierorpreparethemyourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).• –TrpDOSupplement RequiredforselectionofMatchmakerDNA-BDcloningvectorsinyeast• –His/–Leu/–TrpDOSupplement Optional triple-dropout supplement for low stringency screening of yeast two-hybrid

libraries

• –His/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithaDNA-BDplasmidforbackgroundgrowth

onSDminimalmedialackingHis

• –Ade/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithaDNA-BDplasmidforbackgroundgrowth

onSDminimalmedialackingAde

One-HybridLibraryConstruction&ScreeningThefollowingdropout(DO)supplementisnotsuppliedwiththeMatchmakerOne-HybridLibraryConstruction&ScreeningKit(Cat.No.630304).YoumustobtainthissupplementseparatelyfromacommercialsupplierorprepareityourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).• –His/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithapHIS2reporterplasmidforbackground

growthonSDminimalmedialackingHis

PCRColony-Screening• MatchmakerADLD-InsertScreeningAmplimerSet(Cat.No.630433)

III. AdditionalMaterialsRequiredcontinued



Foradditionalinformationonthegrowthandmaintenanceofyeast,seetheYeastProtocolsHandbook(PT3024-1).WealsorecommendGuthrie&Fink’sGuide to Yeast Genetics and Molecular Biology (1991)andHeslot&Gaillardin’sMolecular Biology and Genetic Engineering of Yeasts (1992).

A. Genotypes

table i. matchmaker yeast strain genotypes

Strain Genotypea References

AH109b MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, Jameset al.,1996; gal4Δ,gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, Ourunpublished GAL2UAS -GAL2TATA-ADE2, observations URA3 : : MEL1UAS-MEL1TATA-lacZ, MEL1

Y187 MATα,ura3-52,his3-200, ade2-101, trp1-901, Harperet al.,1993 leu2-3,112,gal4Δ,met –,gal80Δ, URA3 : : GAL1UAS-GAL1TATA-lacZ, MEL1

a TheGAL1,GAL2,andMEL1upstreamactivatingsequences(UASs)areresponsivetotheGAL4transcriptionalactivator.Thetrp1, his3, gal4, and gal80mutationsarealldeletions;leu2-3, 112isadoublemutation.

b AH109isaderivativeofstrainPJ69-2AandincludestheADE2 andHIS3 nutritionalmarkersandanendogenousMEL1 gene(Jameset al.,1996).ThelacZ reportergenewasintroducedintoPJ69-2AtocreatestrainAH109.

B. Phenotypes ItisimportanttoverifythephenotypesoftheAH109andY187strains(TableII).

1.Torecoverstrainsfromfrozenstock,scrapeasmallamountofcellsfromthesurfacewithasterilelooporwoodenstickandstreakthemontoYPDAplates.

2.Incubateplatesat30°Cfor3–5daysuntilcoloniesappear.Propagateadditionalculturesonlyfromisolatedcoloniesonthisworkingstockplate.

Notes: • AH109(andtransformantsderivedfromthisstrain)shouldbemaintainedonadenine-supplementedYPD

(i.e.,YPDA)foroptimalviabilityofthestrainandtopreventselectionofspontaneousade1 orade5 mutations(Guthrie&Fink,1991).

• Ifyoucannotrecoverthestrainbyscrapingthefrozenstock,thecellsmayhavesettledtothebottomofthetubebeforethestockwasfrozen.Ifthishappens,thawthefrozencultureoniceandvortexitbeforerestreaking.

• Althoughnonlibrarystockculturesmaybethawedandrefrozenseveraltimeswithoutsignificantlydecreasingtheviability,werecommendthatyoudividetheonce-thawedstockintoaliquotsbeforeyourefreezeit.Thiswillkeeptheviabilityhigherandwillreducetheriskofbacterialcontamination.

3.TestforthenutritionalrequirementsshowninTableII. a. Usingasterilelooportoothpick,streak3–4coloniesfromtheworkingstockonto

separate,appropriateSDselectionplates. b. Incubateplatesat30°Cfor4–6days.YeastgrowssloweronSDselectionmedium

thanonYPDA. c. CompareyourresultswiththoseshowninTableII.ProceedonlyifAH109andY187

havetheexpectedphenotypes. 4.Usewell-isolatedcoloniesfromtheverifiedworkingstockplatetoinoculateliquidcultures

formatingorforpreparingcompetentcells.SealtheverifiedworkingstockplatewithParafilmandstoreat4°C.table ii. matchmaker yeast strain phenotypes

table ii. matchmaker yeast strain phenotypes

Strain SD/–Ade SD/–Met SD/–Trp SD/–Leu SD/–His SD/–Ura YPDA

AH109 – + – – – + +

Y187 – – – – – + +

Notes:

• AH109andY187cangrowonSD/–Leu/–TrpiffunctionalTRP1 andLEU2genesareintroduced.

• AH109 andAH109/Y187 diploids can grow on SD/–Ade/–His if the ADE2 and HIS3 genes—carried byAH109—areactivated(i.e.,inthepresenceofGAL4).

IV. YeastStrains



C. Matingtypecompatibilities • Y187(MATα)canmatewithAH109,HF7c,CG-1945,Y190,orSFY526(allMATa).

D. ColonyColorandSize • Y187carriestheade2-101mutationandAH109exhibitstheAde–phenotypeintheabsenceof

GAL4.Onmediumwithlowamountsofadenine,thecolonieswillturnpinkafterafewdaysandmayturndarkerasthecolonyages.WhengrownonAde-supplementedmedium,thecolorchangemaynotbenoticeable.Thesecoloniesgrowto>2mmindiameter.However,small(<1mm)whitecolonieswillformatarateof1–2%duetospontaneousmutationsthateliminatemitochondrialfunction(Holm,1993).Avoidthesewhitecolonieswheninoculatingcultures.

• Y187growsmoreslowlyandformsnoticeablysmallercolonies(onaverage)thanAH109.

E. ReportergenesAH109containsfourreporters—ADE2,HIS3,MEL1, and lacZ—underthecontrolof threedistinctGAL4upstreamactivatingsequences(UASs)andTATAboxes(Figure5).TheADE2 reporteraloneprovidesstrongnutritionalselection.Forhigherstringency,andtoreducetheincidenceoffalsepositives,selectforADE2andHIS3(Jameset al.,1996).YoualsohavetheoptionofassayingforMEL1,whichencodesα-galactosidase.MEL1isendogenoustobothY187andAH109.Becauseα-galactosidaseisasecretedenzyme,itsactivitycanbedetectedbyaddingX-α-Gal(Cat.No.630407)totheselectionplate:IfMEL1 isactiveandX-α-Galispresent,thecolonywillturnblue.lacZ inY187exhibitsahighlevelofinducedβ-galactosidaseactivityinapositivetwo-hybridassaybecauseitisunderthecontroloftheintactGAL1UAS.

GAL1 UAS GAL1 TATA lacZ

Y1�� Constructs

AH109 Constructs

GAL1 UAS GAL1 TATA HIS3

GAL2 UAS GAL2 TATA ADE2

lacZMEL1 UAS MEL1 TATA

lacZMEL1 UAS MEL1 TATA MEL1

lacZ

Figure5.ReportergeneconstructsinyeaststrainsAH109andY187.StrainAH109isaderivativeofstrainPJ69-2AandincludestheADE2andHIS3nutritionalmarkers(Jameset al.,1996).MEL1isanendogenousGAL4-responsivegene.ThelacZ reportergenewasintroducedintoPJ69-2AtocreatestrainAH109.TheHIS3, ADE2,andMEL1/lacZ reportergenesareunderthecontrolofthreecompletelyheterologousGAL4-responsiveUASandpromoterelements—GAL1,GAL2,andMEL1,respectively.

IV. YeastStrainscontinued



F. LeakyHIS3 expression • 3-amino-1,2,4-triazole(3-AT)isacompetitiveinhibitoroftheyeastHIS3 protein(His3p).

3-ATisusedtoinhibitlowlevelsofHis3pexpressedinaleakymannerandthustosuppressbackgroundgrowthonSDmediumlackinghistidine(Fields,1993;Durfeeet al.,1993).

• TransformantsderivedfromAH109mayshowslightlyelevatedHIS3 expressionbecauseofintrinsicDNA-bindingpropertiesofthebaitprotein.Asmallamountof3-ATisgenerallysufficienttosuppressbackgroundgrowthonSD/–His.However,ifyouareselectingforbothHIS3andADE2expression,itisgenerallynotnecessarytosuppressHIS3leakinessintheinitiallibraryscreen.

• SomeyeaststrainshaverelativelyhighbasallevelsofHis3p.IfyouuseY190(MATa)asahoststrain,25–45mM3-ATwillberequiredinthemediumtosuppressbackgroundgrowth.

• Tooptimizethe3-ATconcentrationinyourselectionmedium:

Beforestartingthisprocedure,notethat–His/–TrpDropoutSupplementisnotsuppliedwiththiskit.Youmustpurchase–His/–TrpdropoutsupplementseparatelyorprepareyourownusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).

1. Plate yeast transformants on a series of SD/–His/–Trp plates containing differentconcentrationsof3-AT.

• IfyouareworkingwithAH109transformantscontainingDNA-BDplasmidssuchaspGBKT7,werecommendyoustartbytesting[3-AT]intherange0to15mM(e.g.,0,2.5,5,7.5,10,12.5,and15mM).

• IfyouareworkingwithY187transformantscontainingpHIS2reporterplasmids,werecommendyoustartbytesting[3-AT]intherange10to60mM.

Note:Thesearerecommendationsonly.Theoptimalconcentrationmaybeslightlyhigherorlowerdependingontheconstructandstrainused.

2. Usethelowestconcentrationof3-ATthat,afteroneweek,allowsonlysmall(<1mm)coloniestogrow.Toomuch3-ATinthemediumcankillfreshlytransformedcells.

IV. YeastStrainscontinued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 1� Version No. PR6Z2169


A. One-HybridSystem 1.CloningVectors • pHIS2 is a one-hybrid reporter vector that contains the HIS3 nutritional reporter

gene.Ithasamultiplecloningsite(MCS)upstreamoftheHIS3 reportergenesothatacis-actingDNAtargetsequencecanbeinserted,andtherefore,linkedtotheminimalpromoteroftheHIS3 locus(PminHIS3).ItalsocontainsaCEN6/ARS4 sequenceforstable,low-copypropagationinyeast.

• pGADT7-Rec2isacloningvectorthatcanbeusedtoexpressaproteinofinterestasafusionwiththeGAL4activationdomain(AD).Thisvectorisengineeredfor homologousrecombination-mediatedcloninginyeast(Figure4).Thus,youcanconstructcDNA/ADfusionlibrariesbytransformingyeastwithSma I-linearizedpGADT7-Rec2(provided)anddscDNAgeneratedwiththeSMARTlibraryconstructionprotocol(SectionIX).

2.ControlVectors(AppendixC) • p53HIS2isapositivecontrolreportervectorthatcontainsthreetandemcopiesofthe

cis-actingDNAconsensussequencerecognizedbyp53.p53HIS2wasconstructedbyinsertingtheDNAtargetsintothemultiplecloningsiteofpHIS2.Asaresult,theDNAtargetsarepositionedjustupstreamoftheminimalpromoteroftheHIS3locus(PminHIS3)andtheHIS3 reportergene.

• pGAD-Rec2-53isapositivecontrolvectorthatencodesmurinep53asafusionwiththeGAL4AD.Yeastcellsthatcontainbothp53HIS2andpGAD-Rec2-53shouldgrowonminimalSDmedialackinghistidine—i.e.,onSD/–His/–Leu/–Trp.

TABLEIII.ONE-HYBRIDSYSTEMVECTORS

Use Epitopea YeastselectionbBacterialselectionc

Cloningvectors pHIS2 LinkanyDNAtargettoHIS3 TRP1 kanamycin pGADT7-Rec2 ExpresspotentialDNA-binding (Sma I-linearized) proteinsasADfusions HA LEU2 ampicillin

Controlvectors p53HIS2b DetectDNA-bindingactivity TRP1 kanamycin ofp53 pGAD-Rec2-53 Expressp53asanADfusion HA LEU2 ampicillin

a HA=hemagglutinin;Theseepitopetagscanbeusedtoverifyprotein-proteininteractionsin vitrobycoimmunoprecipitation(Co-IP)usingtheantibodiesandprotocolprovidedwiththeMatchmakerCo-IPKit(Cat.No.630449).Theyarenotintendedtobeusedfordetection,affinitypurification,orCo-IPofhybridproteinsexpressedinyeast.

bThereporterandADvectorshavedifferentnutritionalmarkers,sotheycanbe independentlyselectedwhenyeasttransformantsareplatedonSDminimalmediumlackingspecificnutrients.Theselectionmediumyouchoosedependsonwhichplasmidsyouareusing,whetheryouareselectingforoneortwoplasmids,andwhetheryouareselectingforcoloniesinwhichonehybridinteractionsareoccurring.

c ThevectorscarrydifferentantibioticmarkerssothattheycanbeindependentlyselectedinE. coli.

V.YeastVectors



B Two-HybridSystem 1.CloningVectors • pGBKT7:UsedtoexpressaproteinofinterestasafusionwiththeGAL4DNAbinding

domain(DNA-BD). • pGADT7-Rec:UsedtoexpressaproteinofinterestasafusionwiththeGAL4activation

domain (AD).Thisvector isengineered for homologousrecombination-mediatedcloning.pGADT7-RecisprovidedasSma I-digestedlinearDNA.

2.ControlVectors a. PositiveControl • pGBKT7-53encodesafusionbetweentheGAL4DNA-BDandmurinep53. • SV40LargeTPCRFragmentencodesSV40largeT-antigen.UsethisDNAfragment

togetherwithpGADT7-Rectocheckthetransformation-recombinationefficiencyinyeast.In vivo,SV40LargeTPCRFragmentandpGADT7-RecrecombinetoformpGADT7-RecT, which encodes a fusion between the GAL4 AD and largeT-antigen.

• p53andSV40largeT-antigeninteractinayeasttwo-hybridassay(Li&Fields,1993;Iwabuchiet al.,1993).

b. NegativeControl • pGBKT7-Lamencodesa fusionof theGAL4DNA-BDwithhumanlaminCand

providesacontrolforafortuitousinteractionbetweenanunrelatedproteinandeitherthepGADT7-RecTcontroloryourAD/libraryplasmid.LaminCneitherformscomplexesnorinteractswithmostotherproteins(Bartelet al.,1993b;S.Fields,pers.comm.;Ye&Worman,1995).

TABLEIV.TwO-HYBRIDSYSTEMVECTORS

Use Epitopea Yeastselectionc Bacterialselectiond

Cloningvectors pGBKT7 Expressanyprotein c-Myc TRP1 kanamycin asaGAL4DNA-BDfusion pGADT7-Rec Expressanyproteinasa HA LEU2 ampicillin (Sma I-linearized) GAL4ADfusion

Controlvectors pGADT7-RecT b ExpressSV40largeT-antigen HA LEU2 ampicillin asaGAL4ADfusion pGBKT7-53 Expressp53asaGAL4 c-Myc TRP1 kanamycin DNA-BDfusion pGBKT7-Lam ExpresslaminCasaGAL4 c-Myc TRP1 kanamycin DNA-BDfusion

a HA=hemagglutinin;Theseepitopetagscanbeusedtoverifyprotein-proteininteractionsin vitrobycoimmunoprecipitation(Co-IP)usingtheantibodiesandprotocolprovidedwiththeMatchmakerCo-IPKit(Cat.No.630449).Theyarenotintendedtobeusedfordetection,affinitypurification,orCo-IPofhybridproteinsexpressedinyeast.

b Createdbyhomologousrecombinationin vivo bycotransformingyeastwithSV40LargeTPCRFragmentandpGADT7-Rec.

c TheDNA-BDandADvectorshavedifferentnutritionalmarkers,sotheycanbeindependentlyselectedwhenyeasttransformantsareplatedonSDminimalmediumlackingspecificnutrients.Theselectionmediumyouchoosedependsonwhichplasmidsyouareusing,whetheryouareselectingforoneortwoplasmids,andwhetheryouareselectingforcoloniesinwhichtwohybridproteinsareinteracting.

d ThevectorscarrydifferentantibioticmarkerssothattheycanbeindependentlyselectedinE. coli..

V.YeastVectorscontinued



Figure6.MatchmakerOne-HybridLibraryConstruction&Screening.

First-strand synthesis coupled with (dC) tailing by RT

Amplificationby LD-PCR

Poly A+ RNA

poly A 3'

SMART™ IIIOligonucleotide

CDS III oligo(dT) or random primer

Template switching and extension by RT

poly A

poly A


GGG5'CCC

5'

GGG5'

CCC

VI.ProtocolOverview:One-HybridLibraryConstruction&Screening

Protocol No. PT3529-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2169 1�


100 ng of total RNA or 25 ng of poly A+ RNA

Amplify ds cDNA by LD-PCR.

Library Construction [One-Step]

Synthesize first-strand cDNA.

Select for transformants expressing interacting proteins.

Cotransform yeast strain AH109 with: • ds cDNA • pGADT7-Rec • DNA-BD/bait plasmid

Purify (size-select) ds cDNA.

Generate a cDNA LibrarySection IX

AnalysisVerify positive interactions.

Cotransform yeast strain AH109 with: • ds cDNA • pGADT7-Rec

Select transformants on SD/–Leu.

AnalysisVerify positive interactions.

Harvest (Pool) Transformants.

Mate with Y187 pretransformed with DNA-BD/bait plasmid.

Select for yeast diploids expressing interacting proteins.

Active or Toxic

Construct a DNA-BD FusionSection VII

Transform AH109 and Y187 with bait plasmid.Test for autonomous reporter gene activation

and cell toxicity.

See Troubleshooting Guide.

Two-Hybrid Screening

Protocol A• Construct an AD Fusion Library• Screen by Yeast Mating Section XII

Clone bait gene into pGBKT7 or other Matchmaker GAL4-based DNA-BD vector.

Inactive and Nontoxic

Two-Hybrid Screening

Prepare Y187 cells for mating.

Library Construction [Three-Step]

Protocol B• Construct an AD Fusion Library• Screen by Cotransformation Section XIIor

or

Prepare competent AH109 yeast cells

Appendix B

Prepare competent yeast cellsAppendix B

Figure7.MatchmakerLibraryConstruction&ScreeningKit.Two-hybridlibrariesmaybescreenedbyeitheryeastmatingorcotransformation.

VI.ProtocolOverview:Two-HybridLibraryConstruction&Screening



A. Background

To use the Matchmaker One-Hybrid System to screen a cDNA library for DNA-bindingproteins,youmusthaveidentifiedatrueorputativetargetelement.Itmustbepreciselydefinedusing,forexample,deletionand/orpointmutationanalysis.Aconstructcomposedofoneormoretandemcopiesofyourtargetregulatoryelementborderedbyrestrictionsitesisthenpreparedandinsertedintothemultiplecloningsite(MCS)ofpHIS2.ThislinksthetargetelementtotheHIS3 reportergene.InsertingyourtargetelementmayalterthelevelofbackgroundHIS3expression.Therefore,constructsshouldbetestedforbackground(leaky)HIS3 expressionbeforeyoustartaone-hybridanalysis.BackgroundgrowthduetoleakyHIS3expressioniscontrolledbyadding3-ATtotheselectionmedium,asdescribedinSectionIV.F.

B. SynthesizeYourTargetElement

Eachtarget-reporterconstructshouldcontainatleastonecopyoftheDNAtargetelementinsertedupstreamofthereportergene.ManyearlystudiesindicatedthatthereportershouldcontainatleastthreetandemcopiesoftheDNAtarget.However,asWeiet al. (1999)havedemonstrated,asinglecopymaybesufficientinmanycases.Formoreinformationabouttargetcopynumber,seeGhoshet al.,1993.Tandemcopiesmaybegeneratedbyvariousmethods,butwehavefoundthemostconvenientandreliablemethodforgeneratingthemtobeoligonucleotidesynthesis. It works nicely because well-defined regulatory elements are usually <20 bp.

1.Design twoantiparalleloligonucleotides,one representing thesensestrandand theotheritsantisensecomplement.

Note:Thesensestrandshouldconsistofoneormorecopiesofthetargetelementwithadifferentrestrictionsiteoneachend.Whenthetwostrandsareannealed,theresultingdouble-strandedDNAwillhaveadifferentoverhangateachendfordirectionalcloningintopHIS2.SeethepHIS2VectorInformationPacket(PT3705-5)foradiagramofthemultiplecloningsite.

2.Synthesizebothstrandswithout5'phosphates(accordingtotheprotocolofthesynthesizermanufacturer).

C. InsertYourDNATargetintotheMultipleCloningSiteofpHIS2 1.Foreachconstructplanned,mix0.1µgofsense-strandand0.1µgofantisense-strand

oligonucleotidein10µlof50mMNaCl. 2.Annealtheoligonucleotidesbyheatingat70°Cfor5minandthenslowlycoolingto

roomtemperature(~30min). 3.Completelydigest0.1µgofpHIS2ina20-µldoubledigestusinganappropriatepairof

restrictionenzymes.Incubateat37°Cfor2hr,orasdirectedbytheenzymemanufacturer. 4.Electrophoresea2-µlsampleofthedigestona1%agarosegeltoconfirmthattheplasmid

hasbeenefficientlylinearized. 5.Combine5µlofdigestedplasmidwith1µlofannealedoligoand4µlofH2O. 6.Add 1.2 µl of 10XT4 ligation buffer and 0.8 µl (at least 0.8 units) ofT4 DNA ligase,

andincubateatroomtemperaturefor4hr. Note:Sincethemolarratioofoligonucleotidetovectoris100:1orgreater,gelpurificationtoremovethestuffer

fragmentisunnecessary.

7.SeparatelytransformcompetentE. coli cellswitheachconstructusingastandardmethod(Sambrooket al.,1989).WerecommendusingageneralpurposestrainsuchasDH5α orFusion-BlueCompetentCells.

8.PlatetransformantsonLB/kanplates,andincubateat37°Covernight. 9.PrepareplasmidusinganystandardmethodthatyieldshighlypureDNA(Sambrook

et al.,1989).Checkforinsertsbyelectrophoresingona2%agarosegelandsequencingacrossthejunctions.

D. TestyourTarget-ReporterConstructforBackgroundHIS3 Expression 1.TransformY187 with the target-reporter construct using a small-scale protocol. (For

example,seethesmall-scaleprotocolusedinSectionXI.C.) 2.FollowtheprocedureinSectionIV.Ftodeterminetheoptimumconcentrationof3-AT

touseintheselectionmedium.Forexample,wefindthat10mM3-ATissufficienttosuppressbackgroundgrowthofY187cellstransformedwithp53HIS2ControlVector.

VII.ConstructingaReporterVectorforOne-HybridAnalysis



A. ConstructaDNA-BDFusion • YoucangenerateaGAL4DNA-BDfusiongeneifcompatiblerestrictionsitesarepresentinthe

testgeneandthecorrespondingvector(TableV).Ifnot,generatethegenefragmentbyPCRusingprimersthatcontainthedesiredrestrictionsite(Scharf,1990).ArestrictionsiteattheendofagenecanoftenbechangedintoadifferentsiteorputintoadifferentreadingframebyusingaPCRprimerthatincorporatesthedesiredmutation.Alternatively,ifyouhavealreadyclonedyourgeneintoaCreatorTMDonorVector,useCrerecombinasetotransferyourgenetopLP-GBKT7.RefertotheCreatorDNACloningKitsUserManual(PT3460-1)fordetails.

• Formoredetailedinformationoncloning,seeSambrooket al.(1989).

TABLEV.COMPARISONOFMATCHMAKERDNA-BDVECTORS

DNA-BDvector Description Size ProteinExpression

pGBKT7 GAL4(1–147)DNA-BD 7.3kb High TRP1,Kanr

pBridgea GAL4(1–147)DNA-BD 6.5kb Low TRP1,Ampr

pGBT9b GAL4(1–147)DNA-BD, 5.4kb Low TRP1,Ampr

pAS2-1b GAL4(1–147)DNA-BD, 8.4kb High TRP1,Ampr, CYHs2

pLP-GBKT7c GAL4(1–147)DNA-BD, 7.5kb High TRP1,Kanr,loxP aContainstwodistinctexpressioncassettesforinvestigatingternaryproteincomplexes.bDNA-BDvectorsusedinpreviousMatchmakerTwo-HybridSystems.cCreatorAcceptorVector(LP=loxP).AcceptsageneofinterestfromanyCreatorDonorVectorandexpresses

itasaGAL4DNA-BDfusion.

1.Purifythegenefragment. Note:WerecommendtheNucleoSpinExtractionKit(Cat.No.635961)forrapidisolationofDNAfragments.

2.Digest the DNA-BD vector with the appropriate restriction enzyme(s), treat withphosphatase,andpurify.

3.Ligatetheappropriatevectorandinsert.TransformligationmixturesintoE. coli. 4.Identifyinsert-containingplasmidsbyrestrictionanalysisorPCR.

B. TesttheDNA-BDFusionforTranscriptionalActivation 1.TransformAH109andY187withthehybridconstructusingasmall-scaletransformation

protocolsuchastheonegiveninSectionXII.A.7.Platetransformantsonthefollowingmedia*: • SD/–Trp/X-α-Gal • SD/–His/–Trp/X-α-Gal • SD/–Ade/–Trp/X-α-Gal Includeanegativecontrol.Forexample,transformcellswithan"empty"DNA-BDvector. * Note:Thedropoutsupplementsrequiredtomakethesemediaarenotsuppliedwiththe

MatchmakerLibraryConstruction&ScreeningKit(Cat.No.630445).Youmustpurchasethesesupplementsseparatelyfromacommercialsupplier,orpreparethemyourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).

2.Analyzeresults.

• BaitproteinisinactiveifthetransformantcoloniesarewhiteanddonotgrowonSD/–His/–TrporSD/–Ade/–Trp.GotoSteps5–6.

• Baitproteinisactiveiftransformantcoloniesareblue andgrowonSD/–His/–TrporSD/–Ade/–Trp. ContinuewithSteps3–4.

3.Ifabaitstrainexhibitsbackgroundgrowthon–Hismedium,youmaybeabletoeliminate(or reduce) the background by adding 3-AT to the selection medium (Section IV.F).Alternatively,use–Ade/–His/–Leu/–Trpmediumforthelibraryscreening.

4.Ifabaitstrainexhibitsbackgroundgrowthon–Adeand–Hismedium,itwillbedifficulttousethisbaitproteininatwo-hybridlibraryscreening.SeeTroubleshootingGuide.

VIII.ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysis



5.[Optional]Ifaknownproteinpartnerforyourbaitproteinisavailable,useittocheckwhetheratwo-hybridinteractionisdetectablewiththisparticularDNA-BD/baitfusion.

6.[Optional]Verifyproteinexpression: • PrepareWesternblotsfromthelysateoftransformantsandprobetheblotswithantibodies

totheGAL4DNA-BD(Cat.No.630403).Useuntransformedyeastlysateasacontrol. Notes: • Usingpolyclonalantibodiesmayresultinmultiplecross-reactingbands. • The level of expression from pGBT9 or pBridge is too low to permit detection using our GAL4

DNA-BDMonoclonalAntibody.

C. TesttheDNA-BDFusionforToxicity • ComparethegrowthrateinliquidcultureofY187cellstransformedwiththe"empty"

DNA-BDvectorandcellstransformedwiththeDNA-BD/baitplasmid.Ifthebaitstraingrowsnoticeablyslower,yourDNA-BD/baitproteinmaybetoxic.

Note:WealsorecommendcheckingfortoxicityinstrainAH109.

• UseaSmall-ScaleYeastTransformationProtocol(suchastheonegiveninSectionXII.A.7)topreparetransformants.SelecttransformantsonSD/–Trp,thenprepareanovernightcultureasfollows:

1. Inoculate50mlofSD/–Trp/Kan(20µg/ml)withonelarge(2–3mm)colony. 2. Incubateat30°Covernight(16–24hr)withshakingat250–270rpm. 3. ChecktheOD600oftheculture;itshouldbe≥0.8.IftheOD600ismuchlessthan0.8,

theDNA-BDfusionmaybetoxic(seetheTroubleshootingGuide).Ifthefusiondoesnotappeartohamperyeastgrowth—i.e.isnontoxic—andyouplantoscreenyourtwo-hybridlibrarybyyeastmating,continuewithSteps4–7.Ifyouareplanningtoscreenbycotransformation,youmaystophereandproceedtoSectionIX.

4. Centrifugeat600xgfor5min. 5. Removesupernatant. 6. Resuspendin~5mlSD/–Trpliquidmedium.Countcellsusingahemacytometer.The

celldensityshouldbe≥1x109cells/ml. 7. MatethisbaitstrainwithyourADfusionlibraryhoststrain(SectionXII.A.4). Note:Y187(MATα)canmatewithAH109,HF7c,CG-1945,Y190,orSFY526(allMATa).

VIII.ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysiscontinued



A. GeneratingacDNAlibrarywithSMART™technologyMessengerRNAtranscriptsareefficientlycopiedintodscDNAusingSMART(SwitchingMechanism at 5' end of RNA Transcript) technology (Zhu,Y.Y., et al., 2001).This cDNAsynthesisandamplificationsystemisparticularlywellsuitedforone-hybridandtwo-hybridlibraryconstructionbecauseitconsistentlydelivershighyieldsofcDNAwhilemaintainingsequencerepresentation.Bymaintainingthecomplexityoftheoriginaltissue,theSMARTprocedureprovidesyouwiththebestopportunityofdetectingrareandpotentiallynovelinteractionsduringyeastone-hybridandtwo-hybridscreening.

B. HowSMARTcDNASynthesisandAmplificationworksInthefirst-strandcDNAsynthesisstep,MMLV(MoloneyMurineLeukemiaVirus)ReverseTranscriptase(RT)isusedtotranscribeRNAintoDNA.ToprimeRNAforcDNAsynthesis,youmayuseeitheramodifiedoligo(dT)primer(ourCDSIIIPrimer)orarandomprimer(ourCDSIII/6Primer).ThecompositionoftheresultingcDNAlibrarymaydifferdependingonwhichprimeryouchoose.IfyouusetheCDSIIIPrimer,whichhybridizestothe3'-endofpolyA+RNA,sequencesclosetothe5'-endofthetranscriptmaybeslightlyunder-represented.IfinsteadyouusetheCDSIII/6Primer,arandomprimerthatcanhybridizetomanydifferentsequencesontheRNAtemplate,yourlibraryshouldcontainavarietyof5'-and3'-endsequences,whicharerepresentedinnearequalproportions.WhenMMLVRTencountersa5'-terminusonthetemplate,theenzyme’sterminaltransferaseactivityaddsafewadditionalnucleotides,primarilydeoxycytidine,tothe3'endofthecDNA.TheSMARTIIITMOligonucleotide,whichhasanoligo(G)sequenceatits3'end,base-pairswiththedeoxycytidinestretch,creatinganextendedtemplate(Figure8).RTthenswitchestemplatesandcontinuesreplicatingtotheendof theoligonucleotide. In themajorityofsyntheses,theresultingsscDNAcontainsthecomplete5'endofthemRNAaswellasthesequencecomplementarytotheSMARTIIIOligo,whichthenservesasauniversalprimingsite(SMARTanchor)inthesubsequentamplificationbylong-distancePCR(LDPCR;Chenchiket al., 1998).OnlythosesscDNAshavingaSMARTanchorsequenceatthe5'endcanserveasatemplateandbeexponentiallyamplifiedbylong-distancePCR(LDPCR).In the second step, ss cDNA is amplified by LD PCR to produce a ds cDNA library.Werecommend using theAdvantage® 2 PCR Kit (Cat. Nos. 639206 & 639207) to generateandamplifydscDNA.TheAdvantage2PolymeraseMixconsistsofTITANIUMTaqDNAPolymerase (anuclease-deficientN-terminaldeletionofTaqDNApolymerase), TaqStartAntibodytoprovideautomatichot-startPCR(Kellogget al.,1994),andaminoramountofaproofreadingpolymerase.ThispolymerasesystemletsyouamplifycDNA(aslargeas20kb)withafidelityratesignificantlyhigherthanthatofconventionalPCR(Barnes,1994).

First-strand synthesis coupled with (dC) tailing by RT

Amplificationby LD-PCR

Poly A+ RNA

poly A 3'

SMART™ IIIOligonucleotide

CDS III oligo(dT) or random primer

Template switching and extension by RT

poly A

poly A


GGG5'CCC

5'

GGG5'

CCC

Figure8.Synthesisofhigh-qualitydscDNAusingSMARTtechnology.

IX.GeneratingacDNALibrary



C. GoodLaboratoryPractices • WearglovestoprotectyourRNAandcDNAsamplesfromdegradationbynucleases. • Whenresuspendingpelletsormixingreactions,gentlypipetthesolutionupanddown

ortapthebottomofthetube.Spinbrieflytobringcontentstothebottomofthetube.Donotvortexsampleswhenresuspendingpellets;vortexingmayshearyourcDNA.

• Performallreactionsonice,unlessotherwiseindicated. • Addenzymestoreactionmixtureslast.Besuretheenzymeisthoroughlyblendedinto

thereactionmixturebygentlypipettingthemixtureupanddown. • Donotincreasethesize(volume)ofanyofthereactions.Allcomponentshavebeen

optimizedforthevolumesspecified. • Ethidiumbromideisacarcinogen.Useappropriateprecautionsinhandlinganddisposing

thisreagent.Formoreinformation,seeSambrooket al.(1989).BondExEthidiumBromideDetoxificationCartridgesareavailablefromClontech.

• Phenolisacorrosive.Alwayswearglovesandprotectiveclothingwhenhandlingsolutionscontainingthisreagent.Ifpossible,handlesolutionscontainingphenoland/orchloroformunderachemicalfumehood.

• Inpreparingyour reactions,use theDeionizedH2Osupplied.Otherwise,use freshlydeionized(e.g.,Milli-Q-grade)H2O,withouttreatmentwithDEPC(diethylpyrocarbonate).AvoidusingautoclavedH2ObecauserecycledsteaminsomeautoclavescanintroducecontaminantsthatmayinterferewithPCR.

• Rinseallglasswarewith0.5NNaOH,followedbydeionizedH2O.Thenbaketheglasswareat160–180°Cfor4–9hr.

• Useonlysingle-useplasticpipettesandpipettetipswhenhandlingRNA.

D. RNAIsolation • ManyproceduresareavailablefortheisolationoftotalRNAandpolyA+RNA(Chomczynski

&Sacchi,1987;Farrell,1993;Sambrooket al.,1989).ClontechoffersseveralkitsfortheisolationoftotalRNAandsubsequentisolationofpolyA+RNA(seeRelatedProducts).

• TheminimumamountofstartingmaterialforcDNAsynthesisis100ngoftotalRNAor25ngofpolyA+RNA.Ingeneral,themoreRNAyoustartwith,thefewerPCRcycleswillberequiredforamplification(seeTableVI).FewerthermalcyclesarelesslikelytogeneratenonspecificPCRproducts,andthereforearebestforoptimalcDNAandlibraryquality.Thus,ifyourRNAsampleisnotlimiting,usethehigherstartingamountsofRNAshowninthetable.

E. RNAAnalysis • ThesequencecomplexityofthedscDNAsynthesized,andultimatelyofthecDNAlibrary

constructed,dependsonthequalityoftheexperimentalRNAstartingmaterial.Therefore,beforeyouuseitinafirst-strandsynthesis,werecommendyouestimatetheintegrityoftheRNAbyexaminingasampleonadenaturingformaldehyde/agarosegel.TotalRNAfrommammaliansourcesshouldappearastwobrightbands(28Sand18SribosomalRNA)atapproximately4.5and1.9kb.Theratioofintensitiesofthe28Sand18SrRNAbandsshouldbe1.5–2.5:1.IntactmammalianpolyA+RNAshouldappearasasmear(usually0.5–10kb[ormore])withfaint28Sand18SrRNAbands.Thesizedistributionmaybeconsiderablysmaller(0.5–3kb)fornonmammalianspecies(e.g.,plants,insects,yeast,amphibians,etc.).

• Iftheratioofintensityof28SRNAto18SRNA(fortotalRNA)islessthan1:1orifyourexperimentalpolyA+RNAappearssignificantlysmallerthanexpected(e.g.,nolargerthan1–5kb),wesuggestyoupreparefreshRNAaftercheckingyourRNApurificationreagentsforRNaseandotherimpurities.Ifproblemspersist,youmayneedtofindanothersourceoftissueorcells.

IX.GeneratingacDNALibrarycontinued



PLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. • WesuggestyoualsoperformapositivecontrolcDNAsynthesiswithHumanPlacenta

PolyA+RNA.ThiscontrolletsyouverifythatallcomponentsareworkingproperlyandletsyouevaluatetheyieldandsizesofthedscDNAsynthesizedfromyourRNAsample.

• Intheprotocolsthatfollow,youhavetheoptionofprimingfirst-strandcDNAsynthesiswithanoligo(dT)orrandomprimer(SectionsFandG,respectively).Thereactionconditionsvaryslightlydependingontheprimerused.

F. SynthesizeFirst-StrandcDNAusinganOligo(dT)Primer 1.Combinethefollowingreagentsinasterile0.25-mlmicrocentrifugetube: 1–2 µl RNAsample(0.025–1.0µgpolyA+or0.10–2.0µgtotalRNA)

(Forthecontrolreaction,use1µl[1µg]ofthecontrolRNA.) 1.0 µl CDSIIIPrimer

1–2 µl DeionizedH2Otobringvolumeupto4.0µl.

4.0 µl Totalvolume

2.Mixcontentsandspinbriefly. 3.Incubateat72°Cfor2min. 4.Coolonicefor2min. 5.Spinbriefly. 6.Addthefollowingtothereactiontube: 2.0 µl 5XFirst-StrandBuffer 1.0 µl DTT(20mM) 1.0 µl dNTPMix(10mM)

1.0 µl MMLVReverseTranscriptase 9.0 µl Totalvolume

7.Mixgentlybytapping.Spinbriefly. 8.Incubateat42°Cfor10min. 9.Add1.0µlSMARTIIIOligonucleotide. 10.Incubateat42°Cfor1hrinanairincubatororhot-lidthermalcycler. Note:Ifyouuseawaterbathornonhot-lidthermalcyclerforthisincubation,coverthereactionmixturewith

onedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

11.Placethetubeat75°Cfor10mintoterminatefirst-strandsynthesis. 12.Coolthetubetoroomtemperature,thenadd1.0µlRNaseH. 13.Incubateat37°Cfor20min. 14.Ifyouplan toproceeddirectly to theLD-PCRstep, takea2-µlaliquot fromthefirst-

strandsynthesisandplaceitinaclean,prechilled,0.5-mltube.Placethetubeonice,andproceedtoSectionH.Ifyouusedmineraloilinyourfirst-strandreactiontube,besuretotakethe2-µlsamplefromthebottomofthetubetoavoidtheoil.

15.Anyfirst-strandreactionmixturethatisnotusedrightawayshouldbeplacedat–20°C.First-strandcDNAcanbestoredat–20°Cforuptothreemonths.

G. SynthesizeFirst-StrandcDNAusingaRandomPrimer 1.Combinethefollowingreagentsinasterile0.25-mlmicrocentrifugetube: 1–2 µl RNAsample(0.025–1.0µgpolyA+or0.10–2.0µgtotalRNA)

(Forthecontrolreaction,use1µl[1µg]ofthecontrolRNA.) 1.0 µl CDSIII/6Primer

1–2 µl DeionizedH2Otobringvolumeupto4.0µl. 4.0 µl Totalvolume

2.Mixcontentsandspinbriefly. 3.Incubateat72°Cfor2min. 4.Coolonicefor2min. 5.Spinbriefly.




6.Keepthetubeatroomtemperatureandaddthefollowing: 2.0µl 5XFirst-StrandBuffer 1.0µl DTT(20mM) 1.0µl dNTPMix(10mM) 1.0 µlMMLVReverseTranscriptase

9.0µl Totalvolume

7.Mixgentlybytapping.Spinbriefly. 8.Incubateat25–30°Cfor10minatroomtemperature. 9.Incubateat42°Cfor10min. 10.Add1.0µlSMARTIIIOligonucleotide. 11.Incubateat42°Cfor1hrinanairincubatororhot-lidthermalcycler. Note:Ifyouuseawaterbathornonhot-lidthermalcyclerforthisincubation,coverthereactionmixturewith

onedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

12.Placethetubeat75°Cfor10mintoterminatefirst-strandsynthesis. 13.Coolthetubetoroomtemperature,thenadd1.0µl(2units)RNaseH. 14.Incubateat37°Cfor20min. 15.Ifyouplan toproceeddirectly to theLD-PCRstep, takea2-µlaliquot fromthefirst-

strandsynthesisandplaceitinaclean,prechilled,0.5-mltube.Placethetubeonice,andproceedtoSectionH.Ifyouusedmineraloilinyourfirst-strandreactiontube,besuretotakethe2-µlsamplefromthebottomofthetubetoavoidtheoil.

16.Anyfirst-strandreactionmixturethatisnotusedrightawayshouldbeplacedat–20°C.First-strandcDNAcanbestoredat–20°Cforuptothreemonths.

H. AmplifydscDNAbyLongDistancePCR(LD-PCR)TableVIshowstheoptimalnumberofthermalcyclestousebasedontheamountofRNAusedinthefirst-strandsynthesis.FewercyclesgenerallymeanfewernonspecificPCRproducts.TheoptimalcyclingparametersinTableVIweredeterminedusingtheControlPolyA+HumanPlacentaRNA;theseparametersmayvarywithdifferenttemplatesandthermalcyclers.

TABLEVI.RELATIONSHIPBETwEENAMOUNTOFRNAANDOPTIMALNUMBEROFTHERMALCYCLES

TotalRNA(µg) PolyA+RNA(µg) NumberofCycles

1.0–2.0 0.5–1.0 15–20

0.5–1.0 0.25–0.5 20–22

0.25–0.5 0.125–0.25 22–24

0.05–0.25 0.025–0.125 24–26




1.PreheatthePCRthermalcyclerto95°C. 2.TopreparesufficientdscDNAfortransformation,setuptwo100-µlPCRreactionsfor

eachexperimentalsample.SetuponereactionfortheControlsample.Ineachreactiontube,combinethefollowingcomponents:

2 µl First-StrandcDNA 70 µl DeionizedH2O 10 µl 10XAdvantage2PCRBuffer 2 µl 50XdNTPMix 2 µl 5'PCRPrimer 2 µl 3'PCRPrimer 10 µl 10XGC-MeltSolution 2 µl 50XAdvantage2PolymeraseMix

100 µl Totalvolume

3.Mixgentlybyflickingthetube.Centrifugebriefly. 4.Overlaythereactionmixturewith2dropsofmineraloilifnecessary.Capthetubeand

placeitinapreheated(95°C)thermalcycler. 5.Beginthermalcycling.Ifyouhaveahot-lidthermalcycler,usethefollowingprogram:

•95°C30sec •xcyclesa: 95°C 10sec 68°C 6minb •68°C 5min

a RefertoTableVItodeterminetheoptimalnumberofcyclestouse.

b Programthecyclertoincreasetheextensiontimeby5secwitheachsuccessivecycle.Forexample,inthesecondcycle,theextensionshouldlast6minand5sec;inthethird,6minand10sec.Andsoon.

Note:Thesecyclingparametersmaynotbeoptimalfornonhot-lidthermalcyclers. 6.Whenthecyclingiscomplete,analyzea7-µlaliquotofthePCRproductfromeachsample

alongside0.25µgofa1-kbDNAsizemarkerona1.2%agarose/EtBrgel.TypicalresultsobtainedwithHumanPlacentaPolyA+RNAareshowninAppendixA.IfyourPCRproductdoesnotappearasexpected,refertotheTroubleshootingGuide.

7.ProceedwithSectionIorstoredscDNAat–20°Cuntiluse.

I. PurifydscDNAwithaCHROMASPIN™TE-400ColumnCHROMASPINColumnsarepackedwithresinsthatfractionatemoleculesbasedonsize.Moleculeslargerthantheporesizeareexcludedfromtheresin.Thesemoleculesquicklymovethroughthegelbedwhenthecolumniscentrifuged,whilemoleculessmallerthantheporesizeareheldback.Inthefollowingprotocol,aCHROMASPINTE-400ColumnisusedtoselectforDNAmolecules>200bp.FormoreinformationaboutCHROMASPINColumns,pleaserefertotheCHROMASPINColumnsUserManual(PT1300-1),availableatourwebsiteatwww.clontech.com.Note:WerecommendcentrifugingCHROMASPINColumnsinaswingingbucketorhorizontalrotor.Fixed-anglerotorscanalsobeused,butthereisariskthataportionofthesamplewillslidedowntheinnersideofthecolumninsteadofpassingthroughthegelmatrix,resultinginreducedorinconsistentpurification.Performthefollowingstepsforeachexperimentalandcontrolsample.

1.RemovetheCHROMASPINColumnfromtheprotectiveplasticbagandinvertitseveraltimestoresuspendthegelmatrixcompletely.Useonecolumnforeach~95-µlcDNAsample.

2.HoldingtheCHROMASPINColumnupright,graspthebreak-awayendbetweenyourthumbandindexfingerandsnapoff(Figure9).Placetheendofthespincolumnintooneofthe2-mlmicrocentrifuge(collection)tubes,andliftoffthetopcap.Savethetopcapandthewhite-endcap.




3.Centrifugeat700xgfor5min.Aftercentrifugation,thecolumnmatrixwillappearsemi-dry.Thissteppurgestheequilibrationbufferfromthecolumnandre-establishesthematrixbed.

Note:Thecolumnfittedinthe2-mlmicrocentrifugetubecanbeplacedinsidea17x100-mmpolypropylenetubeduringcentrifugationinaswingingbucketrotor.

4.Removethespincolumnandcollectiontubefromthecentrifugerotor,anddiscardthecollectiontubeandcolumnequilibrationbuffer.

Note:AlwaysholdboththeCHROMASPINColumnandthe2-mlmicrocentrifugetubewhenremovingthemfromtherotor.

5.Placethespincolumnintothesecond2-mlmicrocentrifugetube.CarefullyandslowlyapplyyourcDNAsample(~95µlfromStepIX.H.7)tothecenterofthegelbed’sflatsurface.Donotallowanysampletoflowalongtheinnerwallofthecolumn.

Note:Aconventional,tapered1.5-mlmicrocentrifugetubecanbesubstitutedforthe2-mlcollectiontube.Thiswillallowthesampletobeconfinedtoanarrowerareaforeasierhandling.

6.Centrifugeat700xgfor5min. 7.Removethespincolumnandcollectiontubefromtherotoranddetachthemfromeach

other.Thepurifiedsampleisatthebottomofthecollectiontube.Note:Holdthesamplecollectiontubetopreventitfromdetachingfromthespincolumn.

8.Combineduplicateexperimentalsamplesinasingletube. 9.Addthefollowingreagents: 1/10 vol. SodiumAcetate(3M;pH4.8) 2.5 vol. 95%ethanol(–20°C) 10.Mixgentlybyrockingthetubebackandforth. 11.Placethetubeina–20°Cfreezeroradry-ice/ethanolbathfor1hr.(Optional:Youmay

incubateat–20°Covernight,whichmayresultinbetterrecovery.) 12.Centrifugethetubeat14,000rpmfor20minatroomtemperature. 13.Carefullyremovethesupernatantwithapipette.Donotdisturbthepellet. 14.Brieflycentrifugethetubetobringallremainingliquidtothebottom. 15.Carefullyremoveallliquidandallowthepellettoairdryfor~10min. 16.Resuspendthepelletin20µlofDeionizedH2Oandmixgently.ThecDNAisnowready

for in vivo recombination (Library Construction) with pGADT7-Rec or pGADT7-Rec2.ProceedwithOne-HybridorTwo-HybridLibraryConstruction,orstorethecDNAat–20°Cuntilyouareready.

CHROMA SPIN Column main body

2-ml Collection Tubes

Break-away end

White-end cap

Clear top cap

Matrix

Figure9.CHROMASPINColumnandCollectionTubes.Notethataconventional,tapered1.5-mlmicrocentrifugetubecanbesubstitutedforthe2-mlcollectiontube.Thiswillallowthesampletobeconfinedtoanarrowerareaforeasierhandling.




PT� TADH1GAL� AD pGADT�-Rec[2]pGADT�-Rec[2] HAPADH1

pGADT�-Rec or pGADT�-Rec2 Vector (Sma I-linearized)

SMART™ III ds cDNA

Recombination

CDS III

LEU2

Figure10.ConstructingADfusionlibrariesbyrecombination-mediatedcloninginyeast.ToclonecDNAintopGADT7-RecorpGADT7-Rec2,cotransformyeastwithdscDNA(generatedwithourmodifiedSMARTprocedure)andeitherpGADT7-Rec (two-hybrid screen)orpGADT7-Rec2 (one-hybrid screen).Yeast repair enzymeswill restore theSma I-linearizedplasmidtoitscircularformbyrecombiningsequencesattheendsofthecDNAwithhomologoussequencesattheendsofpGADT7-Rec(2).TheoutcomeisafullyfunctionalGAL4AD/cDNAexpressionvector.

A. ConstructingGAL4ADFusionLibrariesforOne-HybridandTwo-HybridScreening 1.Whetheryouplantoscreenforone-hybridortwo-hybridinteractions,themethodsused

toconstructthelibraryarethesame.Inbothcases,recombination-mediatedcloningisusedtofuseSMARTdscDNAwiththeGAL4AD(Figure10).Whilethecloningmethodsarethesame,theGAL4ADcloningvectorsarenot—

• Toconstructaone-hybridlibrary,usepGADT7-Rec2. • Toconstructatwo-hybridlibrary,usepGADT7-Rec.

pGADT7-Rec2andpGADT7-Recdifferintheirmodeofreplication.pGADT7-Recisahigh-copyplasmid;itcontainsa2µoriginofreplication,whichenablesittoreplicatemultipletimesduringthecellcycle.pGADT7-Rec2,ontheotherhand,isalow-copyplasmid;itcontainsanautonomousreplicationsequence,orARSelement,whichallowsthevectortoreplicateonlyonceduringthecellcycle.pGADT7-Rec2alsocontainsacentromericsequence,orCEN element,toensurestablesegregationoftheplasmidduringmitosisandmeiosis. Low-copyplasmidssuchaspGADT7-Rec2arepreferred forone-hybridanalysisbecausetheygeneratefewerfalsepositives.

2.AGAL4ADfusionlibraryisproducedbycotransformingyeastwithSMARTdscDNAand Sma I-linearized pGADT7-Rec or pGADT7-Rec2, depending on whether you areconstructingatwo-hybridorone-hybridlibrary.SMARTdscDNArecombineswiththeADcloningvectorin vivotoyieldacompleteGAL4ADexpressionvector(Figure10).TheresultingconstructwillexpressthecDNAinsertasaGAL4ADfusionprotein.Thisone-stepcloningprocedureispossiblebecausetheSMARTIIIandCDSIIIsequences—incorporatedintothecDNAbyRTandLD-PCR—havebeenengineeredintothepGADT7-RecandpGADT7-Rec2plasmids.Inyeast,thelinearplasmidisrestoredtoitscircularformbyrecombinationwithoverlappingsequencesattheendsoftheSMARTcDNA.Successfulplasmidassemblyresultsinapositive(Leu2+)transformant.

B. ScreeningGAL4ADFusionLibraries 1.One-HybridLibraryScreening(cotransformation) Withrecombination-mediatedcloning(Figure10), libraryconstructionandscreening

can be carried out in the same host strain on the same day. If you prepare a DNAtarget/reporterplasmid—e.g.,pHIS2/DNAtarget—inadvance,youcanincludeitinthecotransformationreactiontogetherwithyourcDNAlibraryandpGADT7-Rec2.Withasingletransformationstep,allthreeDNAcomponentscanbeintroducedintotheyeastreporterstrain(Figure4).ScreeningbeginsassoonasthepGADT7-Rec2ADvectorisassembledbythehost’srecombinationprocesses.Positiveone-hybridscanbeidentifiedimmediatelyaftercotransformationbyplatingthetransformationmixtureonmediumthatselectsfortheHIS3nutritionalreporter.Forprotocoldetails,seeSectionXI.

2.Two-HybridLibraryScreening(yeastmatingorcotransformation) Two-Hybridlibrariescanbescreenedbyeitheryeastmatingorcotransformation.As

describedabove,cotransformationallowsyoutoconstructandscreenyourlibraryinasinglehoststrain.Theprocedureisquickandefficient.FordetailspleasereviewProtocolsAandBinSectionXIIandseetheTwo-HybridLibraryConstruction&ScreeningflowchartinFigure7.

X. Constructing&ScreeningOne-HybridandTwo-HybridLibraries



PLEASE READ ENTIRE PRoToCoL BEfoRE STARTING.Beforestartinglibraryconstruction: • PouranappropriatenumberofSDagarplates: – SD/–His/–Leu/–Trp+optimal[3-AT]toselectforone-hybridinteractions(150-mm) – SD/–Leutomeasurethetransformationefficiencyofthelibraryplasmid(100-mm) – SD/–Trptomeasurethetransformationefficiencyofthereporterplasmid(100-mm) – SD/–Leu/–Trptomeasurethenumberofclonesscreened(100-mmplates) AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr. • PreparePEG/LiAcSolution(SectionIII). • Be sure to run the necessary controls (Part C) in parallel with your experimental

sample.

Important:Notethatyoumustpreparecompetentyeastcellsbeforestartinglibraryconstruction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

A. CotransformYeastStrainY187withdscDNA,pGADT7-Rec2,andpHIS2/targetDNA. 1.Preparecompetentyeastcells(AppendixB). 2.Inasterile,15-mltubecombinethefollowing:

• 20 µl dscDNA(fromSectionIX.I,Step16) • 6 µl pGADT7-Rec2(0.5µg/µl) • 5 µgpHIS2/targetDNA(preparedinSectionVII) • 20 µl HerringTestesCarrierDNA,denatured*

Note:ThecombinedvolumeoftheseDNAcomponentsshouldnotexceed60µl,or1/10thevolumeofthecompetentcellsaddedatStep3,below.

* Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

3.Add600µlofcompetentcellstotheDNA. 4.Gentlymixbyvortexing. 5.Add2.5mlPEG/LiAcSolution. 6.Mixgentlybyvortexingfor3–5sec. 7.Incubateat30°Cfor45min.Mixcellsevery15min. 8.Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. 9.Centrifugeat700xgfor5min. 10.Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumforthisstep.

11.Incubateat30°Cwithshakingat~265rpmfor90min. 12.Centrifugeat700xgfor5min. 13.Discardthesupernatantandresuspendin6mlofNaClSolution(0.9%).

B. SelectforOne-HybridInteractions 1.Todeterminethetransformationefficiencyandtocalculatethenumberofclonesscreened,

spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmSD/–Leu,SD/–Trp,andSD/–Leu/–Trpagarplates.

2.SpreadtheremainingmixtureonSD/–His/–Leu/–Trp+optimal[3-AT]plates(150µlcells/150-mmplate)toselectforone-hybridinteractions.

3.Incubateat30°Cfor3–7daysuntilcoloniesappear. 4.Calculatethetransformationefficiencyandnumberofclonesscreened: a. ColoniesonSD/–Leuxdilutionfactor÷volume(ml)platedx6ml=#transformantsper

3µgpGADT7-Rec2.Expected:≥1x106transformants/3µgpGADT7-Rec2 b. ColoniesonSD/–Leu/–Trpxdilutionfactor÷volume(ml)platedx6ml=#clones

screened Expected:≥3x105clones/library

XI. Constructing&ScreeningaOne-HybridLibrary



5.RestreaktheHis+coloniesonfreshSD/–His/–Leu/–Trp+optimal[3-AT].Incubateat30°Cfor2–4days.SealtheplateswithParafilmandstoreat4°C.Forlong-termstorage,prepareglycerolstocksandstoreat–70°C.

6.AnalyzepositiveinteractionsasdescribedinSectionXIII.

C. One-HybridControls

• pGAD-Rec2-53• p53HIS2

Transform

Y187Y187[pGAD-Rec2-53 + p53HIS2]

One-Hybrid Controls

Control Strain[plasmids]Cotransform Y1�� with:

1. Positive Control

• pGAD-Rec2-53• pHIS2 Y187[pGAD-Rec2-53 + pHIS2]2. Negative Control

Transform

Y187

TABLEVII.SET-UPFORONE-HYBRIDCONTROLCOTRANSFORMATIONS

Component #1PositiveControl(µl)#2NegativeControl(µl)

pGAD-Rec2-53(500ng/µl) 1.0 1.0p53HIS2(500ng/µl) 1.0 —pHIS2(500ng/µl) — 1.0HerringTestesCarrierDNA(10mg/ml),denatured* 5 5Y187CompetentYeastCells 50 50PEG/LiAcSolution 500 500

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe1.5-mlreactiontube.

1.Preparecompetentyeastcells(AppendixB). 2.Setuptwo1.5-mlmicrocentrifugetubes.AddDNA,competentyeastcells,andPEG/LiAc

Solutionusingthevolumesandintheorderindicated(TableVII). 3.Mixthoroughlybygentlyvortexing. 4.Incubateinawaterbathat30°Cfor30min.Vortexgentlyevery10min. 5.Add20µlofDMSOtoeachtube,mix,andthenplacethetubeina42°Cwaterbathfor

15min.Vortexgentlyevery7.5min. 6.Centrifugeathighspeedfor15sec. 7.Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. 8.Shakeat30°Cfor90min. 9.Centrifugeathighspeedfor15sec. 10.Discardthesupernatantandresuspendin1mlofNaClSolution(0.9%)bygentlypipetting

upanddown. 11.Spread100µlofa1:10,1:100,and1:1,000dilutiononSD/–Leu,SD/–Trp,andSD/–Leu/–Trp

tocheckthetransformationefficiency,andonSD/–His/–Leu/–Trp+10mM3-ATtoselectforpositiveone-hybrids.

12.Incubatetheplatesat30°C(facedown)for2–7days,untilcoloniesappear. 13.CompareyourresultswiththoseshowninTableVIII.

TABLEVIII.CONTROLONE-HYBRIDCOTRANSFORMATIONS:EXPECTEDRESULTS

ControlStrain PlatedonSDMinimalMedia Phenotype(Growth)

PositiveControlY187[pGAD-Rec2-53+p53HIS2] –His/–Leu/–Trp+10mM3-AT +

NegativeControlY187[pGAD-Rec2-53+pHIS2] –His/–Leu/–Trp+10mM3-AT –

XI. Constructing&ScreeningaOne-HybridLibrarycontinued



TherearetwowaystoscreenaMatchmakertwo-hybridlibrary:

• Yeastmating

• Cotransformation

Themethodyouchoosedeterminestheprotocolyouwillnowusetoconstruct(andscreen)yourlibrary.Toscreenbyyeastmating,constructyourlibraryusingProtocolA.Toscreenbycotransformation,constructyourlibraryusingProtocolB.Foraquickcomparisonofthesetwoprotocols,referbacktoFigure7.

Important:Notethatyoumustpreparecompetentyeastcellsbeforestartinglibraryconstruction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

ProtocolA:ScreenbyYeastMatingPLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. Beforestarting: • PourSDagarplates.Youwillneed: SD/–Leu ~200 150-mmplates SD/–Leu ~5–10 100-mmplates SD/–Trp(seenotebelow)* ~5–10 100-mmplates SD/–Leu/–Trp ~5–10 100-mmplates SD/–His/–Leu/–Trp(seenotebelow)*~50 150-mmplates SD/–Ade/–His/–Leu/–Trp/X-α-Gal ~50 150-mmplates AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr

beforeplatinganytransformationmixtures. * Note:AsexplainedintheListofAdditionalMaterialsRequired(SectionIII),thedropoutsupplementsrequired

tomakethesemediaarenotsupplied.YoumustobtainthesesupplementsseparatelyfromacommercialsupplierorpreparethemyourselfusingtherecipeinAppendixCoftheYeastProtocolsHandbook(PT3024-1).

• Plancontrols(Steps7–8)Controlsshouldbedoneinparallelwithexperimentalwork. • PrepareFreezingMedium:YPDmediumwith25%(v/v)glycerol. • PreparePEG/LiAcSolution(SectionIII).

1.TransformyeaststrainAH109withdscDNAandpGADT7-Rec. a. Preparecompetentyeastcells(AppendixB). b. Inasterile,prechilled,15-mltubecombinethefollowing: • 20µldscDNA(fromSectionIX.I,Step16) • 6µlpGADT7-Rec(0.5µg/µl) • 20µlHerringTestesCarrierDNA,denatured*

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

c. Add600µlofcompetentcellstotheDNA. d. Gentlymixbyvortexing. e. Add2.5mlPEG/LiAcSolution. f. Gentlymixbyvortexing. g. Incubateat30°Cfor45min.Mixcellsevery15min. h. Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. i. Centrifugeat700xgfor5min. j. Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumfor

thisstep.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolA



k. Incubateat30°Cwithshakingfor90min. l. Centrifugeat700xgfor5min. m. Discardthesupernatantandresuspendin30mlofNaClSolution(0.9%).

2.SelecttransformantsonSD/–Leuplates. a. Spread150µloneach150-mmplate(~200platestotal). Note:Tocheckthetransformationefficiency,spread100µlofa1:10,1:100,1:1,000,and1:10,000dilution

on100-mmSD/–Leuplates.

b. Incubateplatesupsidedownat30°Cuntilcoloniesappear(~3–6days). c. Calculatethetransformationefficiency. Expectedresults:≥1x106transformants/3µgpGADT7-Rec 3.Harvest(pool)transformants. a. Chillplatesat4°Cfor3–4hr. b. Add5mlFreezingMediumtoeachplate. c. Usesterileglassbeadsandgentleswirlingtodislodgethecellsintotheliquid. d. Combineallliquidsinasterileflask.Mixwell. e. Checkthecelldensityusingahemacytometer.Ifthecelldensity≤2x107cells/ml,

reducethevolumeofthesuspensionbycentrifuging. f. Aliquot(1-ml)andstoreat–80°C(notlongerthan1year). g. Todeterminethelibrarytiter,spread100µlofa1:100,1:1,000,and1:10,000dilution

on100-mmSD/–Leuplates.Incubateat30°Cuntilcoloniesappear(~2–3days).Countthecolonies(cfu)andcalculatethenumberofclonesinyourlibrary.

4.Matethelibraryhoststrainwithyourbaitstrain. a. Thawa1-mlaliquot(≥2x107cells)ofyourAH109[library]inaroomtemperature

waterbath. b. Combinethe5-mlY187[bait]culturefromSectionVIII.C.7(≥1x109cells/ml)andthe

1-mlaliquotofAH109[library]cells(≥2x107cells/ml)inasterile2-Lflask. Note:Theflasksizemustbeatleast2Ltopermitsufficientaerationofthematingcultureatlow-speedswirling.

c. Add45ml2XYPDA/Kan(50µg/ml).Swirlgently. d. Rinsecellsfromlibraryvialwithtwo1-mlaliquotsof2XYPDA/Kan(50µg/ml). e. Incubateat30°Cfor20–24hrwithgentleswirling(30–50rpm). Note:Low-speedswirlingisnecessarytokeepcellsfromsettlingtothebottomoftheflask.However,

shakingthecultureatspeeds>50rpmwillsignificantlyreducematingefficiency.

f. After20hrofmating,checkadropofthematingcultureunderaphase-contrastmicroscope(400X).Ifzygotesarepresent,allowmatingtocontinueforfourmorehours.Otherwise,continuetoStepg.

Note:Azygotetypicallyhasathree-lobedshape,thelobesrepresentingthetwohaploid(parental)cellsandthebuddingdiploidcell.

g. Transferthematingmixturetoasterile100-mlcentrifugebottle.Centrifugeat1,000x g for 10 min. Meanwhile, rinse the mating flask twice (50 ml each rinse) with0.5XYPDA/Kan(50µg/ml).Combinetherinsesandusethemtoresuspendthepellet.

h. Centrifugeat1,000xgfor10min.Resuspendthecellpelletin10mlof0.5XYPDA/Kan(50µg/ml).Measurethetotalvolumeofcells+medium.

5.Selectforyeastdiploidsexpressinginteractingproteins a. Todeterminethematingefficiency,spread100µlofa1:10,000,1:1,000,1:100,and

1:10dilutionofthematingmixtureonthreemedia(100-mmplates): •SD/–Leu •SD/–Trp •SD/–Leu/–Trp b. SpreadremainingmatingmixtureonTDOorQDOplates(200µlcells/150-mmplate). •TDOstandsforTripleDropoutMedium:SD/–His/–Leu/–Trp •QDOstandsforQuadrupleDropoutMedium:SD/–Ade/–His/–Leu/–Trp SeeFigure11.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolAcontinued



c. Incubateat30°Cuntilcoloniesappear. After2–3days,somecolonieswillbevisible,butplatesshouldbeincubatedforatleast5

daystoallowslowergrowingcolonies(i.e.,weakpositives)toappear.Ignorethesmall,palecoloniesthatmayappearafter2daysbutnevergrowto>1mmindiameter.TrueAde+,His+coloniesarerobustandcangrowto>2mm.Also,Ade+coloniesarewhiteorlightpink,whereasAde–colonieswillslowlyturnredonadenine-limitedmedium.

d. ScoreforgrowthonSD/–Leu,SD/–Trp,andSD/–Leu/–Trp.CalculateMatingEfficiencyandNumberofColoniesScreened(Part6).

e. ForcoloniesgrowingonTDOmedium:ReplicaplatecoloniesontoQDOmedium.Incubateat30°Cfor3–8days.

f. ChooseAde+,His+coloniesforfurtheranalysis. Not all of the colonies surviving this selection will be true two-hybrid positives.

However,themostcommonclassoffalsepositiveswillbeeliminatedbyscreeningforexpressionofADE2 and HIS3(Jameset al.,1996).OthertypesoffalsepositivescanbeeliminatedasdescribedinSectionXIII.

g. StreakoutAde+/His+coloniesonfreshSD/–Ade/–His/–Leu/–Trp/X-α-Galmasterplatesandgrowfor2–4daysat30°C.HavingX-α-GalpresentinthemediumenablesyoutotestAde+/His+coloniesfortheactivationofathirdreporter:MEL1,which encodesα-galactosidase,asecretedenzymethathydrolyzesX-α-Galtoproduceablueendproduct.

h.SealthemasterplateswithParafilmandstoreat4°C.Ifdesired,prepareglycerolstockculturesofinterestingclonesandfreezeat–70°Cforlong-termstorage.

6.CalculateMatingEfficiency&NumberofClonesScreened a.Countthecolonies(cfu)growingontheSD/–Leu,SD/–Trp,andSD/–Leu/–Trpdilution

platesthathave30–300cfu. b. Calculatetheviablecfu/mloneachtypeofSDmedium: cfu =#viablecfu/ml Vol.plated(ml)xdilutionfactor #cfu/mlonSD/–Leu=viabilityofY187partner #cfu/mlonSD/–Trp=viabilityofAH109partner #cfu/mlonSD/–Leu/–Trp=viabilityofdiploids

Colony growth indicates an interaction between the two-hybrid proteins

Mate

DNA-BD/baitMarker: TRP1

AD/fusion libraryMarker: LEU2

Plate culture on SD/–His/–Leu/–Trp

Plate culture onSD/–Ade/–His/–Leu/–Trp

Medium-stringency

Replica plate to SD/–Ade/–His/–Leu/–TrpHigh-stringency

Y1�� AH109

Figure11.ScreeninganADfusionlibraryfortwo-hybridinteractions.Usethestringencyofyourchoicetoscreenforinteractingproteins.Note:highstringencyselectionsresultinfewercolonies,andreducethenumberoffalsepositives.However,weakinteractionsmaybemissed.




c. Compare theviablecfu/mlof the twomatingpartners.Thestrainwith the lowerviabilityisthe“limiting”partner.Inthislibraryscreeningprotocol,theAH109[library]strainshouldbethelimitingpartnertoensurethatthemaximumnumberoflibrarycellsfindamatingpartner.Inacontrolcross,eitherstraincouldbelimiting.

d. Calculatethematingefficiency(i.e.,%Diploid): #cfu/mlofdiploids x100=%Diploid #cfu/mloflimitingpartner Note:Ifthematingefficiencywas<2%,andifyouobtainedfew(ifany)positiveclones,youmaywishto

repeatthelibraryscreeningwithanother1-mlaliquot.Butfirst,seetheTroubleshootingGuidefortipsonimprovingthematingefficiency.

e. Estimatethenumberofclonesscreened: #cfu/mlofdiploidsxresuspensionvolume(ml)=#ofclonesscreened

7.ControlsforProtocolA:Small-ScaleYeastTransformation Usethissmall-scaletransformationprotocoltoproducethefollowingthreecontrolstrains.

1. SV40 Large T PCR Fragment + pGADT7-RecCotransform

AH109Recombination

in vivoAH109[pGADT7-RecT]

Control Vectors Control Strain[plasmid]

2. pGBKT7-53 Transform

Y187Y187[pGBKT7-53]

3. pGBKT7-Lam Transform

Y187Y187[pGBKT7-Lam]

Two-Hybrid Transformation Controls

TABLEIX.SET-UPFORCONTROLTwO-HYBRIDTRANSFORMATIONS

Component #1(µl) #2(µl) #3(µl)

SV40LargeTPCRFragment(25ng/µl) 5.0 — —pGADT7-Rec(SmaI-linearized;500ng/µl) 0.5 — —pGBKT7-53(500ng/µl) — 0.5 —pGBKT7-Lam(500ng/µl) — — 0.5HerringTestesCarrierDNA(10mg/ml),denatured* 5 5 5AH109competentyeastcells 50 — —Y187competentyeastcells — 50 50PEG/LiAcSolution 500 500 500Selectfortransformantsbyplatingon: SD/–Leu SD/–Trp SD/–Trp


a. Preparecompetentyeastcells(AppendixB). b. Setup three1.5-mlmicrocentrifuge tubes.AddDNA,competentyeastcells,and

PEG/LiAcSolutionusingthevolumesandintheorderindicated(TableIX). c. Mixthoroughlybygentlyvortexing. d. Incubateat30°Cfor30min.Vortexgentlyevery10min. e. Add20µlofDMSOtoeachtube,mix,andthenplacethetubeina42°Cwaterbathfor

15min.Vortexgentlyevery5min. f. Centrifugeathighspeedinamicrocentrifugefor15sec.




g. Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. h. Incubateat30°Cfor90min. i. Centrifugeathighspeedfor15sec. j. Discardthesupernatantandresuspendin1mlofNaClSolutionbygentlypipetting

upanddown. k. Spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmplatescontaining

SD/–LeuorSD/–Trp,dependingonthenutritionalmarkercarriedbytheplasmid(TableIX).

l. Incubatetheplatesat30°C(facedown)for2–4days,untilcoloniesappear. m. Pickthelargestcoloniesandrestreakthemonthesameselectionmedium.Seal

thesemasterplateswithParafilmandstoreat4°C(notlongerthan1month).UsethesecoloniesforthecontrolmatingsinSection8.

8.ControlsforProtocolA:Small-ScaleYeastMating

a. Pick one colony of each type—i.e.,AH109[pGBKT7-RecT],Y187[pGBKT7-53], andY187[pGBKT7-Lam]—touseinthemating.Useonlylarge(2–3-mm),fresh(<2weeksold)coloniesfromthemasterplates.

b. Placebothcoloniesinone1.5-mlmicrocentrifugetubecontaining0.5mlof2XYPDAmedium.Vortexthetubefor1mintocompletelyresuspendthecells.

c. Incubateat30°Covernight(20–24hr)withshaking. Note:Usethelowestshakingspeedpossibletopreventcellsfromsettling.Vigorousshakingcanreduce

thematingefficiency.

d. PlatecellsonSDminimalmedia(TableX;100µlcells/100-mmplate).Usesterile5-mmglassbeadstopromoteevenspreadingofthecells.

e. Incubateplates(colonysidedown)at30°Cfor3–5daystoallowdiploidcellstoformvisiblecolonies.

f. Scoreforgrowth.Calculatethematingefficiency. g. Confirmnutritionalandreporterphenotypesofdiploids(TableX). h. Pickrepresentativecoloniesfromselectionplates.Streakontofreshmedium. i. Aftercolonieshavegrown,sealplateswithParafilmandstoreat4°C.Forlongterm

storage(>2weeks),prepareglycerolstockculturesandfreezeat–70°C.ThesediploidsareusefulasreferencestrainswhenyouwishtocheckanewbatchofSDselectionmedium.

TABLEX.SET-UPFORCONTROLTwO-HYBRIDMATINGS

Cross PlateonSDMinimalMediaa Phenotype (100-mmplates) Mel1 His/Ade

PositiveControlAH109[pGADT7-RecT]xY187[pGBKT7-53] –Leub –Trpb –Leu/–Trpb –Ade/–His/–Leu/–Trp/X-α-Gal Blue +

NegativeControlAH109[pGADT7-RecT]xY187[pGBKT7-Lam] –Leub –Trpb –Leu/–Trpb

–Ade/–His/–Leu/–Trp/X-α-Gal nocolonies –

a Spread100-µlaliquotsof1:10and1:100dilutionsofthematingculture.

bUsetheseplatestocalculatethematingefficiency.




ProtocolB:ScreenbyCotransformationPLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. Beforestarting: • PourSDagarplates. SD/–Leu ~5–10 100-mmplates SD/–Trp ~5–10 100-mmplates SD/–Leu/–Trp ~5–10 100-mmplates SD/–His/–Leu/–Trp ~50 150-mmplates SD/–Ade/–His/–Leu/–Trp/X−α-Gal ~50 150-mmplates AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr

beforeplatinganytransformationmixtures. • Plancontrols(Step3)Controlsshouldbedoneinparallelwithexperimentalwork. • PrepareFreezingMedium:YPDmediumwith25%(v/v)glycerol. • PreparePEG/LiAcSolution(SectionIII).

1.CotransformyeaststrainAH109withdscDNA,pGADT7-Rec,andpGBKT7/bait. Important:Note thatyoumustpreparecompetentyeast cellsbeforestarting library

construction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

a. Preparecompetentyeastcells(AppendixB). b. Inasterile,15-mltubecombinethefollowing: • 20µl dscDNA(fromSectionIX.I,Step16) • 6µl pGADT7-Rec(0.5µg/µl) • 5µg pGBKT7/baitplasmidDNA(≤10µl) • 20µlHerringTestesCarrierDNA,denatured*

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

c. Add600µlofcompetentcellstotheDNA. d. Gentlymixbyvortexing. e. Add2.5mlPEG/LiAcSolution. f. Gentlymixbyvortexing. g. Incubateat30°Cfor45min.Mixcellsevery15min. h. Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. i. Centrifugeat700xgfor5min. j. Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumfor

thisstep.

k. Incubateat30°Cwithshakingfor90min. l. Centrifugeat700xgfor5min. m. Discardthesupernatantandresuspendin6mlofNaClSolution(0.9%).

2.Selectfortransformantsexpressinginteractingproteins a. SpreadthecotransformationmixtureonTDOorQDOplates(150µlcells/150-mm

plate). •TDOstandsforTripleDropoutMedium:SD/–His/–Leu/–Trp •QDOstandsforQuadrupleDropoutMedium:SD/–Ade/–His/–Leu/–Trp SeeFigure11. Note:Determinethetransformationefficiencyasfollows: i. Removea30-µlaliquotfromthe6-mlsuspensionanddilutewith720µlofNaClSolution(forafinal

volumeof750µl).

ii. Spread150-µlaliquotsofthisdilutionontwoseparate150-mmplates:SD/–Leu/–TrpandSD/–Leu.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolB



b. Incubateat30°Cuntilcoloniesappear. After2–3days,somecolonieswillbevisible,butplatesshouldbeincubatedforatleast

5daystoallowslowergrowingcoloniestoappear.Ignorethesmall,palecoloniesthatmayappearafter2daysbutnevergrowto>1mmindiameter.TrueAde+,His+coloniesarerobustandcangrowto>2mm.Also,Ade+coloniesarewhiteorlightpink,whereasAde–colonieswillslowlyturnredonadenine-limitedmedium.

c. ScoreforgrowthontheSD/–LeuandSD/–Leu/–Trpdilutionplates.

i.CountthecoloniesgrowingonSD/–Leu. #coloniesx1,000=#transformants/3µgpGADT7-Rec. Expectedresults:≥1x106transformants/3µgpGADT7-Rec ii.CountthecoloniesgrowingonSD/–Leu/–Trp. #coloniesx1,000=#clonesscreened Expectedresults:≥5x105clones/library d. ForcoloniesgrowingonTDOmedium:ReplicaplatecoloniesontoQDOmedium.

Incubateat30°Cfor3–8days. e. ChooseAde+/His+coloniesforfurtheranalysis. Not all of the colonies surviving this selection will be true two-hybrid positives.

However,themostcommonclassoffalsepositiveswillbeeliminatedbyscreeningforexpressionofADE2 and HIS3 (James et al.,1996).OthertypesoffalsepositivescanbeeliminatedasdescribedinSectionXIII.

f. StreakoutAde+/His+coloniesonfreshSD/–Ade/–His/–Leu/–Trp/X−α-Galmasterplatesandgrowfor2–4daysat30°C.HavingX-α-GalpresentinthemediumenablesyoutotestAde+/His+coloniesfortheactivationofathirdreporter:MEL1,which encodesα-galactosidase,asecretedenzymethathydrolyzesX-α-Galtoproduceablueendproduct.

3.ControlsforProtocolB:Small-ScaleYeastCotransformation Usethissmall-scalecotransformationprotocoltoproducethefollowingtwocontrolstrains.

• SV40 Large T PCR Fragment • pGADT7-Rec• pGBKT7-53

Recombination

in vivoAH109[pGADT7-RecT + pGBKT7-53]

Two-Hybrid Cotransformation Controls

Control Strain[plasmids]Cotransform AH109 with:

1. Positive Control

• SV40 Large T PCR Fragment • pGADT7-Rec• pGBKT7-Lam

Recombination

in vivoAH109[pGADT7-RecT + pGBKT7-Lam]2. Negative Control

TABLEXI.SET-UPFORCONTROLTwO-HYBRIDCOTRANSFORMATIONS

Component #1PositiveControl(µl)#2NegativeControl(µl)

SV40LargeTPCRFragment(25ng/µl 5.0 5.0pGADT7-Rec(SmaI-linearized;500ng/µl) 0.5 0.5pGBKT7-53(500ng/µl) 0.5 —pGBKT7-Lam(500ng/µl) — 0.5HerringTestesCarrierDNA(10mg/ml),denatured* 5 5AH109CompetentYeastCells 50 50PEG/LiAcSolution 500 500


XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolBcontinued



a. Preparecompetentyeastcells(AppendixB). b. Setuptwo1.5-mlmicrocentrifugetubes.AddDNA,competentyeastcells,andPEG/

LiAcSolutionusingthevolumesandintheorderindicated(TableXI). c. Mixthoroughlybygentlyvortexing. d. Incubateinawaterbathat30°Cfor30min.Vortexgentlyevery15min. e. Add20µlofDMSOtoeachtube,mix,andthenplacethetubeina42°Cwaterbathfor

20min.Vortexgentlyevery5min. f. Centrifugeathighspeedfor15sec. g. Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. h. Incubateinawaterbathat30°Cfor90min.Mixevery15minbygentlyvortexing. i. Centrifugeathighspeedfor15sec. j. Discardthesupernatantandresuspendin1mlofNaClSolutionbygentlypipetting

upanddown. k. Spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmSDagarplates:

• SD/–Leu/–Trp Tocheckthecotransformationefficiency

• SD/–Ade/–His/–Leu/–Trp/X−α-Gal Toselectforcotransformantsexpressinginteractingproteins

l. Incubatetheplatesat30°C(facedown)for2–4days,untilcoloniesappear. m. CompareyourresultswiththoseshowninTableXII. n. Pickthelargestcoloniesandrestreakthemonthesameselectionmedium. o. Aftercolonieshavegrown,sealthesemasterplateswithParafilmandstoreat4°C.

Forlongtermstorage(>2weeks),prepareglycerolstockculturesandfreezeat–70°C.ThesestrainsareusefulreferencesforcheckingnewbatchesofSDselectionmedium.

TABLEXII.CONTROLTwO-HYBRIDCOTRANSFORMATIONS:EXPECTEDRESULTS

ControlStrain PlateonSDMinimalMedia Phenotype Mel1 His/Ade

PositiveControlAH109[pGADT7-RecT+pGBKT7-53] –Ade/–His/–Leu/–Trp/X-α-Gal Blue +

NegativeControl

AH109[pGADT7-RecT+pGBKT7-Lam] –Ade/–His/–Leu/–Trp/X-α-Gal nocolonies –

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolBcontinued



This section presents strategies for verifying and analyzing protein-protein and protein-DNAinteractions.Figure12providesadetailedoverview.

A. RetestthePhenotype 1.Library transformants may contain more than one AD/library plasmid, which can

complicatetheanalysisofputativepositiveclones.Thus,itisagoodideatorestreakthepositivecoloniesonSDdropoutplates2–3timestosegregatetheAD/libraryplasmids.YoushouldrestreakonanSDdropoutmediumthatselectsforboththelibraryandbaitplasmidsaswellasfortheone-hybridortwo-hybridinteraction:

a. Restreakpositiveone-hybridclonesonSD/–His/–Leu/–Trp+optimal[3-AT]. Restreakpositivetwo-hybridclonesoneitherTDOorQDOmedium.Keepinmind

thatQDOisamorestringentselection.Reminder: TDOstandsfortripledropoutmedium=SD/–Ade/–Leu/–TrporSD/–His/–Leu/–Trp QDOstandsforquadrupledropoutmedium=SD/–Ade/–His/–Leu/–Trp b. Incubateplatesat30°Cfor4–6days. 2.[Optional]Testthephenotypefurtherbyassayingforathirdreportergeneorbyselecting

fortheinteractionunderdifferentconcentrationsof3-AT. • Two-hybridcolonies:Replicaplateortransferwell-isolatedcoloniestoSD/–Ade/–His/–

Leu/–TrpplatescontainingX-α-GaltoverifythattheymaintainthecorrectphenotypeandtotestfortheexpressionofMEL1.

• One-hybridcolonies:Replicaplateortransferwell-isolatedcoloniestoSD/–His/–Leu/–Trpplatescontainingdifferentconcentrationsof3-ATtoverifythattheymaintainthecorrectphenotypeandtotestthestrengthoftheinteraction.

3.Collecttherestreakedandretestedpositivecoloniesinagridfashiononfreshmasterplates.

4.Incubateplatesat30°Cfor4–6days. 5.Aftercolonieshavegrown,sealplateswithParafilm,andstoreat4°Cforupto4weeks.

B. RescuetheLibrarycDNAInsertToidentifythegene(andthusprotein)responsibleforapositiveone-hybridortwo-hybridinteraction,firstrescuethegenebyPlasmidIsolationorbyPCRColony-Screening:

PlasmidIsolation: 1. IsolateplasmidDNAfromyeastusingtheYeastmakerYeastPlasmid IsolationKit

(Cat.No.630441)orothersuitablemethod. 2. BecausetheplasmidDNAisolatedfromeachyeastcolonywillbeamixtureofthe

baitplasmidandatleastonetypeofAD/libraryplasmid,youwillneedtoseparatetheplasmidsbyselectioninE. coli.

Note:Baitpasmidsusedwithpreviousversionsofourtwo-hybridandone-hybridsystemscontainedthegeneforampicillinresistance.Baitplasmidsusedwithnewerversionsofourkits,however,containthegeneforkanamycinresistance.

Ifyouusedbaitandlibraryplasmidsthatcontaindifferentantibioticselectionmarkers: a. TransformtheisolatedplasmidextractfromyeastintoageneralpurposeE. coli

strain. b. PlatethetransformantsonLBmediumcontainingampicillintoselectfortheAD/

libraryplasmidonly.

Ifyouusedbaitandlibraryplasmidsthatcontainthesameantibioticselectionmarker: a. Transformtheyeast-purifiedplasmidintoKC8E. coli cells. b. PlatethetransformantsonM9mediumlackingleucinetoselectfortheAD/library

plasmidonly. Note:KC8cellshaveadefectinleuBthatcanbecomplementedbyyeastLEU2.

3. PurifytheAD/libraryplasmidusinganysuitablemethod. 4. Amplify the cDNA insert by PCR using the MatchmakerAD LD-Insert Screening

AmplimerSet(Cat.No.630433)andtheAdvantage2PCRKit(Cat.No.639206). 5. AnalyzethePCRproductasdescribedinPartC.

XIII. AnalyzingPositiveInteractions



XIII. AnalyzingPositiveInteractionscontinued

Figure12.Strategiesforanalyzingandverifyingputativepositiveone-hybridandtwo-hybridinteractions.

PCR product consists of more than one band.Yeast colony contains more than one AD plasmid.

Rescue the library cDNA insert using one of the following procedures:

Use one of the following methods to segregate the AD plasmids:

PCR product consists of one band.Yeast colony contains one AD plasmid.

Sequence the PCR product or AD plasmid insert:

Retest the phenotype

Compare the sequence with that of other proteinsin GenBank, EMBL, or other databases.

• Restreak the yeast colony on selective medium.• Isolate the plasmids from yeast and transform E. coli. Select on LB/Amp.• Gel purify each PCR fragment.

Retest the interaction in yeast

Retest the interaction in vitro:CoimmunoprecipitationMatchmaker Co-IP KitGel-shift assay

Y2H→

Y1H→

Retest the interaction in mammalian cells: • Matchmaker Mammalian Two-Hybrid Assay Kit • pCMV-Myc & pCMV-HA Vector Set

Map the interacting domain(s):• Diversify PCR Random Mutagenesis Kit

Isolate the full-length cDNAfrom one of the following SMART cDNA libraries:• SMART™ Libraries in Creator™ Donor Vectors • Large-Insert cDNA Libraries

Gene transfercatalyzed byCre recombinase,15 min, RT

Express the protein in a variety of mammalian cell types usingRevTet-On & RevTet-Off Gene Expression Systems

Localize the protein in vivo in mammalian cells using Living Colors Fluorescent Protein Vectors

Purify the protein using PRO Bacterial Expression Vectors.• Develop antibodies• Determine tissue-specific expression using Protein Medleys• Study the protein's physical properties

PCR Colony Screening using Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433)

Plasmid Isolation(Yeastmaker Yeast Plasmid Isolation Kit)

Amplify the cDNA insert by PCR using the Matchmaker AD LD-Insert Screening Amplimer Set (Cat. No. 630433)

or

Analyze the PCR product by gel electrophoresis.

Analyze protein function using Matchmaker One-Hybrid and Two-Hybrid Systems

Creator™ Acceptor Vectors

in vitro transcriptionand translation

Your own Acceptor Vector

or

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 �0 Version No. PR6Z2169


PCRColonyScreening: This procedure uses the Matchmaker AD LD-Insert Screening Amplimer Set

(Cat. No. 630433) andAdvantage 2 PCR Polymerase Mix.We recommend using theAdvantage 2 Polymerase Mix, rather than any other DNA polymerase formulation,becausewefindthatitperformswellinyeastcellsamples,andbecauseitisoptimizedforapplicationsthatinvolvelongertemplatesandrequirehighfidelity.

1. PreheataPCRthermalcyclerto94°C. 2. PlacetheAdvantage2PCRKitcomponentsandGAL4ADLD-InsertScreeningAmplimers

oniceandallowthemtothawcompletely.Mixeachcomponentthoroughlybeforeuse. 3. PrepareaMasterMixbycombiningthecomponentsasspecifiedinTableXIII.

TABLEXIII:ASSEMBLINGMASTERMIXESFORPCRCOLONYSCREENING

POLYMERASE MIX (Mg 2+ included in the buffer)Reagent 1rxn 10rxns 25rxns

+1extra +1extra

PCR-gradedeionizedH2O 41 µl 451 µl 1066 µl

10XAdvantage2PCRBuffer 5 µl 55 µl 130µl

5'LDAmplimer(20µM) 1µl 11 µl 26 µl

3'LDAmplimer(20µM) 1µl 11 µl 26 µl

50XdNTPMix(10mMea.) 1µl 11 µl 26 µl

50XAdvantage2PolymeraseMix 1µl 11 µl 26 µl

Total 50µl 550µl 1300µl

4. Usingasterilepipettetip,scrapeafewcellsfromacolonythatyouwishtoanalyze.Placethecellsinthebottomofaclean250-µlPCRtube.

5. Add50µlofMasterMixtothetube.Gentlypipetteupanddowntodispersethecells. 6. Overlaythereactionmixturewith2dropsofmineraloilifnecessary.Capthetube

andplaceitinapreheatedthermalcycler. 7. Begin thermal cycling. If you have a hot-lid thermal cycler, use the following

program: Note:Thesecyclingparametersmaynotbeoptimalfornonhot-lidthermalcyclers.

•94°C3min •25–30cycles: 94°C 30sec 68°C 3min •68°C 3min •Soakat15°C 8. AnalyzethePCRproductasdescribedinPartC.

C. AnalyzethecDNAInsertbyAgarose/EtBrGelElectrophoresisAnalyzea5-µlaliquotofthePCRproduct(fromPartB)alongsideDNAsizemarkersona0.8%1XTAEagarose/EtBrgel.Tip:TodistinguishthePCRproductfromsimilarsizeinsertsinotherAD/libraryplasmids,digestthePCRproductwithafrequent-cutterrestrictionenzymesuchasAluIorHaeIII.Runasmallsampleona2%agarose/EtBrgel.Comparethedigestionpatternwiththatofotherinserts.

• IfahighpercentageofthecoloniesappeartocontainthesameAD/libraryinsert,expandyourPCRanalysistoanotherbatchof50colonies.Alternatively,eliminatetheabundantclonesbyperformingyeastcolonyhybridizationoneachmasterplate.RefertotheYPHforthisprocedure.Useavector-freeoligonucleotideprobedesignedfromthesequenceofthemostabundantinsert.Transferarepresentativeofeachtypeofinserttoanewmasterplate.

• IfthePCRproductconsistsofmorethanoneband,seePartE,below. • IfthePCRproductconsistsofasingleband:


Protocol No. PT3529-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2169 �1


1. Prepareanewmasterplatewitharepresentativeclonefromeachgroup. 2. Ifyouaresatisfiedwiththenumberofuniqueclones,prepareaglycerolstockof

eachuniquetype.Storeat–80°C. 3. PurifythePCRproductusinganysuitablemethod.WenormallyuseaNucleoSpin

ExtractionKit(Cat.No.635961) 4. ProceedwithPartD.

D. SequencethecDNAInsertAD/librarycDNAinsertscanbesequencedusingtheMatchmakerADLD-InsertScreeningAmplimerSet(Cat.No.630433),aT7SequencingPrimer,orthe3'ADSequencingPrimerprovidedwithMatchmakerTwo-HybridSystem3(Cat.No.630303).Alwayscheckthevectorsequenceagainsttheprimeryouwishtouse.BeawarethatsomeMatchmakerADplasmids(e.g.,pACT2)donotcontainaT7Promoter.Verifythepresenceofanopenreadingframe(ORF)fusedtotheGAL4ADsequence,andcomparethesequencetothoseinGenBank,EMBL,orotherdatabases.

• Ifyoursequencingresultsrevealaveryshortpeptide(<10aminoacids)fusedtotheAD—ornofusionpeptideatall—keepsequencingbeyondthestopcodon.Youmayfindanother(larger)openreadingframe(ORF).Suchgapscanoccurwhenaportionofthe5'untranslatedregionofanmRNAisclonedalongwiththecodingregion.AWesternblotwillrevealthepresenceandsizeofanADfusionprotein.

• Insomecases,twodifferentORFsmaybeexpressedasafusionwiththeADeventhoughanontranslatedgapcomesbetweenthemdue,forexample,tooccasionaltranslationalread-through.

• IfyoursequencingresultsfailtorevealanyORFinframewiththeADcodingregion,itcouldbethatthepositivelibrarycloneistranscribedinthereverseorientationfromacrypticpromoterwithintheADH1terminator(Chienet al.,1991).Yeastalsoallowtranslationalframeshifts.AlargeORFinthewrongreadingframemayactuallycorrespondtotheexpressedprotein.

E. SegregationofAD/LibraryPlasmidsUse one of the following procedures when a positive one-hybrid or two-hybrid colonycontainsmultiple,non-identicalAD/libraryplasmids.

• SegregationinYeast: 1. RestreakpositivecoloniesonSDdropoutplates2–3timestosegregatetheAD/library

plasmids,asdescribedinPartA,Step1. 2. Replicaplateortransferwell-isolatedcoloniestotheappropriateSDdropoutplates

toverifythattheymaintainthecorrectphenotype. • SegregationinBacteria: 1. Isolatetheplasmidsfromyeastusinganystandardmethod. Note:TheYeastmakerYeastPlasmidIsolationKit(Cat.No.630441)providesacompletesystemforisolating

plasmidDNAfromyeast.

2. TransformE. coliDH5αcellswith theplasmidpreparationandselectonLB/ampplates.Pickindividualcolonies.

• PCRFragmentPurificationusingagarosegelelectrophoresis.

F. RetesttheInteractioninYeast Retestprotein-proteinandprotein-DNAinteractionsinyeastbyeithercotransformationor

yeastmating. 1.Cotransformation a. Transformcompetentyeastcells*withthebaitandAD/libraryplasmids.Alternatively,

transformyeastcells*withbaitplasmid,pGADT7-Rec(2),andthePCRproductofinterest,generatedinPartB.

* Forone-hybridanalysis,useyeaststrainY187.Fortwo-hybridanalysis,useyeaststrainAH109.

Besuretoincludetheappropriatenegativeandpositivecontrols.Forexamples,refertothecontrolsusedinSectionsXIandXII.OnenegativecontrolshouldconsistofyeastcellstransformedwithyourcandidateAD/libraryplasmidandanemptybaitplasmid—eitherpHIS2orpGBKT7,dependingonthesystemyouareusing.




Ifyouareretestingaone-hybridinteraction,andanon-bindingmutantofyourtargetelementisavailable,considerusingitasanadditionalnegativecontrol.Prepareamutant-typeconstructotherwiseidenticaltoyouroriginaltarget-reporterconstructandtransformY187withthemutantreporterplasmid,thecandidatecDNAinsert,andpGADT7-Rec2.Coloniesshouldresultfromtranscriptionalactivationusingthewild-typebutnotthemutant-typetarget.Foranexample,seeLi&Herskowitz,1993.

b. PlateontheappropriateSDdropoutmedium. c.Incubateplatesat30°Cuntilcoloniesappear.

2.YeastMating The followingproceduredescribeshowyeastmatingcanbeusedtoretestpositive

two-hybridinteractions.IfyouhaveaccesstoaMATastrainsuchasAH109,asimilarapproachcouldbeusedfortheanalysisofone-hybridinteractions,inwhichcasetheDNA-BD/baitplasmidisreplacedbythepHIS2reporterplasmid.

Ifyouhavemanypositiveclonestoanalyze,itwillbemoreconvenienttohandletheclonesinbatchesof10orsoeach.

a. TransformAH109withtheAD/libraryplasmid(s),andselectonSD/–Leu. b. TransformY187(orasuitableMATαstrain)withthefollowingthreeplasmids,and

selectonSD/–Trpplates: i. DNA-BD ii. DNA-BD/bait iii. pGBKT7-Lam c. ForeachcandidateAD/libraryplasmidtobetested,setuptheyeastmatingsindicated

inFigure13usingtheTrp+andLeu+transformantsobtainedinStepsa&babove. RefertoSectionXII.A.8forasmall-scaleyeastmatingprocedure. d. Select for diploids by spreading mating mixtures on SD/–Leu/–Trp plates as

directed. e. Streakorreplica-platetoSD/–Ade/–His/–Leu/–Trp/X-α-gal.TruepositivesareAD/library

clones exhibiting reporter gene expression only when theAD/library plasmid isintroducedbymatingwiththeplasmidencodingtheDNA-BD/baitprotein.

AdditionalTwo-HybridTests: • TransferthelibraryinsertfromtheADtotheDNA-BDvectorandviceversa,andthen

repeatthetwo-hybridassay(Chienet al.,1991;vanAelstet al.,1993).Youshouldstillbeabletodetecttheinteraction.

• CreateaframeshiftmutationjustupstreamofthelibraryinsertintheADplasmidbycuttingattheMluIsite,fillingintheoverhangs,andthenreligating(Bendixenet al.,1994).Repeatthetwo-hybridassay;youshouldnotbeabletodetecttheinteraction.

• Generatesite-specificdeletionorsubstitutionmutantsandrepeatthetwo-hybridassay.


DNA-BD DNA-BD/bait DNA-BD/lamin C

Master plate with candidate clones

1. Transform AH109 with AD/library plasmid(s)

2. Plate on SD/–Leu

3. Inoculate 0.5-ml YPD cultures.

5. Plate on SD/–Leu/–Trp

4. Mate to Y187 transformed with:

6. Replica plate or streak onto SD/–Ade/–His/–Leu/–Trp/X-α-Gal

Figure13.Yeastmatingtoverifyprotein-protein(two-hybrid)interactions.



G. RetesttheInteractionIn Vitro • Two-hybrid (Protein-protein) interactions can often be confirmed in vitro using

coimmunoprecipitation(Co-IP).TheMatchmaker™Co-IPKit(Cat.No.630449)includesalltheessentialreagents(includingantibodies,ProteinABeads,andadetailedstep-bystepprotocol)neededtoperformin vitroCo-IP.FollowingisageneralCo-IPprotocolsuitableforMatchmakervectors.

a. Transcribeand translate theepitope-tagged fusionproteins in vitro using theT7promotersintheADandDNA-BDvectors.

Note:MatchmakerSystem3vectors—pGADT7,pGADT7-Rec,pGADT7-Rec2,pLP-GADT7,pGBKT7,andpLP-GBKT7—containaT7RNApolymerasepromoterandeitherac-Mycorhemagglutinin(HA)epitopetagsothatyoucanusethemdirectlyforin vitrotranscriptionandtranslation.(ForallotherGAL4-basedvectors,youmustincorporateaT7promoterandepitopetagusingPCRandanappropriatepairofprimers.)BecausetheT7promoterinSystem3vectorsislocateddownstreamoftheGAL4codingsequence,theGAL4domainsarenottranscribed.Thus,in vitroCo-IPspecificallydetectsinteractionsbetweenbaitandlibraryproteins.

b. Coimmunoprecipitate the epitope-tagged fusion proteins using c-Myc and HAantibodies(Durfeeet al.,1993;Zhanget al.,1993).

Note: If thefusionproteinsdo notcoimmunoprecipitate,useothermeanstoconfirmtheinteraction.Proteininteractionswithweakaffinitiesmayescapedetectionbycoimmunoprecipitation.SeePhizichy&Fields(1995)fordetailsonmoresensitivedetectionmethods.Furthermore,theADfusionproteinsmaypotentiallynotbein-framewiththeepitopetag.

• One-Hybrid(Protein-DNA)interactionscanoftenbeconfirmedandstudiedin vitrousinganelectrophoreticmobility-shiftassay(EMSA;Wuet al., 1994).

a. TranscribeandtranslatetheHAepitope-taggedfusionprotein in vitrousingtheT7promoterintheADvectorpGADT7-Rec2.

Note:TheT7promoterinpGADT7-Rec2islocateddownstreamoftheGAL4codingsequence;thus,theGAL4activationdomainisnottranscribedinvitro.

b. PerformanEMSAassaywithyourwild-typeandmutantDNAtargetstoverifyand,ifdesired,tomaptheDNA-bindingactivityoftheprotein.

H. RetesttheInteractioninMammalianCells Inmammaliancells,proteinsaremore likely tobe in theirnativeconformationsand to

havetheappropriatepost-translationalmodifications;therefore,resultsaremorelikelytorepresentbiologicallysignificantinteractions.

• Two-hybridinteractions Clontechoffersthefollowingproductsfortestingtwo-hybridinteractionsinmammalian

cells: – ThepCMV-Myc&pCMV-HAVectorSet(Cat.No.631604)for in vivocoimmunoprecipitation

in mammalian cells.The CMV promoter in these vectors allows constitutiveexpressionofthebaitandlibrarycDNAinawidevarietyofmammaliancelltypes.

– TheMammalianTwo-HybridAssayKit(Cat.No.630301)isidealforconfirmingproteininteractionsviatwo-hybridinteractionsinmammaliancells.

• One-hybridinteractions Onewaytotestone-hybridinteractionsinmammaliancellsistocloneyourDNAtarget

intoamammalianreportervector.Forexample,ifyourDNAtargetispartoflarger,cis-actingregulatorysequence—apromoterorpromoter/enhancerelement—theelementcanbeclonedintooneofourpromoterlessLivingColors®FluorescentProtein,pSEAP,orpβgalReporterVectors.Similarly,thecDNAencodingtheputativeDNA-bindingproteincanbeclonedintoaconstitutivemammalianexpressionvectorsuchaspIRES2-EGFP.IftheputativeDNA-bindingproteinfunctionsasatranscriptionalactivator,youmaybeabletodetectitsactivitybycotransfectingcellswiththesevectorconstructs.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3529-1 �� Version No. PR6Z2169


A. ConstructingDNA-BDFusionsDNA-BD/baitactivatesreportergenes

• Thebaitproteinhasatranscriptionalactivationdomain.Thisisespeciallylikelyifthebaitproteinisatranscriptionfactor.Acidicamphipathicdomainsareoftenresponsibleforunwantedtranscriptionalactivation(Rudenet al.,1991;Ruden,1992).Removetheactivatingdomainbycreatingspecificdeletionswithin thegene.Retest thedeletionconstructsforactivation.Attheaminoacidlevel,anetnegativechargeper10aminoacidsisaminimalAD.Notethatsuchdeletionsmayalsoeliminateapotentiallyinteractingdomain.

• Iftwotestproteinsarebeingassayed,switchfromtheDNA-BDtotheADvectorandviceversa.

Baitproteinistoxictoyeastcells • Insomecases,strainsthatdonotgrowwellinliquidculturewillgrowreasonablywellon

agarplates.Resuspendthecolonyin1mlofSD/–Trp,thenspreadthecellsuspensiononfive100-mmSD/–Trpplates.Incubatetheplatesat30°Cuntilthecoloniesareconfluent.Scrapethecoloniesfromeachplate,pooltheminonetube,andresuspendinatotalof5mlof0.5XYPDA.Usethecellsuspensioninthenormalmatingprocedure.

B. GeneratingcDNALibrariesLowyieldofdscDNA

• Oneormoreessentialreagentsmayhavebeeninadvertentlyomittedfromthefirst-strandordscDNAsynthesissteps.Repeatbothsteps,beingcarefultocheckoffeveryitemasyouaddittothereaction.

• LowyieldsofdscDNAmaybeduetoPCRundercycling.Ifyoususpectundercycling,incubatethePCRreactionmixturefortwomorecyclesandrechecktheproduct.Ifyoualreadyusedthemaximumrecommendednumberofcycles,increasebythreemorecycles.IfadditionalcyclesdonotimprovetheyieldofPCRproduct,repeatthePCRusingafreshaliquotofthefirst-strandproduct.

• AlowyieldofdscDNAmayalsobeduetoalowyieldoffirst-strandcDNA.Possibleproblemswiththefirst-strandreactionincludeamistakeintheprocedure(suchasusingasuboptimalincubationtemperatureoromittingacomponent)orinsufficientRNAinthereaction.ItisalsopossiblethattheRNAhasbeenpartiallydegraded(bycontaminatingRNases)beforethefirst-strandsynthesis.Problemswiththefirst-strandcDNAsynthesiscanbemoreeasilydiagnosedifyouperformparallelreactionsusingthecontrolRNAprovidedinthekit.IfgoodresultswereobtainedwiththecontrolRNAbutnotwithyourexperimentalRNA,thentheremaybeaproblemwithyourRNA.

SizedistributionofdscDNAislessthanexpected • Your RNA starting material may be degraded, very impure, or too dilute. Check the

qualityandquantityofyourRNAbyrunningasampleonagel.IftheRNAseemstoodilute,butotherwiseofgoodquality,restarttheexperimentusingmoreRNA.IftheRNAseemsdegraded,restarttheexperimentusingafreshlotorpreparationofRNA.Also,checkthestabilityofyourRNAbyincubatingasmallsampleat37°Cfor2hr.Runitonagel,paralleltoafresh(unincubated)sample.IftheRNAappearstobeunstable,itwillyieldpoorresults.Ifthisisthecase,reisolatetheRNAusingadifferentmethod(seeRelatedProducts).ProblemswithyourRNAareeasilydiagnosedifyouperformparallelreactionsusingthecontrolRNAprovidedinthiskit.

Presenceoflowmolecularweight(<0.1kb)materialinthedscDNAproduct • TherawcDNA(e.g.,beforesizefractionation)isexpectedtocontainsomelow-molecular-

weightDNAcontaminants,includingunincorporatedprimers,SMARToligonucleotides,andveryshortPCRproducts.However,thesesmallfragmentsaregenerallyremovedfromthedscDNApreparationinthesizefractionationstepusingthecolumnsprovided.Notethatapreponderanceoflow-molecular-weight(<0.1kb)materialintherawPCRproductmaybeindicativeofovercycling.Ifyoususpectovercycling,thenthePCRstepmustberepeatedwithafreshsampleoffirst-strandcDNA,using2–3fewercycles.

XIV. TroubleshootingGuide



Presenceoflowmolecularweight(<0.1kb)inthesize-fractionateddscDNA • CHROMASPINcolumnsaredesignedtoremovelow-molecular-weightcDNAfragments,

smallDNAcontaminants,andunincorporatednucleotidesfromthecDNA.WhenusingthesecolumnstopurifydscDNA,pleasekeepthefollowingpointsinmind:

– Theresolvingfunctionofthecolumnwillbediminishedifthegelmatrixbecomesdry.Indrying,thematrixbodymayshrinkawayfromtheinnerwallofthecolumncasing.ThedscDNAmixturecanthenflowdownthesidesofthecolumn,allowingsmallcontaminantstoelutewiththecDNA.

– Thecolumnshouldbestoredandusedatroomtemperature.Ifitischilledat4°Candthenwarmedtoroomtemperatureforuse,bubblesmayform,whichinterferewiththeproperfunctioningofthecolumn.

– Extreme,unevendepositionofthedscDNAmixtureonthesurfaceofthecolumncancauseinefficientseparationofdscDNAfromlow-molecular-weightcontaminants.

C. Constructing&ScreeningOne-HybridandTwo-HybridLibrariesLowtransformationefficiency

• CheckthepurityoftheDNAand,ifnecessary,repurifybyethanolprecipitation. • ThefusionproteinencodedbytheDNA-BD/baitplasmid(Two-HybridSystem)maybe

toxic.Tryusingavectorthatexpresseslowerlevelsofthefusion,suchpGBT9. • Impropermediapreparation.Remakemediaandtestwithcontroltransformations. • ChecktheefficiencyusingthepGBT9ControlPlasmid.PlateonSD/–Trp.Thetransformation

shouldyield≥1x105colonies/µgDNA.Lowmatingefficiency(Two-HybridSystem)

• Theremayhavebeenaninsufficientnumberofpretransformedbaitcellsinthemating.Whenyoupreparetheovernightliquidcultureofthebaitstrain,besuretousealarge,freshcolonyfortheinoculum.Aftercentrifugingandresuspending,countthecellsusingahemacytometer.Thedensityshouldbe≥1x109/ml,an~100-foldexcessoverthepretransformedlibrarycells.

• Oneorbothofthefusionproteinsistoxictoyeast.Youmaybeabletoengineerthefusioninawaythatalleviatesitstoxicitybutstillallowstheinteractiontooccur.Alternatively,useaDNA-BDorADvector(e.g.,pBridgeorpGBT9)thatexpresseslowerlevelsofthefusion.Itmaybenecessarytoperformthematingonagarplates(Bendixenet al.,1994)oronfilters(Fromont-Racineet al.,1997).Besuretosetupmatingcontrolsforcomparison.

• Bait proteins may interfere with mating if they share homology to yeast-matingproteins—e.g.,pheromonereceptors(Shultzet al., 1995;Pringleet al.,1992).Ifhomologyissuspected,itmaybenecessarytoscreenyourlibrarybycotransformation.

Excessivebackgroundgrowthonlibraryscreeningmedium • Checktomakesureyouhavepreparedtheselectionmediumcorrectly.Addtheappropriate

amountof3-AT.Performa3-ATtitrationwiththetransformedreporterstraintooptimizetheconcentration.

• Ifyourtarget-pHIS2reporter(One-HybridSystem)growsonSD/–Hismediumeveninthepresenceof≥60mM3-AT,theinsertedtargetelementmaybeinteractingwithyeastendogenoustranscriptionalactivators,ormaynotrequiretrans-actingfactorstoactivatetheHIS3reporter.Itmaybenecessarytoredesignthetargetelementandconstructanewreportervector.

• TheDNA-BDfusionprotein(Two-HybridSystem)mayhavetranscriptionalactivatingproperties.Deletionofcertainportionsofbaitproteinsmayberequiredtoeliminateunwantedactivitybeforetheproteincanbeusedinatwo-hybridscreen(Bartelet al.,1993b).

Failure to detect known protein-protein (Two-Hybrid) or DNA-protein interactions (One-Hybrid)interactions

• Ifexpressionofoneorbothofthehybridproteinsistoxictothecell,transformantswillnotgroworwillgrowveryslowly.Truncationofoneofthehybridproteinsmayalleviatethetoxicityandstillallowtheinteractiontooccur.TryusingvectorssuchaspGBT9orpBridgethatexpresslowerlevelsoftheDNA-BDfusionprotein.

• Thecotransformationefficiencyistoolow.Youmaynotbescreeningasufficientnumberoflibrarycotransformants.Thiscanbecritical,especiallyiftheinteractingtargetproteinisencodedbyararetranscriptinthesourcetissue.

XIV. TroubleshootingGuidecontinued



• Ifoneofthefollowingsituationsisoccurring,itmayinterferewiththeabilityofthetwohybridproteinstointeract:(1)thehybridproteinsarenotstablyexpressedinthehostcell;(2)thefusedGAL4domainsoccludethesiteofinteraction;(3)thehybridproteinfoldsimproperly;or(4)thehybridproteincannotbelocalizedtotheyeastnucleus.Inthesecases,itmayhelptoconstructhybridscontainingdifferentdomainsofthebaitorlibraryprotein.Forexample,tostudyproteinsthatnormallydonotlocalizetothenucleus,itmaybenecessarytogeneratemutantformsoftheproteinthatcanbetransportedacrossthenuclearmembrane.

• Some types of protein interactions may not be detectable in a GAL4-based system.The conditions in yeast cells may not allow the proper folding or post-translationalmodificationsrequiredforinteractionofsomemammalianproteins.

ADfusionlibraryplasmidactivatesthereportersindependentoftheDNA-BD/baitprotein(Two-HybridSystem)

• ArarecategoryoffalsepositivesinwhichanAD/libraryhybridactivatestranscriptioninappropriately.RefertoSectionXIIIformethodstoverifyproteininteractions;seeBartelet al. (1993a)forfurtherdiscussionoffalsepositives.

XIV. TroubleshootingGuidecontinued

Protocol No. PT3529-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2169 ��


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XV. Referencescontinued



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AppendixA:TypicalResultsofdscDNASynthesis

Double-strandedcDNAsynthesizedfromControlHumanPlacentaPolyA+RNAusingtheprotocolinthismanualshouldappearasamoderatelystrongsmearfrom≥0.1kbto4kb(ormore)ona1.2%agarose/EtBrgel(Figure14).

Double-stranded cDNA

M 1 2 3 4 Mkb

10

4 3

2 1.5

1

0.5

Figure14.Double-strandedcDNAsynthesizedfromControlHumanPlacentaPolyA+RNA.1µl(1.0µg)ofControlHumanPlacentaPolyA+RNAwasusedasthetemplateforfirst-strandcDNAsynthesis.Twofirst-strandsampleswereprepared:Onewitharandomprimer(ourCDSIII/6Primer;Lanes1&3),andtheotherwithanoligo(dT)primer(ourCDSIIIPrimer;Lanes2&4).Next,2µlofthesingle-strandedcDNAwasamplifiedbyLD-PCR.EachdscDNAproductwasthenpurifiedwithaCHROMASPIN+TE-400Column.ThedscDNAwasanalyzedona1.2%agarose/EtBrgelbefore(Lanes1&2;7µlcDNAperlane)andafter(Lanes3&4;5µlcDNAperlane)columnpurification.LaneMwasloadedwith250ngofa1-kbDNAsizemarker.



Thisprotocolwillyield1.2mlofcompetentcells.

Beforestarting: • StreakaYPDAagarplatewithasmallportionoffrozenyeaststock—e.g.,AH109orY187. (Ifthetubehasthawedpriortostreaking,vortextoensureevendistributionoftheyeast

cells.)Incubatetheplateupsidedownat30°Cuntilcoloniesappear(~3days). Yeaststrainscanbestoredforupto1monthat4°ConYPDAmediumincultureplates

sealedwithParafilm.

• Prepare1.1XTE/LiAcSolution(SectionIII)

• PrepareYPDAliquidmedium(YeastProtocolsHandbook)

1.Inoculateonecolony(<4weeksold,2–3mmindiameter)into3mlofYPDAmediuminasterile,15-mlcentrifugetube.

2.Incubateat30°Cwithshakingfor8hr. 3.Transfer5µloftheculturetoa250-mlflaskcontaining50mlofYPDA. 4.Incubate at 30°C with shaking at 230–250 rpm for 16–20 hr.The OD600 should reach

0.15–0.3. 5.Centrifugethecellsat700xgfor5minatroomtemperature. 6.Discardthesupernatantandresuspendthecellpelletin100mlofYPDA. 7.Incubateat30°Cfor3–5hr(OD600=0.4–0.5). 8.Centrifugethecellsat700xgfor5minatroomtemperature. 9.Discardthesupernatantandresuspendthecellpelletin60mlofsterile,deionizedH2O. 10.Centrifugethecellsat700xgfor5minatroomtemperature. 11.Discardthesupernatantandresuspendthecellsin3mlof1.1XTE/LiAcSolution. 12.Splittheresuspensionbetweentwo1.5-mlmicrocentrifugetubes(1.5mlpertube). 13.Centrifugeeachtubeathighspeedfor15sec. 14.Discardthesupernatantandresuspendeachpelletin600µlof1.1XTE/LiAcSolution. Note: Competentcellsshouldbeusedfor transformationimmediatelyfollowingpreparation;however, if

necessarytheycanbestoredatroomtemperatureforafewhourswithoutsignificantlyaffectingthecompetency.

AppendixB:PreparationofCompetentYeastCells—LiAcMethod



AppendixC:One-HybridControlVectorInformation

Figure15.Mapofp53HIS2ControlVector.p53HIS2isayeastone-hybridreportervectorthatservesasapositivecontrolintheMatchmakerOne-HybridLibraryConstruction&ScreeningKit(CatNo.630304).Itcontains3tandemcopiesoftheconsensusDNAbindingsiteforp53.ThethreeDNAtargetsarelocatedupstreamoftheminimalpromoteroftheHIS3locus(PminHIS3)andtheHIS3nutritionalreportergene.p53HIS2isdesignedforusewithpGAD-Rec2-53,aplasmidthatencodesmurinep53asafusiontotheGAL4AD.YeastcellsthatcontainbothoftheseplasmidswilldisplaytheHis+phenotypeasaresultoftheinteractionbetweenmurinep53andtheDNAbindingsitesinp53HIS2.WhentheGAL4AD-p53fusioninteractswiththesesites,itstimulatestranscriptionofHIS3,givingyeaststrainssuchasY187andAH109,whicharenormallyauxotrophicforhistidine,theabilitytogrowonhistidinedropoutmedium.

p53HIS2containsanautonomousreplicationsequence(ARS4)andTRP1 nutritionalmarkerforreplicationandselectioninyeast;thecentromericsequenceCEN6ensurespropersegregationoftheplasmidduringmitosisandmeiosis.ThevectoralsocontainsaColE1oriandkanamycinresistancegene(Kanr)forpropagationandselectioninE. coli. Thisvectorhasnotbeencompletelysequenced.

p53HIS2�.2 kbKanr

TRP1Col E1

ori

HIS3PminHIS3

3' UTR&

TminHIS3

CEN6/ARS4

3 x p53 DNA elementsEcoR I

(2)

Xho I (1051)

Figure16.MapofpGAD-Rec2-53ADControlVector.pGAD-Rec2-53encodesafusionoftheGAL4ADandmurinep53,aknownDNA-bindingprotein(Thukral,S.K.,et al., 1994).ThevectorisderivedfrompGADT7-Rec2andwasconstructedatClontechbyhomologousrecombinationinE. coli.Specifically,thevectorwasproducedbytransformingcompetentE. coli cellswithEcoRI/BamHI-linearizedpGADT7-Rec2anddscDNAencodingmurinep53(a.a.73-391).Asaresult,thisvectordoesnotcontaintheT7RNApolymerasepromoterorhemagglutinin(HA)epitopetag,nordoesitshareanyhomologywiththeSMARTIIIorCDSIIIoligonucleotides.

pGAD-Rec2-53isdesignedforuseasapositivecontrolvectorinMatchmakeryeastone-hybridassays.Itisnotintendedtoserveasacloningvector,norisitintendedtobeusedasasourceofmurinep53cDNA.Instead,usepGAD-Rec2-53withp53HIS2toproduceapositivecontrolyeaststrain.YeaststrainsAH109andY187,whicharenormallyunabletogrowonhistidine-deficientmedia,willgrowonmediumlackinghistidinewhentransformedwithpGAD-Rec2-53andp53HIS2.TransformantsacquiretheabilitytosynthesizehistidineasaresultoftheinteractionbetweentheGAL4AD-p53fusion,expressedbypGAD-Rec2-53,andthep53consensusDNA-bindingsequenceinp53HIS2.Uponbindingtheconsensussequence,theGAL4AD-p53fusionstimulatestranscriptionoftheHIS3 reportergeneinp53HIS2andconferstheHis+phenotypetothehost.

pGAD-Rec2-53containsanautonomousreplicationsequence(ARS4)andLEU2 nutritionalmarkerforreplicationandselectioninyeast;thecentromericsequenceCEN6ensurespropersegregationoftheplasmidduringmitosisandmeiosis.ThevectoralsocontainsapUCoriandampicillinresistancegene(Ampr)forpropagationandselectioninE. coli. Thisvectorhasnotbeencompletelysequenced.

pGAD-Rec2-53�.� kb

Ampr GAL4 AD

pUCori

PADH1

SV40 NLS

LEU2

TADH1

murine p53

CEN6/ARS4

Not I(5385)

Hind III (1480)

Hind III (3146)



HA epitope tagpGADT�-RecT

10.0 kb

Ampr

2 micronori

GAL4 AD

pUCori

PADH1

PT7

SV40 NLS

LEU2TADH1

SMART™ III sequence

CDS III sequence

Large TAntigen

Figure17.MapofpGADT7-RecTADControlVector.pGADT7-RecTisaproductofrecombinationthatencodesafusionoftheSV40largeTantigenandtheGAL4AD.Togeneratethiscontrol,cotransformyeastwiththeSV40LargeTPCRFragmentandpGADT7-RecCloningVector(Sma I-linearized).BecausethelinearizedvectorsharessequencehomologywiththeendsoftheLargeTPCRFragment,thesetwocomponentsrecombineviaadouble-crossovermechanismtoproducethecircularcontrolplasmidpGADT7-RecT.TheSV40LargeTDNA(GenBankLocusSV4CG)wasderivedfromaplasmidreferencedinLi&Fields(1993).PCRamplificationwasperformedatClontech.

AppendixD:Two-HybridControlVectorInformation

Nde I Nco I Sfi I EcoR ISma IXma IBamH ISal IPst I

Murine p53 insert

(a.a. 72)

(a.a. 390)

pGBKT�-53�.3 kb

Kanr

2 ori

TRP1

PADH1GAL4

DNA-BDPT7

pUCori

TT7 & ADH1

= c-Myc epitope tag

MCS

f1ori

Δ

Δ

Figure18.MapofpGBKT7-53DNA-BDControlVector.pGBKT7-53isapositivecontrolplasmidthatencodesafusionofthemurinep53protein(a.a.72–390)andtheGAL4DNA-BD(a.a.1–147).Themurinep53cDNA(GenBankAccessionCat.No.K01700)wasclonedintopGBKT7attheEcoRIandBamHIsites.Thep53insertwasderivedfromtheplasmiddescribedinIwabuchiet al.(1993);plasmidmodificationwasperformedatClontech.pGBKT7-53hasnotbeensequenced.



AppendixD:Two-HybridControlVectorInformationcontinued

Nde I Nco I Sfi I EcoR ISma IXma IBamH ISal IPst I

Human lamin C

(a.a. 66)

(a.a. 230)

pGBKT�-Lam�.9 kb

Kanr

2 ori

TRP1

PADH1GAL4

DNA-BDPT7

pUCori

TT7 & ADH1

= c-Myc epitope tag

MCS

f1ori

Δ

Δ

Figure 19. Map of pGBKT7-Lam DNA-BD ControlVector. pGBKT7-Lam is a negative control plasmid that encodes afusionofthehumanlaminCprotein(a.a.66–230)andtheGAL4DNA-BD(a.a.1–147).ThelaminCcDNAinsert(GenBankAccessionCat.No.M13451)wasderivedfromtheplasmidreferencedinBartelet al.(1993a).PlasmidmodificationwasperformedatClontech.YeastcotransformedwithpGBKT7-LamandpGADT7-RecT,provideameasureofthebackgroundthatisduetofalse-positivetwo-hybridinteractions.pGBKT7-Lamhasnotbeensequenced.



Notes

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