duplex-a libraries · libraries constructed in pjg4-5 can be used in both gal4 and lexa systems as...
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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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