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Table of Contents

Package Contents and Storage Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 ListoftheDupLEX-AcDNALibraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Vectorinformationandcloningstrategy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4TheB42domainandincludedelementsinpJG4-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Compatability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5cDNALibraryConstruction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6QualityControl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 ProtocolforAmplfyingDupLEX-AcDNALibraries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Modifiedalkalinelysesmethod. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Citations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

DupLEX-A Libraries

AppLicAtion GuiDE

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pAckAGE contEnts AnD storAGE conDitions

List of DupLEX-ATM cDNA Libraries

DupLEX-ATMcDNALibraries Insert Cat.# DNA(ug)E.coli (mL) GlycerolMDBKCell(bovinekidney) cDNA DLBK100 100 1C.elegans(adult) cDNA DLCE100 100 1D.melanogaster(adult) cDNA DLDM100 100 1HumanLiver cDNA DLH100 100 1HumanFetalBrain cDNA DLH101 100 1WI-38CellLine(lungfibroblastcellline) cDNA DLH102 100 1HeLaCellLine cDNA DLH103 100 1HumanPBL(peripheralbloodleukocytes) cDNA DLH104 100 1HumanFetalLiver cDNA DLH105 100 1HumanFetalKidney cDNA DLH106 100 1HumanProstate,Normal(pooled/8adults)cDNA DLH107 100 1HumanProstate,Tumor(pooled/8adults) cDNA DLH108 100 1LNCaPCell(untreated) cDNA DLH109 100 1LNCaPCell(treated) cDNA DLH110 100 1SKOV3(ovariancancercellline) cDNA DLH111 100 1MCF7Cell(estrogendepleted) cDNA DLH112 100 1MCF7Cell(estrogentreated) cDNA DLH113 100 1HumanAdultOvary cDNA DLH114 100 1JurkatT-cellLine cDNA DLH115 100 1SKBR3Cell(estrogenreceptornegative) cDNA DLH116 100 1MCF7Cell(serumgrown) cDNA DLH117 100 1MG63CellLine(osteosarcomacellline) cDNA DLH118 100 1MouseBrain cDNA DLM100 100 1MouseSpleen cDNA DLM101 100 1MouseLiver cDNA DLM102 100 1MouseOvary cDNA DLM103 100 1MouseProstate,Normal cDNA DLM104 100 1MouseBreast,Normal(lactating) cDNA DLM105 100 1MouseBreast,Normal(involuting) cDNA DLM106 100 1MouseBreast,Normal(Virgin) cDNA DLM107 100 1MouseBreast,Normal(pregnant,12-day) cDNA DLM108 100 1MouseEmbryo(whole,19-day) cDNA DLM110 100 1MouseSkeletalMuscle cDNA DLM111 100 1RatThymus cDNA DLR100 100 1RatTestis cDNA DLR101 100 1RatBrain cDNA DLR102 100 1RatAdipocyte(9weekoldZuckerrat) cDNA DLR103 100 1S.cerevisiae genomicDNADLY100 100 1

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Each DupLEX-ATM cDNA Library package contains:

1tubeofplasmidDNA(100ug/100ul)inTEbuffer(10mMTrisHCI,pH8.0,1mMEDTA)1tubeoftransformedbacterialcells(1ml)in15%Glycerol

Theabovecomponentsareshippedwithdryiceandshouldbekeptat-80oCforstor-age.Ifproperlystored,theywillmaintainactivityforseveralyears.

NOTE:FORRESEARCHPURPOSESONLY.NOTFORDIAGNOSTICORTHERAPEUTICUSE.

Introduction

Yeasttwo-hybridsystemsweredesignedforidentifyingandvalidatingprotein-proteininteractions.Sometranscriptionalfactors,suchasGal4,havetwospecificdomains:aDNAbindingdomainandatranscriptionalactivationdomain,whichareflankedbyalessspecificregion.

Figure 1: Schematic Diagram of the Yeast Two-Hybrid System

DNABinding Activation

DNABinding Activation

DNABinding Activation

Active

Inactive

ActiveX-Fusion Y-Fusion

SingleProtein(fromoneplasmid)

Twopartialproteins(fromtwoplasmids)

Twofusionproteins(fromtwoplasmids)

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FieldsandSongconstructedtwoplasmids,onecontainingtheGal4DNA-bindingdomainandtheothertheGal4activationdomain.YeastcellsexpressingthetwotruncatedproteinslackGal4activation.Subsequentlytheymadetwomoreplasmids.OneplasmidexpressesafusionproteincontainingtheGal4DNAbindingdomainandaproteinX,andtheotherexpressesafusionproteincontainingtheGal4activationdomainandaproteinY.ProteinXandProteinYareknowntointeractwitheachother.YeastcellsexpressingbothfusionsnowgeneratetheGal4activity.Presum-ablytheproteincomplexcontainingthetwofusionproteinslinkedtogetherthroughtheX-YinteractionrestoredtheGal4function.Thisfindingestablishedthebasefortestingwhethertwoproteinsinteractusingthesystem.Todoso,onejustneedstofuseoneproteintotheDNAbindingdomainandtheothertotheactivationdomain,andexaminewhetherthecellswiththetwoconstructscanactivateareportergene:inmostcase,beta-galactosidase.Thissystemhasbeensuccessfullyusednotonlyfortestingprotein-proteininteractioninlaboratories,butithasbeenalsoproventobeefficientinidentifyingnovelproteinsthatinteractwithaknownprotein,byscreeningacDNAlibraryconstructedusingthebindingdomain-containingvector.

Vector information and cloning strategy

OriGene’syeasttwo-hybridlibrariesaredesignedforresearcherstoidentifypro-teinsinteractingwiththeirproteinsofinterest.ThelibrariesaremadefrommRNAsisolatedfromabroadrangeoftissuesinthenormalordiseasedstagetoincreasethechancesofasuccessfulscreen.ThelibrariesareallconstructedinthepJG4-5

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vector(accession#:U89961).pJG4-5isastandardtarget(library)plasmidusedforinducibleexpressionofB42-HAtag-targetfusionproteininyeast.ItcontainstheyeastselectablemarkerTRPI.The2umoriginofreplicationproducesahighcopynumberofplasmidsinyeast.TheB42-HAsequenceencodesapeptidecontainingthreemajorelements.AstretchofpositiveaminoacidsattheN-terminusservesasthenuclearlocalizationsignalfortargetingthefusionproteintothenucleus.TheB42domain,whencomplexedwiththeDNAbindingdomain,activatestranscrip-tionofareportergene.TheHAtagisusedforconvenientdetectionofthefusionprotein.EcoRIandXhoIsitesnearthec-terminusofB42-HAareusedforsubclon-ingatargetgene.TheentirecodingsequenceisdirectedundertheGAL1promoterandterminatedbytheADHterminator.Gal1promoterisastrongpromoterinthepresenceofgalactosebutshowsweakornoactivityinthepresenceofglucose.Thischaracterallowstheexpressionofsometoxicproteins.Thereisaterminationcodoninallthreereadingframestoinsureapropertranslationaltermination.ThepUCoriginofreplicationistomaintaintheplasmidinE.coli,andtheampicillinresistancegeneisforsubcloningselectioninE.coli.

TheB42domainandincludedelementsinpJG4-5:ATGGGTGCTCCTCCAAAAAAGAAGAGAAAGGTAGCTGGTATCAATAAAGATATC54MGAPPKKKRKVAGINKDI18 NLSGAGGAGTGCAATGCCATCATTGAGCAGTTTATCGACTACCTGCGCACCGGACAG108EECNAIIEQFIDYLRTGQ36

GAGATGCCGATGGAAATGGCGGATCAGGCGATTAACGTGGTGCCGGGCATGACG162EMPMEMADQAINVVPGMT54

CCGAAAACCATTCTTCACGCCGGGCCGCCGATCCAGCCTGACTGGCTGAAATCG216PKTILHAGPPIQPDWLKS72

AATGGTTTTCATGAAATTGAAGCGGATGTTAACGATACCAGCCTCTTGCTGAGT270NGFHEIEADVNDTSLLLS90

GGAGATGCCTCCTACCCTTATGATGTGCCAGATTATGCCTCTCCCGAATTCGGC324GDASYPYDVPDYASPEFG108 HAtag EcoRICGACTCGAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAA---RLEXhoI

NLS:NuclearlocalizationsignalB42fusionmoietymolecularweightsize:

Noinsert:163aminoacids(18,398Dalton)Withinsert:107aminoacids(11,835Dalton)+insert

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Compatibility

Therearetwomajoryeasttwo-hybridsystemsfrequentlyusedinlaboratories.OneistheyeastGal4-based,andtheotherisE.coliLexAprotein-based.Ithasbeendemonstratedthattheactivationdomainsofthetwoproteinsareinterchangeableintermsofactivatingtranscriptionofareportergene.Thereforetheyeasttwo-hybridlibrariesconstructedinpJG4-5canbeusedinbothGal4andLexAsystemsaslongastheyeasthostcanaccommodatetheTrpselectablemarkerinpJG4-5.

cDNA library construction

ForeachcDNAlibraryconstruct,thestartingmaterialsarepolyA+RNA.FirststrandcDNAsweresynthesizedusingareversetranscriptaseandoligodT-XhoIlinkedprimer.AftersynthesisofthesecondstrandcDNAs,aEcoRIlinkerwereligatedtothe5’primeoftheDNAs.ThecDNAweredigestedwithEcoRIandXhoI,puri-fiedandthenligatedtotheEcoRIandXhoIsitesofthepJG4-5vector.ThemixedconstructlibraryistransformedinE.coliforlibrarypreparationandplasmidDNApurification.

5’EcoRIlinkersequence:AATTCGGCACGAGGCG-3’GCCGTGCTCCGC-5’

Quality Control

EachDupLEX-ATMcDNALibraryhasbeentestedinastringentqualitycontrolpro-cess.FirstacDNAlibrarymusthaveanadequatenumberofindependentclones.OriGene’scDNAlibrarieshave3.5x106to107independentclones.Second,thepercentageofthevectorswithaninsertmustbeabove95%,andlargesizeinsertsmustbepresentedinthecDNAlibraries.The100ugDNAincludedinthepackageistransformation-readyandissufficientforoneroundofscreening.TheE.coliglyc-erolstockhasbeenamplifiedonceandcanbeusedforalargescaleDNAplasmidpreparation.

MethodsProtocol for Amplifying DupLEX-A cDNA Libraries

Titerfrozenbacteriabydoingserialdilutionplating;theprovidedbacterialcellstockshouldhave~10,000,000pfu/ulglycerolmedium.Whenhandlingfrozenbacteria,gougeachunkofcellswithasterilespatulaortoothpickandkeeptherestfrozen.PlatebacteriaonLBplatescontainingampicillinat100ug/ml,atadensityofaboutahalf-millioncoloniesper150mmpetridish.Atotaloffivemillioncolonies(onten150mmpetridishes)areusuallyenoughtorepresentthewholelibrary.Growbacteriaforabout10hoursat37oCoruntilthecoloniesare1mmindiam-eter.Lowerincubationtemperatures(e.g.32-35oC)arealsosuggested.Note:DONOTLETTHECOLONIESOVERGROW.

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FloodeachplatewithLBmedium(~5ml/150mmplate).Usingasterilecellscraper,rubcoloniesofffromtheplatesandmakeabacterialsuspension.PoolthesuspensionsfromdifferentplatesandpurifyDNAfromthepoolusingacommerciallyavailableplasmidDNApurificationkit,orthemethoddescribedbelow.

Modified alkaline lyses method(CurrentProtocolsinMolecularBiology,p1.6.7):

Asobservedbyresearcherswhoworkonyeasttransformation,inmanycases,some‘crude’plasmidDNApreparationshavemuchhighertransformationefficienciesthancommercial-kitpurifiedplasmidDNAs.ThefollowingDNApreparationmethodgenerallyyieldsDNAswithsatisfactorytransformationefficiencies:

Solutions:

Sol I: Glucose /Tris/EDTA50mMglucose25mMTris.CI,pH8.010mMEDTAAutoclaveandstoreatRT

Sol II: NaOH/SDS solution0.2NNaOH1%(w/vol)sodiumofdodecylsulfate

Sol III: 5 M potassium acetate, pH 4.829.5mlglacialaceticacidKOHpelletstopH4.8H

2Oto100ml

Phenol/chloroform(1:1vol/vol)95%Ethanol70%Ethanol

NOTE: No RNase A should be usedCollectcellsbycentrifuging10minat4,000xgat4oC.Discardthesupernatantandre-suspendthecellsin4mlofSolI.Add8mlofSolIIandmixitbyinvertingthetubeseveraltimes.Add6mlofSolIIIandmixitbygentlevortexing.Putthetubeonicefor10minCentrifugethetubeat12,000xgfor30min.Transferthesupernatanttoafreshpolyethylenetube.Addequalvolumeofcoldphenol/chloroformandmix.Centrifugethetubeat12,000xgfor10min.Transfertheupperlayertoanewcentrifugetube.Add2Xvolumeof100%ethanol(roomtemperature)andmix.

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Centrifugethetubeat12,000xgfor30minDiscardthesupernatantwithoutdisturbingthepellet.Add20mlof70%coldethanoltothepellet.Centrifugethetubeat12,000xgfor15min.Discardthesupernatantanddrythetubebyinvertingitonapieceofpapertowel.Re-suspendthepelletin1mlofTEbuffer(pH8.0).EstimatetheDNAconcentrationbyrunningasampleonanagarosegel(aspectrophotometercannotbeusedformeasuringthequantitybecauseofthepresenceofalargeamountofRNAinthesample).AliquottheDNAsampletoseveraltubesandstoreat–20oC.Useasmallamountforyeasttransformationtotesttheyeasttransformationefficiency.CalculatetheamountofplasmidDNAsneededforalibrary-scalescreening,orfollowrecommendationsfromyouryeasttwo-hybridsystemusermanual.

ReferencesAnovelgeneticsystemtodetectprotein–proteininteractions.StanleyFieldsandOk-kyuSong(1989).Nature340,245-247.Cdi1,ahumanG1andSphaseproteinphosphatasethatassociateswithCdk2.,JenoGyuris,EricaGolemis,HelenChertkovandRogerBrent(1993).Cell75,791-803.

CitationsTumor-suppressiveMaspinRegulatesCellResponsetoOxidativeStressbyDirectInteractionwithGlutathioneS-Transferase.,ShupingYin,XiaohuaLi,YonghongMeng,RussellL.Finley,Jr.,WaelSakr,HengYang,NeelimaReddy,andShijieSheng,J.Biol.Chem.,Oct2005;280:34985-34996.Pias1InteractionandSumoylationofMetabotropicGlutamateReceptor8,ZhongshuTang,OussamaElFar,HeinrichBetz,andAstridScheschonka.J.Biol.Chem.,Nov2005;280:38153-38159.InteractionofMoloneyMurineLeukemiaVirusCapsidwithUbc9andPIASyMedi-atesSUMO-1AdditionRequiredEarlyinInfection.,AndrewYueh,JulianaLeung,SubarnaBhattacharyya,LucyA.Perrone,KeniadelosSantos,Szy-yuanPu,andStephenP.Goff,J.Virol.,Jan2006;80:342-352.Group13HOXproteinsinteractwiththeMH2domainofR-SmadsandmodulateSmadtranscriptionalactivationfunctionsindependentofHOXDNA-bindingcapability,ThomasM.Williams,MelissaE.Williams,JoanneH.Heaton,ThomasD.Gelehrter,andJeffreyW.Innis,NucleicAcidsRes.,Aug2005;33:4475–4484Ric-8B,anOlfactoryPutativeGTPExchangeFactor,AmplifiesSignalTransductionthroughtheOlfactory-SpecificG-ProteinGolf.,LuizEduardoC.VonDannecker,AdrianaF.Mercadante,andBettinaMalnic,J.Neurosci.,Apr2005;25:3793-3800.

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