yeast protocols handbook

Yeast Protocols Handbook

FOR RESEARCH USE ONLY

PT3024-1 (PR13103)Published 14 March 2001


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3024-1 2 Version No. PR13103

I. Introduction 4

II. IntroductiontoYeastPromoters 5

III. CulturingandHandlingYeast 10

IV. PreparationofYeastProteinExtracts 12 A. GeneralInformation 12 B. PreparationofYeastCulturesforProteinExtraction 12 C. PreparationofProteinExtracts:Urea/SDSMethod 13 D. PreparationofProteinExtracts:TCAMethod 15 E. Troubleshooting 17

V. YeastTransformationProcedures 18 A. GeneralInformation 18 B. ReagentsandMaterialsRequired 19 C. TipsforaSuccessfulTransformation 20 D. IntegratingPlasmidsintotheYeastGenome 20 E. Small-scaleLiAcYeastTransformationProcedure 20 F. TroubleshootingYeastTransformation 22

VI. α-andβ-GalactosidaseAssays 23 A. GeneralInformation 23 B. In vivoPlateAssayUsingX-galintheMedium 25 C. Colony-liftFilterAssay 25 D. LiquidCultureAssayUsingONPGasSubstrate 26 E. LiquidCultureAssayUsingCPRGasSubstrate 27 F. LiquidCultureAssayUsingaChemiluminescentSubstrate 28 G. α-GalQuantitativeAssay 32

VII.WorkingwithYeastPlasmids 34 A. GeneralInformation 34 B. PlasmidIsolationFromYeast 34 C. TransformingE. coliwithYeastPlasmids 36

VIII. AnalysisofYeastPlasmidInsertsbyPCR 39 A. GeneralInformation 39 B. TipsforSuccessfulPCRofYeastPlasmidTemplates 39

IX. AdditionalUsefulProtocols 42 A. YeastColonyHybridization 42 B. GeneratingYeastPlasmidSegregants 43 C. YeastMating 44

X. References 46

XI. MatchmakerandRelatedProducts 49

APPENDICES A. GlossaryofTechnicalTerms 50 B. YeastGeneticMarkersUsedintheMatchmakerSystems 52 C. MediaRecipes 53 A. YeastMedia 53 B. E. coliMedia 56 D. SolutionFormulations 57 E. PlasmidInformation 61 F. YeastHostStrainInformation 64

TableofContents


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ListofTablesTableI. YeastPromoterConstructsUsedtoRegulateReporterGeneExpression inMatchmakerPlasmidsandHostStrains 6

TableII. YeastPromoterConstructsintheMatchmakerCloningVectors 9

TableIII. Comparisonofβ-galactosidaseAssays 25

TableIV. SelectedYeastGenesandTheirAssociatedPhenotypes 52

TableV. MatchmakerReporterGenesandTheirPhenotypes 52

TableVI. MatchmakerTwo-HybridSystemCloningVectors 61

TableVII. MatchmakerTwo-HybridSystemReporterandControlPlasmids 62

TableVIII. MatchmakerOne-HybridSystemCloning,Reporter&ControlPlasmids 63

TableIX. YeastReporterStrainsintheMatchmakerOne-andTwo-HybridSystems 64

ListofFiguresFigure1. SequenceofGAL4DNA-BDrecognitionsitesintheGAL1,GAL2, MEL1 6

UASsandtheUASG17-mer

Figure2. Urea/SDSproteinextractionmethod 14

Figure3. TCAproteinextractionmethod 16

TableofContentscontinued

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I. Introduction

The Yeast Protocols Handbook provides background information and general yeast protocols thatcomplementoursystem-specificUserManuals.TheprotocolsinthisHandbookhavebeenoptimizedwithouryeast-basedMatchmaker™Two-HybridandOne-HybridSystems,andMatchmakerLibraries.TheYeastProtocolsHandbookisespeciallyusefulforresearcherswhowishtouseyeastasavehiclefortheirmolecularbiologyexperiments,buthavelittleornopriorexperienceworkingwithyeast.Fornoviceandexperiencedusersalike,theYeastProtocolsHandbookwillhelpyouobtainthebestpossibleresultswithyourMatchmakerandotheryeast-relatedproductsfromClontech.

ThisHandbookincludes: • detailedinformationonculturingandhandlingyeast

• informationontheyeastpromotersusedintheMatchmakerSystems

• twoprotocolsforpreparingproteinextractsfromyeast

• quantitativeandqualitativeβ-galactosidaseassays(forusewithlacZ yeastreporterstrains)

• asimple,optimizedprotocolforisolatingplasmidsfromyeast

• PCRamplificationandyeastcolonyhybridizationprotocolsfortherapidanalysisofpositiveclonesobtainedinalibraryscreening

• asmall-scale,lithiumacetateyeasttransformationprotocol

• additionalprotocolsforworkingwithcertainyeastplasmidsandhoststrains

Thespecialapplicationofyeasttransformationforone-andtwo-hybridlibraryscreeningiscoveredindetailineachproduct-specificUserManual.ThespecialapplicationofyeastmatingforlibraryscreeningiscoveredinthePretransformedMatchmakerLibrariesUserManual.

Aboutouryeast-basedproducts

TheMatchmakerGAL4Two-HybridSystems(CatNo.K1604-1,K1605-1,630303)andLexATwo-HybridSystem(CatNo.K1609-1)arecompletekitsforidentifyingandinvestigatingprotein-proteininteractionsin vivo usingtheyeasttwo-hybridassay.TheMatchmakerOne-HybridSystem(CatNo.K1603-1)providesthebasictoolsforidentifyingnovelproteinsin vivothatbindtoatargetDNAsequencesuchasacis-actingregulatoryelement.MatchmakerTwo-HybridSystemsarecompatiblewithourpBridgeThree-HybridVector(CatNo.630404)fortheinvestigationoftertiaryproteincomplexes.TheMatchmakerLibrariesareconstructedinvectorsthatexpressinsertsasfusionstoatranscriptionalactivationdomain,andarethusaconvenientresourceforresearcherswishingtoscreenalibraryusingtheone-ortwo-hybridassays.PretransformedMatchmakerLibrariesprovideanevengreaterlevelofconvenienceforthosewishingtoperformatwo-hybridlibraryscreeningwithoutusinglarge-orlibrary-scaleyeasttransformations.

Clontech offers an extensive line of kits and reagents that support and complement theMatchmaker Systems and Libraries.TheYeastmakerTMYeastTransformation Kit (Cat No. 630439)includesallthenecessaryreagentsandprotocolsforefficienttransformationusingthelithiumacetatemethod.AlsoavailablefromClontech:aselectionofGAL4DNA-bindingdomain(DNA-BD)andactivationdomain(AD)hybridcloningvectors;thepGildaVectorforusewithLexA-basedtwo-hybridsystems;monoclonalantibodiesandsequencingprimers;andyeastmedia,includingMinimalSDBaseandmanydifferentformulationsofDropout(DO)Supplement.Finally,thepHA-CMVandpMyc-CMVVectorSet(CatNo.631604)canbeusedtoconfirmproteininteractionsinmammaliancells.

Fororderinginformationontheseproducts,pleaseseeChapterXIofthisHandbookortheClontechCatalog.


Protocol No. PT3024-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR13103 �

II. IntroductiontoYeastPromoters

Yeastpromotersandothercis-actingregulatoryelementsplayacrucialroleinyeast-basedexpressionsystemsandtranscriptionalassayssuchastheMatchmakerOne-andTwo-HybridSystems.DifferencesinthepromoterregionofreportergeneconstructscansignificantlyaffecttheirabilitytorespondtotheDNA-binding domain of specific transcriptional activators; promoter constructs also affect the levelofbackground(or leakiness)ofgeneexpressionand the levelof inducedexpression.Furthermore,differencesincloningvectorpromotersdeterminethelevelofproteinexpressionand,insomecases,confertheabilitytoberegulatedbyanutrient(suchasgalactoseinthecaseoftheGAL1 promoter).

Thischapterprovidesabriefintroductiontoseveralcommonlyusedyeastpromotersandcis-regulatoryelements.Forfurther informationontheregulationofgeneexpressioninyeast,werecommendtheGuide to Yeast Genetics and Molecular Biology byGuthrie&Fink(1991;No.V2010-1);Molecular Biology and Genetic Engineering of Yeasts,editedbyHeslot&Gaillardin(1992);Stargell&Struhl(1996);andPringleet al.(1997;No.V2365-1).

UASandTATAregionsarebasicbuildingblocksofyeastpromoters

Theinitiationofgenetranscriptioninyeast,asinotherorganisms,isachievedbyseveralmolecularmechanismsworkinginconcert.Allyeaststructuralgenes(i.e.,thosetranscribedbyRNApolymeraseII)areprecededbyaregioncontainingalooselyconservedsequence(TATAbox)thatdeterminesthetranscriptionstartsiteandisalsoaprimarydeterminantofthebasaltranscriptionlevel.Manygenesarealsoassociatedwithcis-actingelements—DNAsequencestowhichtranscriptionfactorsandothertrans-actingregulatoryproteinsbindandaffecttranscriptionlevels.Theterm“promoter”usuallyreferstoboththeTATAboxandtheassociatedcis-regulatoryelements.Thisusageisespeciallycommonwhenspeakingofyeastgeneregulationbecausethecis regulatoryelementsarerelativelycloselyassociatedwiththeTATAbox(Yoccum,1987).Thisisincontrasttomulticellulareukaryotes,wherecis-regulatoryelements(suchasenhancers)canbefoundveryfarupstreamordownstreamfromthepromoterstheyregulate. In this text, “minimalpromoter”will referspecifically to theTATAregion,exclusiveofothercis-actingelements.

Theminimalpromoter(orTATAbox)inyeastistypicallyapproximately25bpupstreamofthetranscriptionstartsite.YeastTATAboxesarefunctionallysimilartoprokaryoticPribnowboxes,butarenotastightlyconserved.Furthermore,someyeasttranscriptionunitsareprecededbymorethanoneTATAbox.TheyeastHIS3gene,forexample,isprecededbytwodifferentTATAboxes:TR,whichisregulated,andTC,whichisconstitutive(Mahadevan&Struhl,1990).YeastTATAboxescanbemovedtoanewlocation,adjacenttoothercis-regulatoryelements,andstillretaintheirtranscriptionalfunction.

Onetypeofcis-actingtranscriptionelementinyeastisupstreamactivatingsequences(UAS),whicharerecognizedbyspecifictranscriptionalactivatorsandenhancetranscriptionfromadjacentdownstreamTATAregions.TheenhancingfunctionofyeastUASsisgenerallyindependentoforientation;however,itissensitivetodistanceeffectsifmovedmorethanafewhundredbasepairsfromtheTATAregion.TheremaybemultiplecopiesofaUASupstreamofayeastcodingregion.Inaddition,UASscanbeeliminatedorswitchedtochangetheregulationoftargetgenes.

UASandTATAregionscanbeswitchedtocreatenovelpromoters

The“mixandmatch”natureofyeastTATAboxesandUASshasbeenusedtogreatadvantageinyeasttwo-hybridsystemstocreatenovelpromotersforthereportergenes.(Forgeneralreferencesonyeasttwo-hybridsystems,seeChapterX.)Inmostcases,thelacZ, HIS3, ADE2 and LEU2 reportergenesareundercontrolofartificialpromoterconstructscomprisedofaTATAandUAS(oroperator)sequencederivedfromanothergene(TableI).Insomecases,theTATAsequenceandtheUASarederivedfromdifferentgenes;indeed,theLexAoperatorisacis-actingregulatoryelementderivedfromE. coli.

ForGAL4-basedsystems,eitheranativeGAL UASorasyntheticUASG17-merconsensussequence(Heslot&Gaillardin,1992)providesthebindingsitefortheGAL4DNA-BD.ForLexA-basedsystems,multiplecopiesof theLexAoperatorprovidethebindingsitefor theLexAprotein. Ifyouareputtingtogetheryourownone-ortwo-hybridsystem,youmustmakesurethatthereportergene’spromoterwillberecognizedbytheDNA-BDmoietyencodedinyourDNA-BDfusionvector.


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table i. yeast promoter constructs used to regulate reporter gene expression in matchmaker plasmids and host strains

Plasmidor Reporter Originof UAS Originof Expressionlevelb

hoststraina gene UAS regulatedby TATAsequence Induced(uninduced)

CG-1945 lacZ UASG17-mer(x3)c GAL4 CYC1 low

HIS3 GAL1 GAL4 GAL1 h i g h ( s l i g h t l yleaky)

HF7c lacZ UASG17-mer(x3)c GAL4 CYC1 low

HIS3 GAL1 GAL4 GAL1 high(tight)

Y190 lacZ GAL1 GAL4 GAL1 high HIS3 GAL1 GAL4 HIS3 (TC+TR) high(leaky)

Y187 lacZ d GAL1 GAL4 GAL1 high

SFY526 lacZ GAL1 GAL4 GAL1 high

PJ69-2A HIS3 GAL1 GAL4 GAL1 high(tight) ADE2 GAL2 GAL4 GAL2 high(tight)

AH109 HIS3 GAL1 GAL4 GAL1 high(tight) ADE2 GAL2 GAL4 GAL2 high(tight) lacZ MEL1 GAL4 MEL1 low

EGY48 LEU2 LexAop(x6) LexA LEU2 high

p8op-lacZ lacZ LexAop(x8) LexA GAL1e high

pHISi HIS3 (none)f (n.a.) HIS3 (TC+TR) n.a.f(leaky)

pHISi-1 HIS3 (none)f (n.a.) HIS3 (TC+TR) n.a.f(leaky)

pLacZi lacZ (none)f (n.a.) CYC1 n.a.f(tight)

aSeeAppendicesE&Fforreferences.b Wheninducedbyapositivetwo-hybridinteraction;“leaky”and“tight”refertoexpressionlevelsintheabsenceofinduction.c Conserved17-bppalindromicsequencetowhichtheGAL4proteinbinds(Guthrie&Fink,1991).d Y187probablycontainstwocopiesofthelacZ gene,judgingbythestrengthofthesignalinthisstrainandinthestrainsfrom

whichitwasderived(Durfeeet al.,1993;Harperet al.,1993).e ThisistheminimalTATAregionoftheGAL1 promoter;itdoesnotincludetheGAL1UASandthereforeisnotresponsiveto

regulationbyGAL4protein.f TheMatchmakerOne-HybridSystemvectorsdonotcontainaUASbecausetheyareusedtoexperimentallytesttargetelements

insertedupstreamoftheminimalpromoterfortheirabilitytobindspecifictranscriptionalactivators.Intheabsenceofinsertedtargetelements,reportergeneexpressionisnotinduced;however,expressionlevelsmaybeleaky,dependingonthenatureoftheminimalpromoterusedinthatvector.

II. IntroductiontoYeastPromoterscontinued

Figure 1. Sequence of the GAL4 DNA-BD recognition sites in the GAL1, GAL2, and MEL1 UASs and the UASG 17-merconsensussequence(Giniger&Ptashne,1988).

GAL1 UAS

GAL1-bs1 TAGAAGCCGCCGAGCGGGAL1-bs2 GACAGCCCTCCGAAGGAGAL1-bs3 GACTCTCCTCCGTGCGT CGGCCATATGTCTTCCGGAL1-bs4 CGCACTGCTCCGAACAA

GAL2-bs1 CGGAAAGCTTCCTTCCGGAL2-bs2 CGGCGGTCTTTCGTCCGGAL2-bs3 CGGAGATATCTGCGCCG CGGAAGACTCTCCTCCG GAL2-bs4 CGGGGCGGATCACTCCGGAL2-bs5 CGGATCACTCCGAACCG

GAL2 UAS UAS G17-mer

MEL1 UAS




ReportergenesunderthecontrolofGAL4-responsiveelements

Inyeast,thegenesrequiredforgalactosemetabolismarecontrolledbytworegulatoryproteins,GAL4andGAL80,aswellasbythecarbonsourceinthemedium(Guthrie&Fink,1991;Heslot&Gaillardin,1992).Whengalactoseispresent,theGAL4proteinbindstoGAL4-responsiveelementswithintheUASupstreamofseveralgenesinvolvedingalactosemetabolismandactivatestranscription.Intheabsenceofgalactose,GAL80bindstoGAL4andblockstranscriptionalactivation.Furthermore,inthepresenceofglucose,transcriptionofthegalactosegenesisimmediatelyrepressed(Johnstonet al.,1994).

TheUASsofthe20knowngalactose-responsivegenesallcontainoneormoreconservedpalindromicsequences towhich theGAL4proteinbinds(Guthrie&Fink,1991;Ginigeret al. 1985; reviewed inHeslot&Gaillardin,1992).The17-merconsensussequence,referredtohereasUASG17-mer,functionsinanadditivefashion,i.e.,multiplesitesleadtohighertranscriptionlevelsthanasinglesite(Giniger&Ptashne,1988).TheproteinbindingsitesoftheGAL1,GAL2, MEL1 UASs,andtheUASG17-merconsensussequence,areshowninFigure1.

ThetightregulationoftheGAL UASsbyGAL4makesitavaluabletoolformanipulatingexpressionofreportergenesintwo-hybridsystemsthataredependentontheGAL4DNA-BD.However,insuchsystems,theyeasthoststrainsmustcarrydeletionsofthegal4 andgal80 genestoavoidinterferencebyendogenousGAL4andGAL80proteins;thus,nosignificantglucoserepressionisobservedinthesestrainsandnoinductionisobservedunlessatwo-hybridinteractionisoccurring.Therefore,nutritionalregulationofGALUASsisnotafeatureofGAL4-basedtwo-hybridsystems.However,thehoststrainused in theLexAsystemdoessupportgalactose induction,as it iswild type forGAL4andGAL80functions.

IntheGAL4-basedMatchmakerTwo-HybridSystems,eitheranintactGAL1, GAL2 or MEL1 UASoranartificallyconstructedUASconsistingofthreecopiesofthe17-merconsensusbindingsequence,isusedtoconferregulatedexpressiononthereportergenes(TableI).TheHIS3 reporterofAH109,PJ69-2A,HF7c,andCG-1945,andthelacZ reporterofY190,Y187,andSFY526arealltightlyregulatedby the intactGAL1 promoter (including theGAL1UASandGAL1 minimalpromoter). InHF7candCG1945,lacZ expressionisundercontrolofUASG17-mer(x3)andtheextremelyweakminimalpromoterof theyeast cytochromeC1 (CYC1)gene. lacZ under thecontrolof the intactGAL1 promotercanbe expressed at ~10X the level obtained with the UASG 17-mer (x3)/CYC1 minimal promoter constructundersimilar inductionconditons(ClontechLaboratories;unpublisheddata).Therefore,someweakortransienttwo-hybridinteractionsmaynotbedetectableinHF7corCG1945unlessyouuseahighlysensitive β-galactosidase assay (such a liquid culture assay using a chemiluminescent substrate;Chapter VI.F). The ADE2 reporter of PJ69-2A andAH109 is tightly regulated by the intact GAL2promoter,whose inductionpropertiesaresimilar to thoseof theGAL1 promoter. InAH109, lacZ isunder thecontrolof theMEL1UASandminimalpromoter.TheMEL1promoter isstonger than theUASG17-mer(x3)/CYC1 minimalpromoter,butweakerthantheGAL1promoter(Ahoet al.,1997).

ReportergenesunderthecontrolofaminimalHIS3promoter

ThenativeyeastHIS3 promotercontainsaUASsiterecognizedbythetranscriptionalactivatorGCN4,and twoTATAboxes.GCN4 regulatesoneof theTATAboxes (TR),while theotherTATAbox (TC)driveslow-levelconstitutiveexpressionofHIS3 (Iyer&Struhl,1995).TCisnotregulatedbythenativeGCN4-bindingUAS,theGAL1UAS,orartificialUASGconstructs(Mahadevan&Struhl,1990;Hope&Struhl,1986).

TheHIS3 reportergene inyeaststrainY190 isunusualamong theGAL4 two-hybrid reportergeneconstructsinthatitisunderthecontroloftheGAL1 UASandaminimalpromotercontainingbothHIS3 TATAboxes(Flick&Johnston,1990).Theresultishigh-levelexpression(duetotheGAL1UAS)wheninducedbyapositivetwo-hybridinteraction;thisconstructalsoexhibitsasignificantlevelofconstitutiveleakyexpression(duetotheHIS3TC).Incontrast,inHF7c,CG-1945,PJ69-2A,andAH109theentireHIS3promoter(includingbothTATAboxes)wasreplacedbytheentireGAL1promoter,leadingtotightregulationoftheHIS3 reporterinthosestrains(Feilotteret al.,1994).


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TheHIS3 reporterplasmidspHISiandpHISi-1usedintheMatchmakerOneHybridSystemalsohavebothoftheHIS3 TATAboxespresentintheminimalpromoter.Byinsertingacis-actingelementintheMCS,theregulatedTATAbox(TR)canbeaffected,butthereisstillasignificantamountofconstitutive,leakyexpressionduetotheHIS3TC.TheleakyHIS3expressionoftheseone-hybridplasmidsisfirstusedtohelpconstructHIS3 reporterstrains,andlateriscontrolledbyincluding3-aminotriazoleinthemediumtosuppressbackgroundgrowth.

ReportergenesunderthecontrolofLexAoperators

InLexA-based two-hybridsystems, theDNA-BD isprovidedby theentireprokaryoticLexAprotein,whichnormallyfunctionsasarepressorofSOS genesinE. coliwhenitbindstoLexAoperators,whichareanintegralpartofthepromoter(Ebinaet al.,1983).Whenusedintheyeasttwo-hybridsystem,theLexAproteindoesnotactasarepressorbecausetheLexAoperatorsareintegratedupstream oftheminimalpromoterandcodingregionofthereportergenes.LEU2 reporterexpressioninyeaststrainEGY48isunderthecontrolofsixcopiesoftheLexAoperator(op)sequenceandtheminimalLEU2promoter.InthelacZ reporterplasmids,lacZ expressionisundercontrolof1–8copiesoftheLexAop(Estojaket al.,1995)andtheminimalGAL1 promoter.BecausealloftheGAL1 UASsequenceshavebeenremovedfromthe lacZ reporterplasmids(Westet al.,1984),thispromoterisnotregulatedbyglucoseorgalactose.

Promotersusedtodrivefusionproteinexpressionintwo-hybridcloningvectors

The ADH1 promoter (or a truncated version of it) is the promoter used to drive expression of thefusionproteins inmostof theMatchmakercloningvectors.The1500-bp full-lengthADH1 promoter(Ammerer,1983;Vainio,GenBankaccessionnumber:Z25479)leadstohigh-levelexpressionofsequencesunderitscontrolinpGADT7,pGADGH,pLexA,andpAS2-1duringlogarithmicgrowthoftheyeasthostcells.Transcriptionisrepressedinlatelogphasebytheethanolthataccumulatesinthemediumasaby-productofyeastmetabolism.

Several Matchmaker cloning vectors contain a truncated 410-bp ADH1 promoter (Table II).At onepoint, itwasbelievedthatonlythisportionwasnecessaryforhigh-levelexpression(Beier&Young,1982).Inmostvectorconstructs,however,thistruncatedpromoterleadstoloworverylowlevelsoffusionproteinexpression(Ruohonenet al.,1991;Ruohonenet al.,1995;Tornow&Santangelo,1990).ThisobservationhasbeenconfirmedatClontechbyquantitativeWesternblots (unpublisheddata).Thehigh-levelexpressionreportedbyBeier&Young(1982)wasapparentlyduetoasegmentofDNAderivedfrompBR322,whichwaslaterfoundtocoincidentallyenhancetranscriptionalactivityinyeast(Tornow&Santangelo,1990).IntheMatchmakervectorpACT2,strongconstitutivefusionproteinexpressionisdrivenbythe410-bptruncatedADH1 promoteradjacenttothisenhancingpBR322segment.

TheDNA-BDcloningvectorpGBKT7used inMatchmakerTwo-HybridSystem3containsa700-bpfragmentoftheADH1promoter.Thistrucatedpromoterleadstohigh-levelexpression,butnoethanolrepression(Ruohonenet al.,1991;Ruohonenet al.,1995).

TheADcloningvectorpB42ADandthealternativeDNA-BDvectorpGildausedintheMatchmakerLexATwo-HybridSystemutilizethefull-lengthGAL1 promotertodrivefusionproteinexpression.BecausetheLexAsystemhoststrainiswild-typeforGAL4andGAL80,fusionproteinexpressionisregulatedbyglucoseandgalactose.



table ii. yeast promoter constructs in the matchmaker cloning vectors

Regulation/ Signal RelativeProtein StrengthonVectorsa Promoter ExpressionLevel Westernblotb

pLexA,pGADGH, ADH1 (full-length) Ethanol-repressed/High +++pAS2-1,pAS2,pGADT7

pACT2,pACT ADH1 (410bp+)c Constitutive/medium ++

pGADGL ADH1(410bp) Constitutive/low +/–(weak)

pGAD424,pGAD10 Constitutive/verylow (notdetectable)pGBT9

pGBKT7 ADH1(700bp) Consitutive/high +++

pB42AD,pGilda GAL1 (full-length) Repressedbyglucose; (notdetectable)d induced(high-level)bygalactose +++d

p8op-lacZ GAL1(minimal) Notregulatedbyglucose (nodata) orgalactosea SeeAppendixEforvectorreferences.bUnpublisheddataobtainedatClontechLaboratoriesusing theappropriateGAL4domain-specificmAb(CatNo.630402or

CatNo. 630403).Solubleproteinextractswereprepared fromCG-1945 transformedwith the indicatedplasmid.Samplesequivalentto~1OD600unitofcellswereelectrophoresedandthenblottedtonitrocellulosefilters.TheblotswereprobedwitheitherGAL4DNA-BDmAb(0.5µg/ml)orGAL4ADmAb(0.4µg/ml)using1mlofdilutedmAbper10cm2ofblot,followedbyHRP-conjugatedpolyclonalGoatAnti-MouseIgG(JacksonImmunologicalResearch;diluted1:15,000inTBST).Signalsweredetectedusingachemiluminescentdetectionassayanda2.5-minexposureofx-rayfilm.SignalintensitieswerecomparedtothatofknownamountsofpurifiedGAL4DNA-BD(a.a.1–147)orGAL4AD(a.a.768–881).

c ThetruncatedADH1 promoterinpACT2isadjacenttoasectionofpBR322whichactsasatranscriptionalenhancerinyeast.dDataobtainedusingEGY48[p8op-lacZ]transformedwithpGildaandgrowninthepresenceofglucoseorgalactose,respectively

(April1997Clontechniques);nodataavailableforpB42AD.




III.CulturingandHandlingYeast

Foradditionalinformationonyeast,werecommendGuthrieandFink(1991)Guide to Yeast Genetics and Molecular Biology(CatNo.V2010-1).

A. YeastStrainMaintenance,RecoveryfromFrozenStocks,andRoutineCulturing 1.Long-termstorage • Yeaststrainscanbestored indefinitely inYPDmediumwith25%glycerolat–70°C.For

storage>1year,thetemperaturemustbemaintainedbelow–55°C. • TransformedyeaststrainsarebeststoredintheappropriateSDdropoutmediumtokeep

selectivepressureontheplasmid.(SeeAppendixC.AforrecipesandAppendixEforplasmidinformation.)

Topreparenewglycerolstockculturesofyeast: a. Useasterileinoculationlooptoscrapeanisolatedcolonyfromtheagarplate. b. Resuspendthecellsin200–500µlofYPDmedium(ortheappropriateSDmedium)ina

1.5-mlmicrocentrifugetube.Vortextubevigorouslytothoroughlydispersethecells.Addsterile50%glyceroltoafinalconcentrationof25%.

c. Tightlyclosethecap.Shakethevialbeforefreezingat–70°C. 2.Torecoverfrozenstrainsandprepareworkingstockplates: a. StreakasmallportionofthefrozenglycerolstockontoaYPD(orappropriateSD)agar

plate. b. Incubatetheplateat30°Cuntilyeastcoloniesreach~2mmindiameter(thistakes3–5

days).Usethesecoloniesasyourworkingstock. c. SealplateswithParafilmandstoreat4°Cforuptotwomonths.Streakafreshworking

stockplatefromthefrozenstockat1–2-monthintervals. d. Ifyoucannotrecoverthestrain,thecellsmayhavesettled;inthiscase,thawtheculture

onice,vortexvigorously,andrestreak.Theglycerolstocktubemayberefrozenafewtimeswithoutdamagingthecells.

3.Toprepareliquidovernightcultures: a. Use only fresh (<2-months old) colonies from the working stock plate. Use one large

(2–3-mmdiameter)colonyper5mlofmedium.Ifcoloniesaresmall,orifyouareinoculatingalargervolume,useseveralcolonies.Important:Vigorouslyvortexthemediumfor~1mintothoroughlydispersethecells.

Notes: • Liquidcultureswillgrowslowerthanexpectedifclumpsarepresentintheinoculum;cellsintheinteriorof

theclumpsdonothaveaccesstothenutrientsinthemedium. • Ifyouareinoculatingavolumegreaterthan1ml,itiseasiertodispersetheclumpsifthecoloniesarefirst

placedin1mlofmediuminamicrocentrifugetube,vortexed,andthentransferredtothedesiredvolume. • Whengrowingovernightculturesofyeasttransformants,usetheappropriateSDminimalmediumtokeep

selectivepressureonextrachromosomalplasmid(s). • Thegrowth inYPDofyeaststrainscarryingtheade2-101mutationwillbeenhancedbyaddingadenine

hemisulfate(0.003%finalconcentration)tothemedium(AppendixC.A).Allofthehoststrains(exceptEGY48)usedintheMatchmakerSystemscarrythisauxotrophicmutation.

• ThegrowthoftransformedPJ69-2AcellsinSD/–Trpmayalsobeenhancedbyaddingexcessadeninetothemedium(AppendixC.A).

b. Incubateat30°Cfor16–18hrwithshakingat230–270rpm.Withmoststrains,thiswillyieldastationaryphaseculture(OD600>1.5).

Note:Differentyeaststrainsgrowatdifferentrates.Growthratesmayalsobeaffectedbythepresenceoffusionproteinsincertaintransformants.Inaddition,thedoublingtimeofmoststrainsgrowinginSDminimalmediumistwiceaslongasinYPD.

c. Ifyouneedamid-logphaseculture,transferenoughoftheovernightcultureintofreshmedium to produce an OD600 = 0.2–0.3. Incubate at 30°C for 3–5 hr with shaking(230–250rpm).Thiswill,withmoststrains,produceaculturewithanOD600~0.4–0.6.

Note:Generally,YPDorYPDAmaybeusedinthisincubation.Becauseoftheshorterincubationtime,plasmidlosswillnotbesignificant.However,donotuseYPDifyouwanttoinduceproteinexpressionfromtheyeastGAL1 promoterofaLexAsystemplasmid,e.g.,pB42ADorpGilda;YPDcontainsglucose,whichrepressestranscriptionfromtheGAL1 promoter.



III. CulturingandHandlingYeastcontinued

B. GrowthSelectionforTransformationMarkersandReporterGeneExpression

Mostyeastcloningvectorsandcontrolplasmids (including thoseprovided inourMatchmakerSystems)carryatleastonenutritionalmarkertoallowforselectionofyeasttransformantsplatedon SD minimal medium lacking that specific nutrient. Furthermore, if you are cotransformingyeastwithtwoormoredifferentplasmidsbearingdifferentnutritionalmarkers,theplasmidscanbeindependentlyselected.Thus,theSDselectionmediumyouchooseforplatingtransformantsdependsgenerallyonthepurposeoftheselection.SpecificfactorstoconsiderinchoosingtheappropriateSDselectionmediumare:

• theplasmid(s)usedandwhetheryouareselectingforoneormoreplasmids • whetheryouareselectingforcoloniesinwhichtwohybridproteinsareinteracting • whether—andtowhatextent—thehoststrainisleakyforreportergeneexpression • whetheryouwanttoinduceproteinexpressionfromtheregulatedGAL1 promoter • whether you intend to perform in-vivo, agar-plate β-galactosidase assays (for lacZ

reporterexpressionintheLexATwo-HybridSystem). Please refer to your system-specific User Manual for further information on choosing the

appropriateSDselectionmediaforparticularplasmids,hoststrains,andapplications.



IV.PreparationofYeastProteinExtracts

A. GeneralInformation

Weprovidetwoalternativeprotocolsforthepreparationofproteinextractsfromyeast.Theresults(i.e.,proteinyieldandquality)willvarydependingontheproteinandmaybemoresuccessfulwithoneprotocolthanwiththeother.Becauseitisdifficulttopredictwhichprocedurewillgivebetterresults,weprovidetwoprotocolsforcomparison.Thecellculturepreparationmethod(SectionB)isthesameforbothproteinextractionprocedures.

Bothextractionproceduresaddressthetwomostchallengingaspectsofisolatingproteinsfromyeast:1)disruptingyeastcellwalls;and2)inhibitingthemanyendogenousyeastproteases.Yeastcellwallsaretoughandmustbedisruptedbyacombinationofphysicalandchemicalmeans;methodsthatutilizeglycolyticenzymesarenotrecommendedforthisapplicationbecausetheyare oftencontaminatedwithproteases.Endogenousproteasesmustbecounteractedwithacocktailofstrongproteaseinhibitors(recipeinAppendixD.A).Ifyouknowyourproteinofinterestissusceptibletoaproteasenotinhibitedbytherecommendedcocktail,addtheappropriateinhibitorbeforeusingthemixture.Youmayalsowishtoaddotherinhibitorssuchassodiumfluoridetopreventdephosphorylation,ifthatisappropriateforyourprotein.

B. PreparationofYeastCulturesforProteinExtraction

ReagentsandMaterialsRequired: • YPDandappropriateSDliquidmedium(RecipesinAppendixC.A) • 20-and50-mlculturetubes • Ice-coldH2O • Dryiceorliquidnitrogen

1.ForeachtransformedyeaststrainyouwishtoassayinaWesternblot,preparea5-mlovernightcultureinSDselectionmediumasdescribedinSectionIII.A,exceptuseasingleisolatedcolony(1–2mmindiameter,noolderthan4days).UsetheSDmediumappropriateforyoursystemandplasmids(AppendixE).Alsopreparea10-mlcultureofanuntransformedyeastcolonyinYPDor(ifpossible)appropriateSDmediumasanegativecontrol.

2.Vortex theovernight cultures for0.5–1min todispersecell clumps.Foreachclone tobeassayed(andthenegativecontrol),separatelyinoculate50-mlaliquotsofYPDmediumwiththeentireovernightculture.

3.Incubateat30°Cwithshaking(220–250rpm)untiltheOD600reaches0.4–0.6.(Dependingonthefusionprotein,thiswilltake4–8hr.)MultiplytheOD600(ofa1-mlsample)bytheculturevolume(i.e.,55ml)toobtainthetotalnumberofOD600units;thisnumberwillbeusedinSectionsC&D.(Forexample,0.6x55ml=33totalOD600units.)

Note: During late log phase the ADH1 promoter shuts down and the level of endogenous yeast proteasesincreases.

4.Quicklychilltheculturebypouringitintoaprechilled100-mlcentrifugetubehalfwayfilledwithice.

5.Immediatelyplacetubeinaprechilledrotorandcentrifugeat1000xgfor5minat4°C. 6.Pouroffsupernatantandresuspendthecellpelletin50mlofice-coldH2O.(Anyunmeltedice

poursoffwiththesupernatant.) 7.Recoverthepelletbycentrifugationat1,000xgfor5minat4°C. 8.Immediatelyfreezethecellpelletbyplacingthetubeondryiceorinliquidnitrogen.Storecells

at–70°Cuntilyouarereadytoproceedwiththeexperiment.



IV. PreparationofYeastProteinExtractscontinued

C. PreparationofProteinExtracts:Urea/SDSMethod(Figure2;Printen&Sprague,1994)

ReagentsandMaterialsRequired: • 1.5-mlscrew-capmicrocentrifugetubes • Glassbeads(425–600µm;SigmaCatNo.G-8772) • Proteaseinhibitorsolution(AppendixD.A) • PMSFstocksolution(AppendixD.A) • Crackingbufferstocksolution(AppendixD.A) • Crackingbuffer,complete(AppendixD.A)

Note:Unlessotherwisestated,keepproteinsamplesonice. 1.Preparecompletecrackingbuffer(AppendixD.A)andprewarmitto60°C.BecausethePMSF

degradesquickly,prepareonlytheamountofcrackingbufferyouwillneedimmediately.Use100µlofcrackingbufferper7.5OD600unitsofcells.(Forexample,for33totalOD600unitsofcells,use0.44mlofcrackingbuffer.)

2.Quickly thawcellpelletsbyseparatelyresuspendingeachone in theprewarmedcrackingbuffer.

• Ifcellpelletsarenotimmediatelythawedbytheprewarmedcrackingbuffer,placethetubesbrieflyat60°Ctohastenmelting.Toavoidriskofproteolysis,donotleavethemlongerthan2minat60°C.

• BecausetheinitialexcessPMSFinthecrackingbufferdegradesquickly,addanadditionalaliquotof the100XPMSFstocksolutiontothesamplesafter15minandapproximatelyevery7minthereafteruntilStep9,whentheyareplacedondryiceoraresafelystoredat–70°Corcolder.(Use1µlof100XPMSFper100µlofcrackingbuffer.)

3.Transfereachcellsuspensiontoa1.5-mlscrew-capmicrocentrifugetubecontaining80µlofglassbeadsper7.5OD600unitsofcells.

Note:Thevolumeoftheglassbeadscanbemeasuredusingagraduated1.5-mlmicrocentrifugetube.

4.Heatsamplesat70°Cfor10min. Note:Thisinitial incubationat70°Cfreesmembrane-associatedproteins.Thus, ifyouskipthisstep,membrane-

associatedproteinswillberemovedfromthesampleatStep6(high-speedcentrifugation).

5.Vortexvigorouslyfor1min. 6.Pelletdebrisandunbrokencellsinamicrocentrifugeat14,000rpmfor5min,preferablyat

4°C,otherwiseatroomtemperature(20–22°C). 7.Transfer the supernatants to fresh 1.5-ml screw-cap tubes and place on ice (first

supernatants). 8.Treatthepelletsasfollows: a. Placetubesina100°C(boiling)waterbathfor3–5min. b. Vortexvigorouslyfor1min. c. Pelletdebrisandunbrokencellsinamicrocentrifugeat14,000rpmfor5min,preferably

at4°C,otherwiseatroomtemperature. d. Combineeachsupernatant(secondsupernatant)withthecorrespondingfirstsupernatant

(fromStep7). Note:Ifnosupernatantisobtained,addmorecrackingbuffer(50–100µl)andrepeatSteps8.b&c.

9.Boilthesamplesbriefly.Immediatelyloadthemonagel.Alternatively,samplesmaybestoredondryiceorina–70°Cfreezeruntilyouarereadytorunthemonagel.




Figure2.Urea/SDSproteinextractionmethod.

Cell pellets

First supernatant

Secondsupernatant

Combined supernatants

Pellet

Pellet(discard)

• Thaw and resuspend cell pellets in prewarmed Cracking buffer• Add cells to glass beads• Heat at 70°C for 10 min• Vortex vigorously for 1 min• Centrifuge at 14,000 rpm for 5 min

• Boil for 3–5 min• Vortex vigorously for 1 min • Centrifuge at 14,000 rpm for for 5 min

• Place on ice

• Combine with second supernatant• Place on ice

• Immediately load gel or freeze at –70°C or colder


Protocol No. PT3024-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR13103 1�


D. PreparationofProteinExtracts:TCAMethod(Figure3;Horecka,J.,personalcommunication)

ReagentsandMaterialsRequired: • 1.5-mlscrew-capmicrocentrifugetubes • Glassbeads(425–600µm;SigmaCatNo.G-8772) • Proteaseinhibitorsolution(AppendixD.A) • PMSFStocksolution(AppendixD.A;Add as necessary throughout the protocol.) • [Recommended]BeadBeater(BioSpec,Bartlesville,OK)

Note:IfyoudonothaveaccesstoaBeadBeater,ahigh-speedvortexercanbeusedinstead.However,vortexingisnotaseffectiveasbead-beatingatdisruptingthecells.

• TCAbuffer(AppendixD.A) • Ice-cold20%w/vTCAinH2O(seeSambrooket al.[1989]fortipsonpreparingTCAsolutions) • TCA-Laemmliloadingbuffer(AppendixD.A)

Note:Unlessotherwisestated,keepproteinsamplesonice. 1.Thawcellpelletsonice(10–20min). 2.Resuspendeachcellpelletin100µlofice-coldTCAbufferper7.5OD600unitsofcells.(For

example,for33totalOD600unitsofcells,use0.44mlofTCAbuffer.)Placetubesonice. 3.Transfereachcellsuspensiontoa1.5-mlscrew-capmicrocentrifugetubecontainingglass

beadsandice-cold20%TCA.Use100µlofglassbeadsand100µlofice-cold20%TCAper7.5OD600unitsofcells.

Note:Thevolumeoftheglassbeadscanbemeasuredusingagraduated1.5-mlmicrocentrifugetube.

4.Todisruptcells,placetubesinaBead-Beaterandsetspeedathighestsetting.Bead-beatthecellsfor2X30sec,placingtubesonicefor30secinbetweenthetwobead-beatings.Placetubesonice.

Note:IfyoudonothaveaccesstoaBead-Beater,youcanvortexthetubesvigorouslyat4°Cfor10min;alternatively,youcanvortexatroomtemperatureforshorterperiods(of1mineach)at least4times,placingtubesonicefor30secinbetweeneachvortexing.Placetubesonice.

5.Transferthesupernatantabovethesettledglassbeadstofresh1.5-mlscrew-captubesandplacetubesonice.Thisisthefirstcellextract.

Note:Theglassbeadssettlequickly,sothereisnoneedtocentrifugetubesatthispoint.

6.Washtheglassbeadsasfollows: a. Add500µlofanice-cold,1:1mixtureof20%TCAandTCAbuffer. b. PlacetubesinBeatBeaterandbeatforanother30secatthehighestsetting.(Alternatively,

vortexfor5minat4°C,orvortex2X1minatroomtemperature,placingthetubeonicefor30secinbetweenthetwovortexings.)

c. Transfertheliquidabovetheglassbeads(secondcellextract)tothecorrespondingfirstcellextractfromStep5.

7.Allowanycarryoverglassbeadstosettleinthecombinedcellextracts~1min,thentransfertheliquidabovetheglassbeadstoafresh,prechilled1.5-mlscrew-captube.

8.Pellettheproteinsinamicrocentrifugeat14,000rpmfor10minat4°C. 9.Carefullyremovesupernatantanddiscard. 10.Quicklyspintubestobringdownremainingliquid.Removeanddiscardliquidusingapipette

tip. 11.ResuspendeachpelletinTCA-Laemmliloadingbuffer.Use10µlofloadingbufferperOD600

unitofcells. Note:Iftoomuchacidremainsinthesample,thebromophenolblueinthebufferwillturnyellow.Generally,thiswill

notaffecttheresultsoftheelectrophoresis.

12.Placetubesina100°C(boiling)waterbathfor10min. 13.Centrifugesamplesat14,000rpmfor10minatroomtemperature(20–22°C). 14.Transfersupernatanttofresh1.5-mlscrew-captube. 15.Loadthesamplesimmediatelyonagel.Alternatively,samplesmaybestoredondryiceorin

a–70°Cfreezeruntilyouarereadytorunthemonagel.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3024-1 1� Version No. PR13103


Figure3.TCAproteinextractionmethod.

Cell pellets

First Cell Extract(liquid above beads)

Second Cell Extract(liquid above beads)

Combined Cell Extracts(liquid above beads)

Supernatant (Discard)

Supernatant (Protein extract)

Beads and unbroken cells

Beadsand unbroken cells

(discard)

Beads and unbroken cells

(discard)

Pellet(Protein and contaminants)

Pellet (discard)

• Thaw and resuspend cell pellets in cold TCA buffer• Add cells to glass beads and ice-cold, 20% TCA• Bead-beat cells 2 x 30 sec (or vortex vigorously for 10 min at 4°C)

• Place on ice

Immediately load gel or freeze at –70°C or colder

• Add ice-cold 20% TCA• Bead-beat cells 1 x 30 sec (or vortex for 5 min at 4°C)

• Combine Cell Extracts• Allow glass beads to settle ~1 min

• Resuspend in TCA-Laemmli loading buffer • Boil 10 min• Centrifuge at 14,000 rpm for 10 min

• Centrifuge at 14,000 rpm for 10 min



E. Troubleshooting

Optimalelectrophoreticseparationofproteinsdepends largelyonthequalityof theequipmentand reagentsused in thegel system, themanner inwhich theprotein samplesarepreparedpriortoelectrophoresis,theamountofproteinloadedonthegel,andthevoltageconditionsusedduringelectrophoresis.Thesesameconsiderationsareimportantforthesubsequenttransferofproteinstothenitrocellulosemembranewheretransferbuffercomposition,temperature,durationoftransfer,andtheassemblyoftheblottingapparatuscanallhaveprofoundeffectsonthequalityoftheresultantproteinblot.Thefollowingtroubleshootingtipspertaintotheisolationofproteinfromyeast.InformationonrunningpolyacrylamideproteingelsandperformingWesternblotsisavailableinpublishedlaboratorymanuals(e.g.,Sambrooket al.,1989,orAusubelet al.,1987–96).

1.Fewornoimmunostainedproteinbandsontheblot • ThetransferofproteinbandstotheblotmaybeconfirmedbystainingtheblotwithPonceau

S. • Thepresenceofproteinbandsinthegel(beforetransfer)maybeconfirmedbystaininga

parallellaneofthegelwithCoomassieblue.(NotethatonceagelhasbeenstainedwithCoomassieblue,theproteinbandswillnottransfertoablot.)

• Theextentofcellwalldisruptioncanbedeterminedbyexaminingasampleoftreatedcellsunderthemicroscope.Incompletecelllysiswilllowertheproteinyield.

2.Severalbandsappearontheblotwhereasingleproteinspeciesisexpected • Proteindegradationand/orproteolysismayhaveoccurredduringsamplepreparation.

Additional protease inhibitorsmaybeusedasdesired.Also,makesure that inStepsC.8.aandD.12(boilingtheproteinextracts),thesamplesareplacedintoawaterbaththatisalreadyboiling.Ifsamplesareplacedinthewaterbeforeithasreachedboilingtemperature,amajoryeastprotease(ProteinaseB)willbeactivated.(ProteinaseBisaserineproteaseofthesubtilisinfamily.)

• Dephosphorylationofanormallyphosphoryatedfusionproteinmayhaveoccurredduringsamplepreparation.Sodiumfluoride(NaF)maybeaddedtotheproteaseinhibitorstocksolutiontohelppreventdephosphorylation(Sadowskiet al.,1991).

3.Ifyouarerunningareducinggel,makesurethattheproteinsamplehasbeencompletelyreducedwitheitherdithiothreitolor2-mercaptoethanolpriortoloadingthegel.




V. YeastTransformationProcedures


LiAc-mediatedyeasttransformation

ThereareseveralmethodscommonlyusedtointroduceDNAintoyeast,includingthespheroplastmethod,electroporation,andthelithiumacetate(LiAc)-mediatedmethod(reviewedinGuthrie&Fink,1991).AtClontech,wehavefoundtheLiAcmethod(Itoet al.,1983),asmodifiedbySchiestl&Gietz(1989),Hillet al.(1991),andGietzet al.(1992),tobesimpleandhighlyreproducible.ThischapterprovidesdetailedprotocolsforusingtheLiAcprocedureinastandardplasmidtransformationandinamodifiedtransformationtointegratelinearDNAintotheyeastgenome.

IntheLiActransformationmethod,yeastcompetentcellsarepreparedandsuspendedinaLiAcsolutionwiththeplasmidDNAtobetransformed,alongwithexcesscarrierDNA.Polyethyleneglycol(PEG)withtheappropriateamountofLiAcisthenaddedandthemixtureofDNAandyeastisincubatedat30°C.Aftertheincubations,DMSOisaddedandthecellsareheatshocked,whichallowstheDNAtoenterthecells.Thecellsarethenplatedontheappropriatemediumtoselectfortransformantscontainingtheintroducedplasmid(s).Because,inyeast,thisselectionisusuallynutritional,anappropriatesyntheticdropout(SD)mediumisused.

Simultaneousvs.sequentialtransformations

TheLiAcmethodforpreparingyeastcompetentcellstypicallyresultsintransformationefficienciesof105perµgofDNAwhenusingasingle typeofplasmid.When theyeast is simultaneouslycotransformedwithtwoplasmidshavingdifferentselectionmarkers,theefficiencyisusuallyanorderofmagnitudelowerduetothelowerprobabilitythataparticularyeastcellwilltakeupbothplasmids.(Yeast,unlikebacteria,cansupportthepropagationofmorethanoneplasmidhavingthesame replicationorigin, i.e., there isnoplasmid incompatibility issue in yeast.)Thus, inacotransformationexperiment,theefficiencyoftransformingeachtypeofplasmidshouldremainat~105perµgofDNA,asdeterminedbythenumberofcoloniesgrowingonSDmediumthatselectsfor onlyoneof theplasmids.Thecotransformationefficiency isdeterminedby thenumberofcoloniesgrowingonSDmediumthatselectsforbothplasmidsandshouldbe~104cfu/µgDNA.

Simultaneous cotransformation is generally preferred because it is simpler than sequentialtransformation—andbecauseoftheriskthatexpressionofproteinsencodedbythefirstplasmidmaybetoxictothecells.Iftheexpressedproteinistoxic,clonesarisingfromspontaneousdeletionsinthefirstplasmidwillhaveagrowthadvantageandwillaccumulateattheexpenseofclonescontainingintactplasmids.However,ifthereisnoselectivedisadvantagetocellsexpressingthefirstclonedprotein,sequentialtransformationmaybepreferredbecauseitusessignificantlylessplasmidDNAthansimultaneouscotransformation.Insomecases,suchaswhenoneofthetwoplasmidsisthesameforseveraldifferentcotransformations,sequentialtransformationsmaybemoreconvenient.

Scalingupordown

Thesmall-scaleyeasttransformationproceduredescribedherecanbeusedforupto15paralleltransformations,anduses0.1µgofeachtypeofplasmid.Dependingontheapplication,thebasicyeasttransformationmethodcanbescaledupwithoutadecreaseintransformationefficiency.Ifyouplantoperformatwo-hybridlibraryscreening,youwillneedalargeorlibrary-scaletransformationprocedure,whichwillrequiresignificantlymoreplasmidDNA.PleaserefertoyourMatchmakersystem-specificUserManualforfurtherinformationonlibraryscreeningstrategiesandspecificprotocols.

Integrationvs.nonintegrationofyeastplasmids

FormostyeasttransformationsperformedwhileusingtheMatchmakerSystems,itisnotnecessaryordesirabletohavetheplasmidintegrateintotheyeastgenome.(Infact,yeastplasmidsdonotefficientlyintegrateiftheycarryayeastoriginofreplicationandareuseduncut.)However,therearetwoexceptionstothisgeneralrule,asexplainedintherespectivesystem-specificUserManuals:(a)IntheMatchmakerOne-HybridSystem,theresearchermustconstructtheirowncustomreporterplasmidandthenintegrateitintotheyeasthoststrainbeforeperformingtheone-hybridassay.(b)IntheMatchmakerLexATwo-HybridSystem,thep8op-lacZreporterplasmidcanbeusedeitherasanautonomouslyreplicatingplasmidorasanintegratedplasmid,dependingonthedesiredlevel



ofreportergeneexpression.TheprimaryreasonforintegratingaplasmidinsomeMatchmakerapplicationsistogenerateastableyeastreporterstraininwhichonlyonecopyofthereportergeneispresentpercell,andtherebycontrolthelevelofbackgroundexpression.Ifyouhaveanapplicationthatrequiresintegrationofaplasmidintotheyeastgenome,pleaseseeSectionV.D.

Transformationcontrols

When setting up any type of transformation experiment, be sure to include proper controlsfor transformationefficiencies. In thecaseofsimultaneouscotransformation, it is important todeterminethetransformationefficienciesofbothplasmids together,aswellasofeach type ofplasmid independently.Thatway, if thecotransformationefficiency is low,youmaybeable todeterminewhetheroneoftheplasmidtypeswasresponsible(seeTroubleshootingGuide,SectionF).Therefore,besuretoplateanaliquotofthetransformationmixtureontheappropriateSDmediathatwillselectforonlyonetypeofplasmid.ExamplecalculationsareshowninSectionV.E.Whenscreeningalibraryorperformingaone-ortwo-hybridassay,youwillneedadditionalcontrols,asexplainedyoursystem-specificUserManual.

B. ReagentsandMaterialsRequiredNote:TheYeastmakerYeastTransformationSystem(CatNo.630439)containsallthesolutions(exceptmedia,H2O,andDMSO)requiredforyeasttransformation.YeastmakerreagentshavebeenoptimizedforuseintheMatchmakerOne-andTwo-HybridSystems.

• YPDortheappropriateSDliquidmedium • Sterile 1XTE/1X LiAc (Prepare immediately prior to use from 10X stocks; stock recipes in

AppendixD.B) • Sterile1.5-mlmicrocentrifugetubesforthetransformation • AppropriateSDagarplates(100-mmdiameter)

Notes: • Preparetheselectionmediaandpourtherequirednumberofagarplatesinadvance.(Seeyoursystem-specificUser

ManualorAppendixEformediarecommendations.)Besuretoplanforenoughplatesforthecontroltransformationsandplatings.

• AllowSDagarplatestodry(unsleeved)atroomtemperaturefor2–3daysorat30°Cfor3hrpriortoplatinganytransformationmixtures.Excessmoistureontheagarsurfacecanleadtoinaccurateresultsduetounevenspreadingofcellsorlocalizedvariationsinadditiveconcentrations.

• AppropriateplasmidDNAinsolution(checkamountsrequired)• Appropriateyeastreporterstrainformakingcompetentcells(checkvolumeofcompetentcellsrequired;

Steps1–11ofSectionV.Ewillgiveyou1.5ml,enoughfor14–15small-scaletransformations) • HerringtestescarrierDNA(AppendixD.B) • SterilePEG/LiAcsolution(Prepareonlythevolumeneeded,immediatelypriortouse,from 10Xstocks;AppendixD.B) • 100%DMSO(Dimethylsulfoxide;SigmaCatNo.D-8779) • Sterile1XTEbuffer(Preparefrom10XTEbuffer;AppendixD.B) • Sterileglassrod,bentPasteurpipette,or5-mmglassbeadsforspreadingcellsonplates.

V. YeastTransformationProcedurescontinued




C. TipsforaSuccessfulTransformation • Fresh(one-tothree-week-old)colonieswillgivebestresultsforliquidcultureinoculation.Asingle

colonymaybeusedfortheinoculumifitis2–3mmindiameter.Scrapetheentirecolonyintothemedium.Ifcoloniesonthestockplatearesmallerthan2mm,scrapeseveralcoloniesintothemedium.SeeChapterIII.Aforfurtherinformationonstartingliquidculturesfromcoloniesandfromaliquidcultureinoculum.

• Vigorouslyvortexliquidculturestodispersetheclumpsbeforeusingtheminthenextstep. • Thehealthandgrowthphaseofthecellsatthetimetheyareharvestedformakingcompetent

cellsiscriticalforthesuccessofthetransformation.Theexpansionculture(StepE.6)shouldbeinlog-phasegrowth(i.e.,OD600between0.4and0.6)atthetimethecellsareharvested.Iftheyarenot,seetheTroubleshootingguide(SectionV.F).

• Whencollectingcellsbycentrifugation,aswingingbucketrotorresultsinbetterrecoveryofthecellpellet.

• For thehighest transformationefficiency (as isnecessary for libraryscreening),usecompetentcellswithin1hroftheirpreparation.Ifnecessary,competentcellscanbestored(afterStepE.11)atroomtemperatureforseveralhourswithaminorreductionincompetency.

• Toobtainanevengrowthofcoloniesontheplates,continuetospreadthetransformationmixturesovertheagarsurfaceuntilallliquidhasbeenabsorbed.Alternatively,use5-mmsterileglassbeads(5–7beadsper100-mmplate)topromoteevenspreadingofthecells.

D. IntegratingPlasmidsintotheYeastGenome

Important: Please read Section V.A for guidelines on when it is appropriate to use thisprocedure.

Topromoteintegrationofyeastplasmids,followthesmall-scaleLiActransformationprocedure(SectionV.Ebelow)withthefollowingexceptions:

• Beforetransformation,linearize1–4µgofthereportervectorbydigestingitwithanappropriaterestrictionenzymeinatotalvolumeof40µlat37°Cfor2hr.Electrophoresea2-µlsampleofthedigestona1%agarosegeltoconfirmthattheplasmidhasbeenefficientlylinearized.Notes:• Ifthevectorcontainsayeastoriginofreplication(i.e.,2µori),itwillbenecessarytoremoveitbeforeyouattempt

tointegratethevector.• Thevectorshouldbe linearizedwithin thegeneencoding the transformation (i.e.,nutritionalselection)marker.

However, if thedigestionsite iswithinaregionthat isdeletedinthehoststrain, theplasmidwillnotbeabletointegrate.Pleaserefertoyourproduct-specificUserManualforrecommendedlinearizationsites.

• AtStep12,add1–4µgofthelinearizedreporterplasmid+100µgofcarrierDNA;foreachreporterplasmid,alsosetupacontroltransformationwithundigestedplasmid(+100µgcarrierDNA).

• AtStep20,resuspendcellsin150µlofTEbuffer. • PlatetheentiretransformationmixtureononeplateoftheappropriateSDmediumtoselectfor

colonieswithanintegratedreportergene.

E. Small-scaleLiAcYeastTransformationProcedure 1.Inoculate1mlofYPDorSDwithseveralcolonies,2–3mmindiameter. Note:Forhoststrainspreviouslytransformedwithanotherautonomouslyreplicatingplasmid,usetheappropriateSD

selectionmediumtomaintaintheplasmid(AppendixE).

2.Vortexvigorouslyfor5mintodisperseanyclumps. 3.Transferthisintoaflaskcontaining50mlofYPDortheappropriateSDmedium. 4.Incubateat30°Cfor16–18hrwithshakingat250rpmtostationaryphase(OD600>1.5). 5.Transfer30mlofovernightculturetoaflaskcontaining300mlofYPD.ChecktheOD600of

thedilutedcultureand,ifnecessary,addmoreoftheovernightculturetobringtheOD600upto0.2–0.3.

6.Incubateat30°Cfor3hrwithshaking(230rpm).Atthispoint,theOD600shouldbe0.4–0.6. Note:IftheOD600is<0.4,somethingiswrongwiththeculture(seeTroubleshootingSectionF.6).

7.Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature(20–21°C).

8.DiscardthesupernatantsandthoroughlyresuspendthecellpelletsinsterileTEordistilled




H2O.Poolthecellsintoonetube(finalvolume25–50ml). 9.Centrifugeat1,000xgfor5minatroomtemperature. 10.Decantthesupernatant. 11.Resuspendthecellpelletin1.5mloffreshlyprepared,sterile1XTE/1XLiAc. 12.Add0.1µgofplasmidDNAand0.1mgofherringtestescarrierDNAtoafresh1.5-mltube

andmix. Notes: • Forsimultaneouscotransformation(usingtwodifferentplasmids),use0.1µgofeachplasmid(anapproximately

equalmolarratio),inadditiontothe0.1mgofcarrierDNA. • Fortransformationstointegrateareportervector,useatleast1µgoflinearizedplasmidDNAinadditiontothe

carrierDNA.

13.Add0.1mlofyeastcompetentcellstoeachtubeandmixwellbyvortexing. 14.Add0.6mlofsterilePEG/LiAcsolutiontoeachtubeandvortexathighspeedfor10sectomix. 15.Incubateat30°Cfor30minwithshakingat200rpm. 16.Add70µlofDMSO.Mixwellbygentleinversion.Donotvortex. 17.Heatshockfor15minina42°Cwaterbath. 18.Chillcellsonicefor1–2min. 19.Centrifugecellsfor5secat14,000rpmatroomtemperature.Removethesupernatant. 20.Resuspendcellsin0.5mlofsterile1XTEbuffer. 21.Plate100µloneachSDagarplatethatwillselectforthedesiredtransformants.Toensurethatyou

willobtainaplatewithwell-separatedcolonies,alsospread100µlofa1:1000,1:100,and1:10dilutionon100-mmSDagarplates.Thesewillalsoprovidecontrolsfor(co)transformationefficiency.

Note:Ifyouareperformingacotransformation,platecontrolstochecktransformationefficiencyandmarkersofeachplasmid.Onseparate100-mmplates,spread1µl(dilutedin100µlH2O)onmediumthatwillselectforasingletypeofplasmid.

22.Incubateplates,up-side-down,at30°Cuntilcoloniesappear(generally,2–4days). 23.Tocalculatethecotransformationefficiency,countthecolonies(cfu)growingonthedilution

platefromStep22abovethathas30–300cfu. cfuxtotalsuspensionvol.(µl) =cfu/µgDNA Vol.plated(µl)xdilutionfactorxamt.DNAused(µg)* *Inacotransformation,thisistheamountofoneoftheplasmidtypes,notthesumofthem.Ifyouhaveusedunequal

amountsoftwoplasmids,usetheamountofthelesserofthetwo.

Samplecalculation: •100coloniesgrewonthe1:100dilutionplate(dilutionfactor=0.01) • platingvolume:100µl • resuspensionvolume=0.5ml • amountoflimitingplasmid=0.1µg 100cfux0.5mlx103µl/ml =5x105cfu/µgDNA 100µlx0.01x0.1µg

24.Pickthelargestcoloniesandrestreakthemonthesameselectionmediumformasterplates.SealplateswithParafilmandstoreat4°Cfor3–4weeks.

F. TroubleshootingYeastTransformation

Theoveralltransformationefficiencyshouldbeatleast104cfu/µgfortransformationwithasingle



typeofplasmid,and103cfu/µgforsimultaneouscotransformationwithtwotypesofplasmids.Ifyourcotransformationefficiencyislowerthanexpected,calculatethetransformationefficiencyofthesingleplasmidsfromthenumberoftransformantsgrowingontheappropriatecontrolplates.If the two typesofplasmidsseparatelygave transformationefficiencies>105cfu/µg,switch tosequentialtransformation.

Ifthetransformationefficiencyforoneorbothoftheseparateplasmidsis<105cfu/µg,severalcausesarepossible.

1.Suboptimalplasmidpreparation • Repeat the transformationusingmore (up to 0.5µg) of theplasmidDNA that had the low

transformationefficiency. • CheckthepurityoftheDNAand,ifnecessary,repurifyitbyethanolprecipitationbeforeusing

itagain.

2.SuboptimalcarrierDNA • Ifyouarenotalreadydoingso,useYeastmakerCarrierDNA,which isavailableseparately

(CatNo.630440)oraspartoftheYeastmakerYeastTransformationSystem(CatNo.630439),andhasbeenoptimizedforhightransformationefficienciesinthissystem.

• Iftransformationefficienciesaredeclininginsuccessiveexperiments,thecarrierDNAmayberenaturing.ReboilthecarrierDNAfor20min,andthenchillitquicklyinanice-waterbath.

3.Suboptimalyeastcompetentcells

• Makesurethattheexpansionculture(StepE.6)wasinlog-phasegrowthatthetimethecellswereharvestedformakingcompetentcells. If theovernightculture(StepE.4)orexpansionculture(StepE.6)grewslowerthanexpected(ornotatall),startoveratStepE.1bypreparingafreshovernightculture.Failuretothoroughlydispersethecolonyusedfortheinoculumwillresultinslowgrowth;seeSectionIII.A.3.Ifyoustillhaveproblemsobtainingahealthyliquidculture,streakafreshworkingstockplate(fromthefrozenglycerolstock)andinoculatewithafreshcolony.

• Checktheliquidmediumtomakesureitwasmadecorrectly.Ifyoususpectthatthemediumorcarbonsourcestocksolutionshavebeenover-autoclaved,remakefreshsolutionsandeitherfiltersterilizethemoradjusttheautoclavesettingsappropriatelybeforeautoclaving.

• TheadditionofadeninehemisulfatetoYPD(inStepsE.3andE.5)willenhancethegrowthofyeaststrainsthatcontaintheade2-101mutation.AllofourMatchmakerhoststrains(exceptEGY48)carrythismutation.

• Check the concentration of the resuspended competent cells (after Step E.11) using ahemocytometer.Ifthecellconcentrationis<1x109/ml,spinthecellsdownagain(at1,000xgfor5min)andresuspendtheminasmallervolumeof1XTE/LiAcbuffer.

• Occasionally,thereisacontaminantinthewaterthatcanaffecttransformationefficiencyand/orcellgrowth.Prepareallreagentsusingsterile,deionized,distilledwatersuchasMilli-QTM-filtered.Confirmthatyourwaterpurificationsystemisfunctioningproperly.




VI. α-andβ-GalactosidaseAssays


Considerations • Toreducevariabilityinliquidassays,assayfiveseparatetransformantcolonies,andperform

eachassayintriplicate. • Itisimportantthatthecoloniestobeassayedforα-andβ-galactosidaseactivityaregrowing

on the appropriate SD minimal medium. SD (dropout) medium is used to keep selectivepressureonthehybridplasmidsand,inthecaseoftheMatchmakerLexATwo-HybridSystem,the lacZ reporterplasmidup to the time thecellsare lysed for theassay.The typeofSDmediumneededdependsontheplasmidsandhoststrainsused.Furthermore,whenworkingwithalacZ reporterunderthecontroloftheinducibleGAL1 promoter(suchasintheLexASystem),theSDmediummustcontaingalactose(notglucose)asthecarbonsource.Seethesystem-specificUserManualformediarecommendations.

β-GalactosidaseAssays • X-galmustbeusedastheβ-galactosidasesubstrateforsolid-supportassaysbecauseofitshigh

degreeofsensitivity.(X-galis~106-foldmoresensitivethanONPG.)AlthoughmoresensitivethanX-gal,Galacton-StarTMisnotrecommendedforagarplateandfilterassaysbecauseitgivestroublesomebackground.

• Thefilterandliquidβ-galactosidaseassaysdescribedhereuseatleastonefreeze/thawcycleinliquidnitrogentolysetheyeastcellwalls.Freeze-thawcyclesarearapidandeffectivecelllysismethodwhichpermitsaccuratequantificationofβ-galactosidaseactivity(Schneideret al.,1996).

• Thecolony-liftfilterassay(Breeden&Nasmyth,1985)usedtomeasureβ-galactosidaseactivityisprimarilyusedtoscreenlargenumbersofcotransformantsthatsurvivetheHIS3 growthselectioninaGAL4two-hybridorone-hybridlibraryscreeening.ItcanalsobeusedtoassayforaninteractionbetweentwoknownproteinsinaGAL4two-hybridsystem.

• Thein vivo, agarplateassayisprimarilyusedtoscreenlargenumbersofcotransformantsfortheexpressionofthelacZ reportergeneinaLexAtwo-hybridlibraryscreeningwhenthereportergeneismaintainedonanautonomouslyreplicatingplasmid.Thein vivo assayworksforLexAtransformantsbecauseofthelacZ reporterplasmid’shighcopynumberandbecauseofthepreamplificationstepthatnormallyprecedestheβ-galactosidaseassayinthissystem.(PleaserefertotheMatchmakerLexATwo-HybridUserManualformoreinformationonlibraryscreening.)Becauseofitsrelativelylowsensitivity,thein vivo,agarplateassayisnotsuitableforscreeningtransformantsinaGAL4-basedtwo-hybridassay,orinaLexA-basedtwo-hybridassaywhenthereportergenehasbeenintegratedintothehostgenome.

• Liquidculturesareassayedforβ-galactosidasetoverifyandquantifytwo-hybridinteractions.Becauseoftheirquantitativenature,liquidassayscanbeusedtocomparetherelativestrengthoftheprotein-proteininteractionsobservedinselectedtransformants.However,thereisnodirectcorrelationbetween β-galactosidaseactivityandtheKdofaninteraction(Estojaket al.,1995).Furthermore,quantitativedatacannotbecomparedbetweendifferenthoststrainshavingdifferentlacZ reporterconstructs.Infact,duetopromoterstrengthdifferences,itmaybepossibletoquantitatetherelativestrengthofinteractionsinsomeyeaststrains(e.g.,Y190,Y187),butnotinothers(e.g.,CG-1945orHF7c).(SeeChapterIIforadiscussionofthepromoters.)

• The liquid assays described here use one of three substrates: ONPG, CPRG, or achemiluminescentsubstrate(Galacton-Star).Thethreesubstratesdifferintheirrelativecost,sensitivity,andreproducibility.SeeTableIII.

α-GalactosidaseAssays • The α-Gal Quantitative Assay is a sensitive colorimetric method for the detection and

quantitationofyeast α-galactosidaseactivityresultingfromexpressionoftheMEL1reportergeneinourGAL4-basedMatchmakertwo-hybridsystems.

MEL1 isamemberof theGAL gene family,which,asagroup, facilitates theuptakeandutilizationofgalactosebythecell.UponbindingofGAL4totheMEL1upstreamactivatingsequence(MEL1 UAS),theMEL1geneproduct,α-galactosidase,isactivelyexpressedandsecretedtotheperiplasmicspaceandculturemediumwhereitcatalyzesthehydrolysisofmelibiosetogalactoseandglucose.Theα-GalQuantitativeAssayallowsyoutospecificallyidentifyandmeasurethiscatalyticactivityusingp-nitrophenyl-α-d-galactoside (PNP-α-Gal),acolorlesscompoundthatyieldsayellowproduct(p-nitrophenol)uponhydrolysis.

The α-Gal QuantitativeAssay can be used to measure the extracellular α-galactosidase



activityproducedduringthecultureofanyyeaststrainthatcarriestheMEL1 gene.MEL1 isendogenoustomanybutnotallyeaststrains.(Liljeström,1985;Post-Beittenmilleret al.,1984).TableIXprovidesalistoftheGAL4-basedMatchmakeryeaststrainsknowntocarrytheMEL1gene.ThislistincludesstrainAH109usedinClontech’sMatchmakerTwo-HybridSystem3(CatNo.630303)andincludedwithClontech’sGAL4-basedcDNAlibraries.

MEL1hasbeenshowntobeasensitivein vivoreporterforGAL4-basedtwo-hybridassays(Ahoet al.,1997).Thequantitativenatureoftheα-GalAssaymakesitpossibletocomparethedegreeofMEL1expressionindifferentMatchmakertwo-hybridhostcellpopulationscontainingdifferentpairsofinteractingproteins,ortomeasuredifferencesintherelativestrengthofbindingbetweenmutantformsofinteractingproteins.

Principleoftheα-GalQuantitativeAssay In the α-Gal Quantitative Assay, the catalytic activity of α-galactosidase is monitored

colorimetrically by measuring the rate of hydrolysis of the chromogenic substrate,p-nitrophenyl-α-d-galactoside(PNP-α-Gal).Oneoftheproductsofthisreaction,p-nitrophenol,displaysastrongabsorptionbandat410nm.

PNP-α-Gal+H2Oα-galactosidase p-nitrophenol+D-galactose

(λmax=410nm)

Because yeast naturally secrete α-galactosidase into the surrounding medium, it is moreconvenienttoassaythanβ-galactosidase,anintracellularenzymeencodedbythelacZreportergene.Theα-galactosidaseassayiscarriedoutbysimplycombiningasmallaliquotofcell-freeculturemediawithafixedvolumeofAssayBuffer;celllysisisnotnecessary.Afteraprescribedincubation time(usually60min), theabsorbanceat410nm(OD410),which isproportionaltomolesp-nitrophenol liberated, isrecordedandusedtocalculatetheconcentrationofα-galactosidaseinmilliunits/(mlxcell).

• TomonitorMEL1 expressiondirectlyonnutritionalselectionplates,useX-α-Gal(CatNo.630407).X-α-Galcanbeincludedinthemediumbeforepouringplatesorspreadontopofthemediumbeforeplatingliquidcultures.Asα-galactosidaseaccumulatesinthemedium,ithydrolyzesX-α-Galcausingyeastcoloniestoturnblue.InstructionsforpreparingX-α-Galindicatorplatesaregiven in theX-α-GalProtocol-at-a-Glance (PT3353-2)suppliedwitheachpurchaseofthesubstrate.Directionsforusecanalsobedownloadedfromourwebsiteatwww.clontech.com.

VI. α-andβ-GalactosidaseAssayscontinued



table iii. comparison of β-galactosidase assays

ProtocolTypeofassay Substrate Section Applications/Comments

In vivo, X-galinmedium VI.B •Lesssensitivethancolony-liftassays;recommendedagarplate onlywhenthecellstobeassayedcontainmany copiesofthelacZ reportergene(suchasonahigh- copy-numberplasmid) •Convenientforlarge-scaleexperiments;screen manyplatesandcoloniesatthesametime •Potentialdrawbacks: –Qualitativeresultsonly –Expensiveifassayingmanyplates –Needtocheckforbluecolordevelopmentat severaltimeintervalsbetween24and96hr. –Backgroundcanbetroublesome

Colony-lift, X-galonfilter VI.C •Relativelysensitive;recommendedwhenthecells filtertobeassayedcontainoneoronlyafewcopies ofthelacZ reportergene •Convenientforlarge-scaleexperiments;screen manyplatesandcoloniesatthesametime •Relativelyinexpensivetoscreenmanyplates •Getresultsquickly(inmostcases,withinafew hours) •Potentialdrawbacks: –Qualitativeresultsonly –Moremanipulationsrequiredthanforin vivo assay

Liquidculture ONPG VI.D •Forassayingasmallnumberofselectedtransformants •LessexpensivethanCPRGorGalacton-StarTM

•Potentialdrawbacks: –Maynotbesensitiveenoughtoquantifyweakor transienttwo-hybridinteractions

Liquidculture CPRG VI.E •Forassayingasmallnumberofselectedtransformants •10-timesmoresensitivethanONPG •Potentialdrawbacks: –LessreproduciblethanONPGforstrongpositive coloniesbecauseofCPRG’sfastreactionrate

Liquidculture Chemiluminescent VI.F •Forassayingasmallnumberofselectedtransformants (Galacton-StarTM) •Themostsensitiveβ-galsubstrate •Potentialdrawbacks: –Relativelyexpensive –Requiresluminometerorscintillationcounter –Cangivehighbackground

Summary:Relativesensitivityofthefivetypesofβ-galactosidaseassays:

[Leastsensitive] [Mostsensitive]

X-gal ONPG CPRG X-gal GalactonStar(inagarplates) (liquidassay) (liquidassay) (filterassay) (liquidassay)




ReagentsandMaterialsRequired:

• AppropriateSDagarplatescontainingX-gal(80mg/L)and1XBUsalts(AppendixC.A). Notes: • BUsaltsareincludedinthemediumtomaintaintheoptimumpHforβ-galactosidaseandtoprovidethephosphate

neededfortheassay. • TheX-galshouldbeincorporatedintothemediumbeforetheplatesarepoured.IftheX-galisspreadoverthe

surfaceoftheagarplates,itcanresultinunevendistributionandthuslocalizedvariationsinX-galconcentration.Also,theextraliquidontheplatesurface(fromspreadingtheX-gal)mayleadtounevenspreadingofthecellsuspensionandwilldelayabsorptionoftheliquid.

• X-galisheat-labileandwillbedestroyedifaddedtohot(i.e.>55°C)medium. • Preparetherequirednumberofplatesinadvance.Allowplatestodry(unsleeved)atroomtemperaturefor2–3days

orat30°Cfor3hrpriortospreadingorstreakingthecells.Excessmoistureontheagarsurfacecanleadtounevenspreadingofcells.

1.Streak,replicaplate,orspreadthetransformantstobeassayedonselectionmediumcontainingX-galandBUsalts.

• Whenperformingatwo-hybridlibraryscreeningwhereveryfewofthecotransformantsareexpectedtobepositiveforlacZ expression(orwhereitisdifficulttopredictthenumberofinteractors),platethecellsatahighdensity.Werecommendplatingattwodifferentdensitiestocoverarange;e.g.,0.5x106cfuonsome(150-mmplates)and2x106onothers.

• Whenperformingatwo-hybridassaywheremostoralloftheindividualcoloniesmaybeLacZ+,spread200–400cfuper100-mmplate.

2.Incubateplatesat30°Cfor4–6days. 3.Checkplatesevery12hr(upto96hr)fordevelopmentofbluecolor. Notes: • Ifyouareperformingatwo-hybridlibraryscreeningusingtheMatchmakerLexASystem,pleaseseetheUser

ManualforfurtherinformationonidentifyingandstoringLacZ+colonies. • ColoniesgrownonX-gal-containingmediumwillbesomewhatsmallerthanthosegrownwithoutX-gal.

C. Colony-liftFilterAssay

ReagentsandMaterialsRequired:

• WhatmanNo.5orVWRGrade410paperfilters,sterile Notes: • 75-mmfilters(e.g.,VWRCatNo.28321-055)canbeusedwith100-mmplates;125-mmfilters(e.g.,VWRCatNo.28321-113)

canbeusedwith150-mmplates • Alternatively,85-and135-mmfilterscanbespeciallyorderedfromWhatman. • Nitrocellulosefiltersalsocanbeused,buttheyarepronetocrackwhenfrozen.

• Forcepsforhandlingthefilters

• Zbuffer(AppendixD)

• Zbuffer/X-galsolution(AppendixD)

• X-galstocksolution(AppendixD)

• Liquidnitrogen

1.Forbestresultsusefreshcolonies(i.e.,grownat30°Cfor2–4days),1–3mmindiameter. Notes: •Ifonlya fewcoloniesare tobeassayed,streak them(orspread them insmallpatches)directlyontomaster

SDselectionagarplates. Incubate theplatesat30°C foranadditional1–2days,and thenproceedwith theβ-galactosidaseassaybelow.

• UsetheSDselectionmediumappropriateforyoursystemandplasmids.WhentestingLexAtransformants,besuretousegal/raffinductionmedium.

2.PrepareZbuffer/X-galsolutionasdescribedinAppendixD. 3.Foreachplateoftransformantstobeassayed,presoakasterileWhatmanNo.5orVWRgrade

410filterbyplacingitin2.5–5mlofZbuffer/X-galsolutioninaclean100-or150-mmplate. 4.Usingforceps,placeaclean,dryfilteroverthesurfaceoftheplateofcoloniestobeassayed.

Gentlyrubthefilterwiththesideoftheforcepstohelpcoloniesclingtothefilter. 5.Pokeholesthroughthefilterintotheagarinthreeormoreasymmetriclocationstoorientthe




filtertotheagar. 6.Whenthefilterhasbeenevenlywetted,carefullyliftitofftheagarplatewithforcepsandtransfer

it(coloniesfacingup)toapoolofliquidnitrogen.Usingtheforceps,completelysubmergethefiltersfor10sec.

Note:Liquidnitrogenshouldbehandledwithcare;alwayswearthickglovesandgoggles.

7.Afterthefilterhasfrozencompletely(~10sec),removeitfromtheliquidnitrogenandallowittothawatroomtemperature.(Thisfreeze/thawtreatmentistopermeabilizesthecells.)

8.Carefullyplacethefilter,colonysideup,onthepresoakedfilter(fromStepC.3).Avoidtrappingairbubblesunderorbetweenthefilters.

9.Incubatethefiltersat30°C(orroomtemperature)andcheckperiodicallyfortheappearanceofbluecolonies.

Notes: • Thetimeittakescoloniesproducingβ-galactosidasetoturnbluevaries,typicallyfrom30minto8hrinalibrary

screening.Prolongedincubation(>8hr)maygivefalsepositives. • Yeasttransformedwiththeβ-galactosidasepositivecontrolplasmidwillturnbluewithin20–30min.Mostyeast

reporterstrainscotransformedwiththepositivecontrolsforatwo-hybridinteractiongiveapositivebluesignalwithin60min.CG-1945cotransformedwiththecontrolplasmidsmaytakeanadditional30mintodevelop.Ifthecontrolsdonotbehaveasexpected,checkthereagentsandrepeattheassay.

10.Identifytheβ-galactosidase-producingcoloniesbyaligningthefiltertotheagarplateusingtheorientingmarks.Pickthecorrespondingpositivecoloniesfromtheoriginalplatestofreshmedium.Iftheentirecolonywasliftedontothefilter,incubatetheoriginalplatefor1–2daystoregrowthecolony.

D. LiquidCultureAssayUsingONPGasSubstrate ReagentsandMaterialsRequired: • Appropriateliquidmedium(AppendixC.A) • 50-mlculturetubes • Zbuffer(AppendixD) • Zbuffer+β-mercaptoethanol(AppendixD) • ONPG(AppendixD) • 1MNa2CO3

• Liquidnitrogen

1.Prepare5-mlovernightculturesinliquidSDselectionmediumasdescribedinChapterIII.A.3.UsetheSDmediumappropriateforyoursystemandplasmids.

Note:BesuretouseSDmediumthatwillmaintainselectionontheplasmidsused.

2.Onthedayoftheexperiment,dissolveONPGat4mg/mlinZbuffer(AppendixD)withshakingfor1–2hr.

3.Vortextheovernightculturetubefor0.5–1mintodispersecellclumps.Immediatelytransfer2mloftheovernightcultureto8mlofYPD(exceptfortheLexASystem).

Note:FortheLexASystem,usetheappropriateSD/Gal/Raffinductionmediumforthestrainsbeingassayed.

4.Incubatethefreshcultureat30°Cfor3–5hrwithshaking(230–250rpm)untilthecellsareinmid-logphase(OD600of1ml=0.5–0.8).RecordtheexactOD600whenyouharvestthecells.

Note:BeforecheckingtheOD,vortextheculturetubefor0.5–1mintodispersecellclumps.

5.Place1.5mlofcultureintoeachofthree1.5-mlmicrocentrifugetubes.Centrifugeat14,000rpm(10,000xg)for30sec.

6.Carefullyremovesupernatants.Add1.5mlofZbuffertoeachtubeandvortexuntilcellsareresuspended.

7.Centrifugecellsagainandremovesupernatants.Resuspendeachpelletin300µlofZbuffer.(Thus,theconcentrationfactoris1.5/0.3=5-fold).

Note:Differencesincellrecoveriesafterthiswashstepcanbecorrectedforbyre-readingtheOD600oftheresuspendedcells.

8.Transfer0.1mlofthecellsuspensiontoafreshmicrocentrifugetube. 9.Placetubesinliquidnitrogenuntilthecellsarefrozen(0.5–1min). 10.Placefrozentubesina37°Cwaterbathfor0.5–1mintothaw.




11.Repeatthefreeze/thawcycle(Steps9&10)twomoretimestoensurethatthecellshavebrokenopen.

12.Setupablanktubewith100µlofZbuffer. 13.Add0.7mlofZbuffer+β-mercaptoethanoltothereactionandblanktubes.DonotaddZ

bufferpriortofreezingsamples. 14.Starttimer.Immediatelyadd160µlofONPGinZbuffertothereactionandblanktubes. 15.Placetubesina30°Cincubator. 16.Aftertheyellowcolordevelops,add0.4mlof1MNa2CO3tothereactionandblanktubes.

Recordelapsedtimeinminutes. Notes:

• Thetimeneededwillvary(3–15minforthesingle-plasmid, β-gal-positivecontrol;~30minforatwo-hybridpositivecontrol;andupto24hrforweakerinteractions).

• Theyellowcolorisnotstableandwillbecomemoreintensewithtime.Youwillneedtorunanewblanktubewitheverybatch.

17.Centrifugereactiontubesfor10minat14,000rpmtopelletcelldebris. 18.Carefullytransfersupernatantstocleancuvettes. Note:Thecellulardebris,iftransferredwiththesupernatant,willstronglyinterferewiththeaccuracyofthistest.

19.CalibratethespectrophotometeragainsttheblankatA420andmeasuretheOD420ofthesamplesrelativetotheblank.TheODsshouldbebetween0.02–1.0tobewithinthelinearrangeoftheassay.

20.Calculateβ-galactosidaseunits. 1unit ofβ-galactosidase isdefinedas theamountwhichhydrolyzes1µmolofONPGtoo-nitrophenolandD-galactoseperminpercell(Miller,1972;Miller,1992):

β-galactosidaseunits = 1,000xOD420/(txVxOD600)

where: t = elapsedtime(inmin)ofincubation V = 0.1mlxconcentrationfactor* OD600 = A600of1mlofculture

*Theconcentrationfactor(fromStepD.7)is5.However,itmaybenecessarytotryseveraldilutionsofcellsatthisstep(hencedifferentconcentrationfactors)toremainwithinthelinearrangeoftheassay.

E. LiquidCultureAssayUsingCPRGasSubstrate

ReagentsandMaterialsRequired: • Appropriateliquidmedium(AppendixC.A) • 50-mlculturetubes • Buffer1(AppendixD) • Buffer2(AppendixD) • CPRG(chlorophenolred-β-D-galactopyranoside;BMCCatNo.884308) • 3mMZnCl2(Filtersterilizedtopreservefor~3months) • Liquidnitrogen

1.Prepare5-mlovernightculturesinliquidSDmediumasdescribedinChapterIII.A.3.UsetheSDselectionmediumappropriateforyoursystemandplasmids.

Note:BesuretouseSDmediumthatwillmaintainselectionontheplasmidsused.

2.Vortextheovernightculturetubefor0.5–1mintodispersecellclumps.Immediatelytransfer2mloftheovernightcultureto8mlofYPD(exceptforLexASystem).


3.Incubatefreshcultureat30°Cfor3–5hrwithshaking(230–250rpm)untilthecellsareinmid-logphase(OD600of1ml=0.5–0.8).RecordtheexactOD600whenyouharvestthecells.

Note:BeforecheckingtheOD,vortextheculturetubefor0.5–1mintodispersecellclumps.

4.Place 1.5 ml of culture into each of three 1.5-ml microcentrifuge tubes. Centrifuge at14,000rpm(16,000xg)for30sectopelletthecells.

5.Carefullyremovethesupernatant,add1.0mlofBuffer1,andvortexuntilcellsarethoroughlyresuspended.




6.Centrifugeat14,000rpm(16,000xg)for30sectopelletthecells. 7.Carefully remove the supernatant and resuspend the cells in 300 µl of Buffer 1.

(Theconcentrationfactoris1.5/0.3=5-fold.) Note:Differencesincellrecoveriesafterthiswashstepcanbecorrectedforbyre-readingtheOD600oftheresuspended

cells.

8.Transfer0.1mlofthecellsuspensiontoafreshmicrocentrifugetube. 9.Placetubesinliquidnitrogenuntilthecellsarefrozen(0.5–1min). 10.Placefrozentubesina37°Cwaterbathfor0.5–1mintothaw. 11.Repeatthefreeze/thawcycle(Steps9and10)twotimestoensurethatallcellsarebroken

open. 12.Add0.7mlofBuffer2toeachsampleandmixbyvortexing.Thoroughmixingiscriticalto

theassay. 13.RecordthetimewhenBuffer2wasadded.Thisisthestartingtime. 14.Add1mlofBuffer2toaseparatetube(thiswillbethebufferblank). 15.When thecolorof thesamples isyellow/grey to red,add0.5mlof3.0mMZnCl2toeach

sampleandthebufferblanktostopcolordevelopment.Recordthestoptime.(Forverystrongβ-galactosidase-positivecolonies,colordevelopmentoccurswithinseconds;weak-to-moderatereactionstakeseveralhourstodevelop).

16.Centrifugesamplesat14,000rpmfor1mintopelletcelldebris. 17.Transfersamplestofreshtubes. 18.ZerothespectrophotometerusingthebufferblankandmeasuretheOD578ofthesamples.(An

OD578between0.25and1.8iswithinthelinearrangeoftheassay.) 19.Calculateβ-galactosidaseunits. 1unit ofβ-galactosidase isdefinedas theamountwhich

hydrolyzes1µmolofCPRGtochlorophenolredandD-galactoseperminpercell (Miller,1972;Miller,1992):

β-galactosidaseunits = 1000xOD578/(txVxOD600)

where: t = elapsedtime(inmin)ofincubation V = 0.1xconcentrationfactor* OD600 = A600of1mlofculture *Theconcentrationfactor(fromStepE.7)is5.However,itmaybenecessarytotryseveraldilutionsofcellsat

thisstep(hencedifferentconcentrationfactors)toremainwithinthelinearrangeoftheassay.

F. LiquidCultureAssayUsingaChemiluminescentSubstrate

ReagentsandMaterialsRequired: • Appropriateliquidmedium(AppendixC.A) • 50-mlculturetubes • Zbuffer(AppendixD) • Galacton-Starreactionmixture(ProvidedwiththeLuminescentβ-galactosidaseDetectionKitII) • Liquidnitrogen • Luminometer[orscintillationcounterwithsingle-photon-countingprogram] • Optional:96-well,opaquewhite,flat-bottommicrotiterplates[XenoporeCatNo.WBP005] • Optional:Purifiedβ-galactosidase(forastandardcurve)

Note:Forbestresults,werecommendusingtheLuminescentβ-galactosidaseDetectionKitII(CatNo.631712),whichincludesareactionbuffercontainingtheGalacton-StarsubstrateandtheSapphireIITMaccelerator,positivecontrolbacterial β-galactosidase,andacompleteUserManual.

Chemiluminescentdetectionofβ-galactosidase




Itisimportanttostaywithinthelinearrangeoftheassay.High-intensitylightsignalscansaturatethephotomultipliertubeinluminometers,resultinginfalselowreadings.Inaddition,lowintensitysignalsthatarenearbackgroundlevelsmaybeoutsidethelinearrangeoftheassay.Ifindoubt,determine the linear range of the assay and, if necessary, adjust the amount of lysate usedtobring thesignalwithin the linear range.SeeCampbellet al. (1995) forachemiluminescent β-galactosidaseassayusedinayeasttwo-hybridexperiment.

1.Prepare5-mlovernightculturesinliquidSDmediumasdescribedinChapterIII.A.3.UsetheSDmediumappropriateforyoursystemandplasmids.

Note:Forqualitativedata,awholecolony,resuspendedinZbuffer,maybeusedfortheassaydirectly.Seeinstructionsfollowingthissection.

2.Onthedayoftheexperiment,preparetheGalacton-Starreactionmixture.Keepbufferoniceuntilyouarereadytouseit.

3.Vortextheovernightculturetubefor0.5–1mintodispersecellclumps.Immediatelytransferatleast2mloftheovernightculturetonomorethan8mlofYPD(exceptfortheLexASystem).


4.Incubatethefreshcultureat30°Cfor3–5hrwithshaking(230–250rpm)untilthecellsareinmid-logphase(OD600of1ml=0.4–0.6).

5.Vigorouslyvortextheculturetubefor0.5–1mintodispersecellclumps.RecordtheexactOD600whenyouharvestthecells.

6.Place1.5mlofcultureintoeachofthree1.5-mlmicrocentrifugetubes.Centrifugeat14,000rpm(10,000xg)for30sec.

7.Carefullyremovesupernatants.Add1.5mlofZbuffertoeachtubeandthoroughlyresuspendthepellet.

8.Centrifugeat14,000rpm(10,000xg)for30sec. 9.Remove the supernatants. Resuspend each pellet in 300 µl of Z buffer. (Thus, the

concentrationfactoris1.5/0.3=5-fold.) 10.ReadtheOD600oftheresuspendedcells.TheOD600shouldbe~2.5.Ifthecelldensityislower,

repeatSteps5–9,exceptresuspendthecellsin<300µlofZbuffer. 11.Vortexeachcellsuspensionandtransfer100µltoafreshtube. Note:Theremainingcellsuspensioncanbestoredat–70°Cto–80°C.

12.Placetubesinliquidnitrogenfor0.5–1mintofreezethecells. 13.Placefrozentubesina37°Cwaterbathfor0.5–1mintothaw. 14.Repeat freeze/thaw cycle (Steps 12 & 13) once to ensure that cells have been cracked

open. 15.Warmtoroomtemperatureenoughreactionbufferfortheentireexperiment. 16.Setupablanktubewith25µlofZbuffer. 17.[Optional] If you wish to obtain absolute as well as relative data, set up a series of

β-galactosidase standard tubes containing 0.0005, 0.001, 0.003, 0.010, and 0.020 unit of β-galactosidasein25µlofZ-buffer.

18.Place20–30µlofeachcell lysate inaseparatesample tube (or intowellsofanopaque96-well,flat-bottommicrotiterplatesuitableforplateluminometers).Ifyouareusingasampletube,thetubeshouldholdatleast0.5ml.

Note:Theamountofyeastextractrequiredmayvarydependinguponthelevelofβ-galexpressionandthedetectiondeviceused.Use10–30µlofextractforpositivecontrolsand20–30µlforexperimentalsampleswithpotentiallylowlevelsofenzymeactivity.Itisimportanttovarytheamountofextracttokeepthesignalwithinthelinearrangeoftheassay.

19.Add200µlofGalacton-Starreactionmixturetoeachsampletubeorwellandmixgently. 20.Incubateatroomtemperature(20°–25°C)for60min. Note: Light signals produced during this incubation are stable for >1 hr; therefore, detection can be performed

1–2hraftertheincubation.

21.Centrifugetubesat14,000rpm(16,000xg)for1minat4°C.(Ifyouareusingmicrotiterplates,centrifugeplatesat1,000xgfor5mininaspeciallyadaptedrotor.)Proceeddirectlytothe




appropriatedetectionstepsforyourassay:Step22,23,24,or27. 22.Detectionusingatubeluminometer a. Turnonthetubeluminometer.Settheintegrationtimefor5sec. b. Calibratetheluminometeraccordingtothemanufacturer’sinstructions. c. Ifthesampleisnotalreadyinatubesuitableforluminometerreadings,transfertheentire

solutionfrom(Step21)toanappropriatetube.Donotdisturbthepellet. d. Placeonesampleatatimeintheluminometercompartmentandrecordthelightemission

(RLU)asa5-secintegral.Useyourblanksampleasareferencewheninterpretingthedata. 23.Detectionusingaplateluminometer AfterStep21,simplyrecordlightsignalsas5-secintegrals. 24.Detectionusingascintillationcounter a. TransfertheentiresolutionfromStep21toa0.5-mlmicrocentrifugetube. Note:Plantousescintillationcounteradaptorsthatkeepthetubesupright.

b. Placethetubeinthewasherofthescintillationcounteradaptorandplacetheadaptorinthemachine’scountingrack.Settheintegrationtimeforatleast15sec.

Note:Integrationtimes<15secmaynotproduceaccurateresults.

c. Todetectchemiluminescentsignals,useasingle-photon-countprogram.Consultyourscintillationcounter’smanufacturerforfurtherinformationaboutthissoftware.

25.Fordetectionmethodsdescribed inSteps22–24:Calculate theβ-galactosidaseactivity intermsofRLU/OD600unitofcellculture.(NotethatMillerunitcalculationsarenotpossibleusingthesemethods.)

26.[Optional] Ifyouhavesetupβ-galactosidasestandards,prepareastandardcurveofRLUvs.theamountofβ-galactosidase.Estimatethequantityofβ-galactosidaseintheunknownsamplesusingthestandardcurve.DeterminetheamountofenzymeperOD600unitofcellculture.ThefinalOD600unitsofcellsassayedpersampleiscalculatedasfollows:

OD600(fromStep5)xvol(fromStep18) xconc.factor(fromStep9)

27.Detectionbyexposureofx-rayfilm Lightemissioncanalsoberecordedbyexposureofx-rayfilmtoreactionsamplesinopaque

96-wellflat-bottommicrotiterplates.Therelativeintensityoftheresultingspotsonthefilmcanbeestimatedbycomparisontopositiveandnegativecontrols.Notethatx-rayfilmisseveralordersofmagnitudelesssenstivethanaluminometerorscintillationcounter.

Overlaythemicrotiterplatewithx-rayfilm,coverthefilmwithplasticwrap,andplaceaheavyobjectsuchasabookontoptoholdthefilminplace.Exposethefilmatroomtemperaturefor5–30min.

Note:Tocomparesamplesaccurately,theymustbewithinthelinearresponsecapabilityofthex-rayfilm.Wethereforerecommendthatyouobtainseveraldifferentexposures.

QualitativeLiquidAssayUsingGalacton-StarastheSubstrate

Thisalternativecellpreparationmethoddirectlydetectsβ-galactosidaseactivityinresuspendedyeastcolonies.ItisrecommendedfordetectingextremelyweaklacZtranscriptionalsignalsthatcannotbedetectedbyX-galfilterassays.Fora+/–result,itismorelabor-intensivethanafilterassay.However,becauseofitsgreatersensitivity,itislesslikelytogiveafalse-negativeresult.

1.GrowcoloniesontheappropriateSDselectionmedium. 2.Transferanentire large (2–3mm), fresh (2–4-day-old) colony toa0.5-ml tubecontaining

50µlofZbuffer.Ifcoloniesaresmall,useseveral.Atthesametime,prepareamasterorreferenceplateofthecoloniestobeassayed.

3.CompletelyresuspendthecolonyintheZbufferbyrepeatedlypipettingupanddown. 4.Placetubesinliquidnitrogenfor0.5–1mintofreezethecells. 5.ContinuewithStep13ofthemainprocedure(VI.F)above. 6.Compareresultswiththoseofthenegativecontrol.

G. α-GalQuantitativeAssay ReagentsandMaterialsRequired:




• Appropriateliquidsyntheticdropout(SD)culturemedium(AppendixC.A) • 50-mlculturetubes • PNP-α-GalSolution(100mM) • 10XStopSolution(AppendixD.F) • 1XNaOAcBuffer(AppendixD.F) • AssayBuffer(AppendixD.F) • 1.5-mlcuvettesor96-well,flat-bottommicrotiterplatesforOD410measurements

PreparationofSamples 1.Inoculate2–5mlofliquidsyntheticdropout(SD)medium,containingtheappropriatedropout

supplements,withayeastcolonyexpressingthepairofproteinsbeinganalyzed.Itisadvisabletosetuptriplicateculturesforeachtypeofyeastcolonybeinganalyzed.

Fresh(one-tothree-week-old)colonieswillgivebestresultsforliquidcultureinoculation.Asinglecolonymaybeusedfortheinoculationifitis2–3mmindiameter.Scrapetheentirecolonyintothemedium.Ifthecoloniesonthemasterplatearesmallerthan2mm,transferseveralcoloniesintothemedium.

Examples: • ForAH109andY190cotransformantsexpressinginteractingpairsofGAL4BDandADfusionproteins,inoculate

intoSD/–His/–Leu/–Trp. • ForAH109andY190cotransformantsexpressingnon-interactingpairsoffusionproteinconstructs,inoculateinto

SD/–Leu/–Trp. • ForY187tranformantsexpressinginteractingornon-interactingproteins,inoculateintoSD/–Leu/–Trp. • Fornon-transformedAH109,Y187,andY190hoststrains,inoculateintoSD/–Ura.

2.Incubateat30°Covernight(~16–18hr)withshaking(250rpm). 3.Vortexthecellculturetubefor0.5–1mintodispersecellclumps,thentransfer1mlofthe

suspensiontoacleancuvetteandrecordtheOD600.Foraccuracy,theOD600shouldliebetween0.5–1.0.Dilutethecellsuspensionifnecessary;remembertoaccountforthedilutionfactorwhenmakingyourfinalcalculations(Step11,below).

4.Place 1.0 ml of culture into a 1.5-ml microcentrifuge tube. Centrifuge at 14,000 rpm(10,000xg)for2minoruntilthecellsarecompletelypelleted.

Note:Itisimportanttoensurethatcellsandcellulardebrisarecompletelypelletedtominimizeintereferencefromlightscatteringinthecolorimetricassaybelow.

5.CarefullytransferthesupernatanttoacleantubeandstoreatroomtemperatureforuseinStep6,below.

Note:Tominimizethelossofenzymeactivity,wesuggestproceedingwiththecolorimetricassayimmediatelyoncethecell-freesuperantanthasbeenisolated.

ColorimetricAssay Belowweprovideprotocolsfor1-mland200-µlassays.Ifyouhaveaccesstoaspectrophotometer

equippedtoreadmicrotiterplates,youmayfinditmoreconvenienttousethe200-µlassayprotocol,whichisintendedforusewith96-well,flat-bottommicrotiterplates.Note:Theexperimental conditionsandvolumesof reagentsused throughout theα-GalactosidaseQuanitativeAssayhavebeencarefullytestedandoptimizedforuseinthe1-mland200-µlassayformatsdescribedbelow.Pleasefollowasdirected.Thoughwedonotrecommendchangingtheactualvolumesofreagentsusedinthecolorimetricassay,ifthesignalfromanexperimentalsampleexceedsthelinearrangeoftheassay,youcandilutethemediasupernatantbeforetransferringanaliquottothereactiontubeorwell.RemembertocorrectforindividualsamplevolumesanddilutionswhentabulatingfinalresultsatStep11.

5.PrepareasufficientamountofAssayBufferforallsamplesincludingcontrols,andallowtoequilibratetoroomtemperature.

• Foreach1-mlassay,youwillneed24µlAssayBuffer. • Foreach200-µlassay(96-wellmicrotiterplateformat),youwillneed48µlAssayBuffer.

Note:Werecommendassayingeachsampleintriplicate.Besuretoincludepositiveandnegativecontrolsinyourassay.AlsoincludeareagentblankineachassaytocalibratethespectrophotometerpriortoreadingtheODofyoursamples.Areagentblankiscomposedofsterile,unusedculturemedia,AssayBuffer,andStopSolutioncombinedaccordingtoSteps6–9.

AssayScale





200-µl 1-ml 6.Transfercellculturemediumsupernatant(fromStep4) 16µl 8µl intoa1.5-mlmicrocentrifugetubeorintoawellofa clearmicrotiterplatea.

7.AddAssayBuffertoeachsample. 48µl 24µl

8.Incubateat30°Cfor60min.Besuretocover microtiterplateswithalidorparafilmtoprevent evaporation.

9.TerminatethereactionbyaddingStopSolution. 136µlof10X 960µlof1X Note:Use1XStopSolutionfor1-mlassaysand 10XStopSolutionfor200-µlassays.

10.Recordtheopticaldensityofeachsampleat 96-wellplate 1.5-mlcuvette410nm(OD410)

b. a Addacorrespondingvolumeofsterile,unusedculturemediatothereagentblank. b ZerothespectrophotometerusingthereagentblankandmeasuretheOD410oftheexperimentalandcontrolsamples

relativetotheblank.

11.Calculate α-galactosidase units. One unit of α-galactosidase is defined as the amountof enzyme that hydrolyzes 1 µmole p-nitrophenyl-α-d-galactoside to p-nitrophenol and d-galactosein1minat30°Cinacetatebuffer,pH4.5(Lazoet al.,1978).

α-galactosidase [milliunits/(mlxcell)] = OD410xVfx1,000/[(εxb)xtxVixOD600]

t = elapsedtime(inmin)ofincubation

Vf = finalvolumeofassay(200µlor992µl)

Vi = volumeofculturemediumsupernatantadded(16µlor8µl)

OD600 = opticaldensityofovernightculturea

ε xb = p-nitrophenolmolarabsorbtivitybat410nmxthelightpath(cm) = 10.5(ml/µmol)for200-µlformatb,c

= 16.9(ml/µmol)for1-mlformatwhereb=1cmb,d

a Theopticaldensityat600nm,recordedinStep2,isusedtonormalizetheOD410ofdifferentmediasamplestothenumberofcellsineachculture.

bThe molar absorbtivity, though independent of concentration and light path, varies with the chemical properties(e.g.,pH)ofthesolution.BecausedifferentstrengthsandquantitiesofStopSolutionareusedtoterminatethe200-and1-mlreactions,thefinalpHsand,therefore,themolarabsorbtivitiesaredifferentforthetwoformats.

cDetermined at Clontech using a SPECTRAmax® Microplate Spectrophotometer and Corning CostarUV-transparent, flat-bottom plates (Corning Cat No. 3635); Some microplate readers have pathlength correctioncapabilitiestonormalizeabsorbancevaluestothoseobtainedwhenthelightpathlengthis1-cm(e.g.,using1.5-mlcuvettes).Withpathlengthcorrectionon,themolarabsorbtivity,ε,ofp-nitrophenolat410nminthe200-µlformatwasdeterminedtobe20.3ml/µmol.

Thewell-diameterand,therefore,thelightpath(b)inotherbrandsof96-wellplatesmaydifferfromthatoftheCorningplatesusedforthesedeterminations.Todetermineε xbinotherplates,constructaplotofA410versusconcentrationofp-nitrophenol(PNP)asfollows.Usinga10µmol/mlstandardsolutionofp-nitrophenol(SigmaCatNo.104-1),make1:2serialdilutionsinwaterdownto0.02µmol/mlPNP.UsetheseserialdilutionsinplaceofmediumsupernatantinStep6,above.ThenfollowSteps7,9,and10asdirected;omitStep8.AtStep7,useAssayBufferthathasbeenpreparedbycombining2volumesof1XNaOAcwith1volumeofH2O,notPNP-α-Gal.Finally,plotA410versusconcentrationofPNP.AccordingtotheBeer-LambertLaw,theproportionalityconstant,ε xb,isequaltotheslopeofthestraightlinedefinedbythesedata.

dDeterminedatClontechusing1.5-mlcuvettes



VII.WorkingWithYeastPlasmids


IsolatingplasmidDNAfromyeastisnottrivial,primarilybecauseofthetoughcellwall.Furthermore,therelativelylargesize(>6kb)andlowcopynumber(~50/cell)ofsomeyeastplasmidsresultsinverylowDNAyields,regardlessoftheplasmidisolationmethodused.Inaddition,plasmidDNAisolatedfromyeastisoftencontaminatedbygenomicDNAbecauseyeastcontain~3XasmuchgenomicDNAasE. coli,andtheisolationmethodbreakstheyeastchromosomesandreleasesthemfromcellularmaterial.

Thereareseveralyeastplasmidisolationprocedurescurrentlyinuse.Thevariousprotocolsdifferprimarilyinthemethodusedtobreakthecellwalls.HereweprovidetheprotocolthatweoptimizedforourYeastmakerYeastPlasmidIsolationKit(CatNo.630441).Thisprocedure,whichwasmodifiedfromthemethodofLinget al. (1995),usesextensivedigestionwithlyticasetoweakenthecellwallsandSDStobursttheresultingspheroplasts.TheDNAprepscanbecleanedupusingeitherCHROMASPINTMColumnsorphenol:chloroformextractionfollowedbyethanolprecipitation.IfCHROMASPINTMColumnsareused,thismethodtakes<2hrfromcellpelletstopurifiedplasmid,andissimpleenoughtobeeasilyadaptedforprocessingmanysamplessimultaneously.

ThispurificationmethodyieldsDNAofsufficientpurityforuseasaPCRtemplate(ChapterVIII)orfortransformingE. coli(ChapterVII.C).However,ifyouneedalargequantityofplasmid,orverypureplasmidDNA,suchasforsequencingorrestrictionenzymedigestion,youwillhavetotransformE. coliandprepareplasmidusingstandardmethods(Sambrooket al.,1989).

PlasmidrescueviacomplementationofE. coli mutations

Plasmid isolation fromyeastcotransformants iscomplicatedby thepresenceof two(ormore)typesofplasmidsinasingleyeastcolony.NutritionalselectionofE. coli transformantsbearingtheyeastplasmidofinterestcanbeanefficientwayto“rescue”onetypeofplasmidfromamixtureofplasmidsbearingdifferentnutritionaltransformationmarkers.FormoreinformationonplasmidrescueviatransformationofE. coli,seeSectionVII.C.

B. PlasmidIsolationfromYeast

ReagentsandMaterialsRequiredThe Yeastmaker Yeast Plasmid Isolation Kit (Cat No. 630441) provides the SDS and lyticase solutions, CHROMASPIN-1000DEPC-H2OColumns,and2-mlcentrifugetubesforusewiththecolumns.

• Appropriate SD liquid or agar medium to keep selection on the plasmids (Appendix C.A;AppendixE).

• Sterile,1.5-mlmicrocentrifugetubes(ora96-tubemicrotiterarray,multichannelpipettors,andcentrifugeadaptorformultiwellplates).

• 20%SDS

• LyticaseSolution(5units/µlinTEbuffer;storeat4°Cforupto2monthsorat–20°Cforupto6months.Ifcolloidalmaterialprecipitates,mixthesolutionbyinversionbeforeusing.)

• Recommended:CHROMASPIN-1000DEPC-H2OColumns(CatNo.636093)and2-mlcentrifugetubesforusewiththecolumns

• IfyoudonotuseCHROMASPINColumns,youwillneedmaterialstoperformphenol:chloroformextractionandethanolprecipitation:

• Phenol:chloroform:isoamylalcohol(25:24:1;SeeSambrooket al.,1989,forinformationonpreparingneutralizedphenolsolutions)

• 10Mammoniumacetate

• 95–100%ethanol



1.Prepareyeastculturesforlysis(Stepa,b,orcbelow). a. Fromasolidpatchofgrowth:

i. Spreadathinfilmofyeastcells(~2-cm2patch)ontotheappropriateSDagarmedium.

ii. Incubateplateat30°Cfor3–4days.(Thepatchshouldshowabundantyeastgrowth.)

iii. Scrapeupaportionofthepatch(~10mm2)andresuspendthecellsin50µlofsterileH2OorTEina1.5-mlmicrocentrifugetube.

b. Fromaliquidculture:

i. Inoculate a large (2–4-mm), fresh (2–4-day-old) yeast colony into 0.5 ml of theappropriate SD liquid medium. Vortex tube vigorously to completely break up thecolonyandresuspendthecells.

ii. Incubateat30°Covernightwithshakingat230–250rpm.

iii. Spindownthecellsbycentrifugingat14,000rpmfor5min.

iv. Carefullypouroff thesupernatantandresuspendpellets in the residual liquid (totalvolume~50µl).

c. Forsemi-automatedhandlingofalargenumberofsamples:

i. Placealarge(2–4-mm),fresh(2–4-day-old)yeastcolonyinto0.5mloftheappropriateSD liquidmedium in separatewellsofa96-tubemicrotiterarray.Vortexeach tubevigorouslytoresuspendthecells.(Alternatively,use0.5mlofanovernightSDliquidcultureinsteadofayeastcolony.)

ii. Usingacentrifugeadaptedformultiwellplates,centrifugetheentirearrayat1,000xgfor5mintopelletthecells.

iii. Carefullypour(ordraw)offsupernatantsandresuspendpelletsintheresidualmedium(~50µl)byvortexingorpipettingupanddown.

2. Add10µloflyticasesolutiontoeachtube.Thoroughlyresuspendthecellsbyvortexingorrepeatedlypipettingupanddown.

3. Incubatetubesat37°Cfor30–60minwithshakingat200-250rpm. [Optional]Checkadropofthecellsuspensionunderaphasecontrastmicroscope(400X)fortheprogressofcell

lysisbyaddingadropof20%SDStothesideofthecoverslip.AstheycomeintocontactwiththeSDS,mostcellsshouldlosetheirrefractileappearanceandappearas“ghost-like”spheroplasts.Iftherearestillmanyintactcellspresent,incubatethesamplesforanother30min.

4. Add10µlof20%SDStoeachtubeandvortexvigorouslyfor1mintomix. 5. Put thesamplesthroughonefreeze/thawcycle(at–20°C)andvortexagaintoensure

completelysisofthecells. 6. Ifnecessary,samplescanbestoredfrozenat–20°C.Ifsampleshavebeenfrozen,vortex

themagainbeforeusingthem. 7. PourtheentirecontentsofthetubefromStep5aboveontoaprespunCHROMASPIN-

1000ColumnandpurifytheplasmidDNAaccordingtotheCHROMASPINUserManual.PurifiedplasmidDNAwillelutefromthecolumn.

IfyoudonotuseCHROMASPINColumns,cleanuptheprepasfollows: a. Bringthevolumeofthesampleupto200µlinTEbuffer(pH7.0).

b. Add200µlofphenol:chloroform:isoamylalcohol(25:24:1).

c. Vortexathighestspeedfor5min.

d. Centrifugeat14,000rpmfor10min.

e. Transfertheaqueous(upper)phasetoafreshtube.

f. Add8µlof10Mammoniumacetateand500µlof95–100%Ethanol.

h. Placeat–70°Corinadry-ice/ethanolbathfor1hr.

i. Centrifugeat14,000rpmfor10min.

j. Discardsupernatantanddrythepellet.

k. Resuspendpelletin20µlofH2O. Note:TheamountofplasmidDNArecoveredissmallrelativetothecontaminatinggenomicDNA;

therefore,itcannotbemeasuredbyA260orseenonanagarosegel.

VII.WorkingWithYeastPlasmidscontinued



C. TransformingE. coli withYeastPlasmids

We recommend using electroporation (Section C.1) when transforming E. coli with plasmidsisolatedfromyeastbecauseoftherelativelyhightransformationefficiencythatcanbeobtained.ThisisimportantbecauseoftheyeastgenomicDNAthatispresentinyeast-isolatedplasmids;thepresenceofgenomicDNAreducesthetransformationefficiencyoftheplasmids.However,ifyouchoosetousechemicallycompetentcells(SectionC.2),itisessentialthatthecellsbeabletoyieldatransformationefficiencyofatleast107cfu/µg(ofpUC19DNA).

NutritionalselectionofE. colitransformants

IntheMatchmakertwo-hybridsystems,cloningvectorscarryingHIS3, LEU2 , orTRP1markerscan be selectively rescued by complementation of the E. coli hisB, leuB, or trpC mutations,respectively.(TheyeastHIS3, LEU2 , andTRP1genesareexpressedwellenoughinE. colitoallowthiscomplementation.)Furthermore,duetoincompatibilityoftheE. coliplasmidreplicationoriginsusedonthedifferentvectors,onlyoneplasmidconstructwillpropagateinagivenE. colitransformantplatedonselectionmedium.Thus,thereisnoneedtoscreeneveryE. coli transformantforthepresenceoftheother(unwanted)plasmids.

Ifyouplantoperformanutritionalselectionforplasmidrescue,werecommendusingE. colisrainKC8,whichcarriesthehisB, leuB, andtrpCmutations(K.Struhl,personalcommunication).KC8ChemicallyCompetent(CatNo.630434)andElectrocompetent(CatNo.630435)CellsareavailablefromClontech.HB101,whichcarriestheleuBmutation(Bolivar&Backman,1979),maybeusedtoselectforyeastplasmidsbearingtheLEU2 markeronly.

FornutritionalselectionofKC8andHB101transformantsonM9minimalmedium,adda1Xmixtureofaminoacids(i.e.,dropout[DO]supplement)lackingthespecificnutrientthatwillallowselectionofthedesiredplasmid(AppendixE).(ThesameDOsupplementsusedforyeastSDmediumcanbeusedtosupplementM9minimalmedium;seeAppendixCforrecipes).

• Becauseofitsauxotrophicmutations,KC8requiresHis,Leu,Trp,andthiamineforgrowthonminimalmedium,unlessoneofthesenutrientsisspecificallyomittedfortheselection.

• HB101requiresleucine,proline,andthiamineforgrowthonminimalmedium,unlessoneofthesenutrientsisspecificallyomittedfortheselection;notethatHB101isstreptomycinresistant.

• Although optional, we recommend including ampicillin (50 µg/ml) in the medium to reducebackgroundgrowth.

AnyofthecommonE. colihoststrains(e.g.,DH5α;JM109)maybeusedifyouprefertoselecttransformantsbyresistancetoampicillinratherthanusinganutrionalselection.However,becauseboththeDNA-BDandADplasmidswillberepresentedintheE. colitransformantpopulation(andnotnecessarily inequalproportions),manytransformantcolonieswillneedtobescreenedforthepresenceofthedesiredplasmid(s).TheplasmidscanbedistinguishedbyrestrictionenzymedigestionorPCRamplificationusingADvector-specificinsert-screeningprimers.

VII. WorkingWithYeastPlasmidscontinued



Reagentsandmaterialsrequired

• E. coli competentcells(chemicallycompetentorelectrocompetent) Notes: • FormethodstoprepareelectrocompetentE. coli cells,seeKaiser&Auer(1993),Doweret al.(1988),Chuanget al.

(1995),andSambrooket al.(1989).Alternatively,purchasepremadechemicallycompetentorelectrocompetent E. coli cellsfromClontech.

• IfyouusethedirectelectroporationmethodofMarcil&Higgins(1992),theE. coli competentcellsmustbetransformedatanefficiencyof109cfu/µg(ofpUC19DNA)toworksatisfactorilywithyeastplasmids.

• Fortransformationofelectrocompetentcells,youneedanelectroporatorandacuvettewitha0.1-cmgap.

• YeastplasmidDNA(fromSectionBabove)

• Sterile,14-mlpolypropyleneconicaltubes(e.g.,FalconTMCatNo.2059)

• Hanahan’sSOCmediumorLBbroth(Sambrooket al.,1989)

• LB/amp(50µg/ml)agarplatesforantibioticselectionorappropriatelysupplementedM9/ampplatesfornutritionalselection(AppendixC.B)

• MaterialsforisolatingplasmidDNAfromE. coli.

1.ProcedurefortransformingelectrocompetentE. coli KC8 a. PrepareorthawelectrocompetentE. colicells. b. Add1–2µlofyeastplasmidsolutionto40µlofelectrocompetentcellsonice. c. Transfersamplestoaprechilledcuvettehavinga0.1-cmgap.Performtheelectroporation

accordingtothemanufacturer’sinstructions. d. Add1mlofLBor(preferably)SOCmediumwithnoantibiotictothecuvette.Transferthe

cellsuspensiontoa14-mlconicalFalcontube. e. Incubateat37°Cfor1hrwithvigorousshaking(250rpm). e. Pelletcellsbycentrifugingat2,500rpmfor5mininatabletopcentrifuge. f. Discardsupernatantandresuspendpelletinresidualliquid. g. PlatecellsonsupplementedM9/ampagarmedium. h. Incubateplatesat37°Cfor24hr(LB/ampselectiononly),orfor36–48hr(fornutritionalselection

onM9medium).Ifyoudonotrecoveranycolonies,seetheTroubleshootingtipsbelow. i. SeeSectionC.3fortipsonplasmidisolation. 2.ProcedurefortransformingchemicallycompetentE. coli KC8 Transformation efficiency is significantly affected by temperature. Therefore, prechill the

14-ml Falcon tubes and pipette tips to 4°C before using them. a. Preparethechemicallycompetentcellsorthawthemonice. b. Add10µlofyeastplasmidsolutiontoaprechilledFalcontube. c. Add100µlofcompetentcellstothetubeandmixwellbygentlytappingthetube. d. Incubateonicefor30min. e. Heatshockbytransferringthetubetoa42°Cwaterbathandincubatingfor45–50sec. f. Chillonicefor2min. g. Add1mlofLBbrothor(preferably)SOCmediumwithnoantibiotic. h. Incubateat37°Cfor1hrwithvigorousshaking(250rpm). i. Pelletcellsbycentrifugingat2,500rpmfor5mininatable-topcentrifuge. j. Discardsupernatantandresuspendpelletinresidualliquid. k. Platecellsonappropriatemedium(LB/amporsupplementedM9). l. Incubateplatesat37°Cfor24hr(LB/ampselectiononly),orfor36–48hr(fornutritional

selectiononM9medium).Typically,10–100colonieswillbeseenontheplateforasuccessfultransformationusingplasmidisolatedfromyeast.Ifyoudonotrecoveranycolonies,seetheTroubleshootingtipsbelow.




m. Ifyouperformedaparallel transformationusingthecontrolpUC19DNA,calculatethetransformationefficiency. (Thecompetentcellsshouldhavebeen transformedwithanefficiencyof≥1x107cfu/µg.SeeSectionV.E.23forasamplecalculation.)

n. SeeSectionC.3fortipsonplasmidisolation. 3.TipsonIsolatingplasmidDNAfromtheE. coli transformants a. Use a standard plasmid mini-prep procedure to isolate plasmid DNA from the E. coli

transformants(Sambrooket al.,1989). Notes: •IfyouareusinganendA+bacterialstrainsuchasKC8orHB101asthehoststrain,extracaremustbetakenwhen

preparingplasmidDNAbecauseofthepresenceofendonucleaseA.(SeeSambrooket al.[1989]1:1.22–1.23.) •BoilinglysisisnotrecommendedforisolationofplasmidsfromendA+bacteria. •Ifyouareusingacommercialplasmidpreparationkit,followthemanufacturer’sdirectionsforhoststrainsthat

areendA+. •IfyouplantousetheplasmidforsequencingorotherapplicationsrequiringhighlypurifiedDNA,theplasmid

shouldbeextractedwithphenol:chloroform:isoamylalcoholandprecipitatedwithethanolbeforeuse.Alternatively,CHROMASPINTM+TE-400Columns(CatNo.K1323-1)maybeusedtopurifytheplasmid.

b. Toverifythatyouhaveobtainedthecorrectplasmid,amplifytheinsertbyPCR,digestitwithAluIorHaeIII,andrunasmallsampleonanagarose/EtBrgel.Comparetherestrictiondigestionpatternwiththatoftheoriginalcloneisolatedfromyeast.

4.Troubleshootingtips a. Ifyoudonotobtainanytransformants,youmayneedtoimprovethetransformationeffiency

ofthecells.

• IfyouperformedanutritionalselectiononM9minimalmedium,repeatthetransformation,butplatethecellsonLB/ampinstead.(TherecoveryofnewtransformantsisgenerallybetterononLB/ampthanonM9medium.)ThenreplicaplatetheAmprtransformantstotheappropriateM9minimalmediumforselectionofthedesiredplasmidandtoverifythattheundesiredplasmid(s)havebeenlost.NotethatittakessomewhatlongertoseecoloniesonM9mediumthanonLB.

• If you are not already doing so, use electrotransformation rather than chemicaltransformation; higher transformation efficiencies are usually obtained withelectroporation.

• Usecompetentcellsthatareknowntobetransformedwithaveryhighefficiency.(BothchemicallycompetentandelectrocompetentcellsareavailablefromClontech.)

b. IfyoutrythemeasuresrecommendedinSection4.aaboveandstilldonotrecoverany E. coli transformantstheproblemmaybetheplasmidpreparationortheplasmiditself.

• TheyeastplasmidpreparationmayhavenoplasmidDNAinit.Checkthemediumyouusedfortheovernightcultures.Itisimportanttouseamediumthatmaintainsselectiononthedesiredplasmid.Theworkingstockplateusedasyourinoculumsourceshouldalsokeepselectionontheplasmid.Whenyourepeattheplasmidisolationprocedure,besuretoincludethefreeze/thawcycleatStepVII.B.5toensurecompletecelllysis.

• ChecktheconcentrationoftotalDNAinyourplasmidprepusingabsorbanceat260nmorbyrunningasmallsample(10µl)onagel.AlthoughplasmidDNAmakesuponlyasmallfractionofthetotalDNA,youcanatleastconfirmthatyouhaveDNAinyourprep.ThelargerchromosomalDNAfragmentsshouldbevisibleona1%agarose/EtBrgel.(ThelimitofdetectionwithEtBrstainingis~4ng[Sambrooket al.,1989,AppendixE.5].)

• EvenifyouhaveasubstantialamountofDNAinyourprep,thereisaremotepossibilitythattheplasmidofinteresthasintegratedintotheyeastchromosomeandthereforecannotreplicateautonomouslywhenintroducedintoE. coli.Iftheplasmid’sinsertcanbeamplifiedbyPCR(ChapterVIII),itmaybepossibletorecovertheinsertbysubcloningfromthePCRproduct.

• TheplasmidmayencodeaproteinthatistoxictoE. coli. Again,itmaybepossibletorecovertheinsertbysubcloningthePCR-amplifiedfragment.





Sometimesatwo-hybridlibraryscreeningresultsinmany,evenhundreds,ofpositivecandidateclones.However,afewabundantinsertsequencesmayaccountforthemajority.Sortingcoloniesintogroupswilleliminateduplicatesbearing thesameplasmid insertandwillsave time in thesubsequentanalysis.ThecDNAinsertsfromallplasmidsencodingcandidateinteractingproteinscanbeamplifiedbyPCRandsorted intogroupsbasedon restrictiondigestionpatterns.Aftercolonieshavebeensorted,arepresentativeclonefromeachgroupcanbetransferredtoanewmasterplateforfurtheranalysis.

Toensureefficientamplificationofallinserts,regardlessofsize,westronglyrecommendtheuseoflong-distance(LD)PCR(Barnes,1994;Chenget al.,1994)withtheAdvantage®2PolymeraseMix (CatNo.639201).TheAdvantage®2PCRKit (CatNo.639206)providesaAdvantage®2PolymeraseMix(whichincludesTaqStartTMAntibody),a10XAdvantage®2PCRBuffer,50XdNTP,apositivecontroltemplate,amixofpositivecontrolprimers,andacompleteUserManual.

ClontechoffersPCRprimersdesignedtoamplifyinsertsclonedintoMatchmakerTwo-HybridSystemvectors.Theinsert-screeningamplimershybridizetosequencesflankingthemultiplecloningsite(MCS)oftherespectivevectors.IfyoupurchaseMatchmakerLD-InsertScreeningAmplimers,werecommendthatyouusetheLD-PCRprotocolthataccompaniesthatproduct.However,LD-InsertScreeningAmplimerscanalsobeusedinconventionalPCRusingasingleDNApolymerasetoamplifyinsertsupto3kb(e.g.,Ausubelet al.,[1995]Chapters15.1&15.3).

• MatchmakerADLD-InsertScreeningAmplimers(CatNo.630433)areforamplifyinginsertsintheGAL4ADcloningvectorspGAD10,pGAD424,pGADGL,pGADGH,pGADT7,pACT,andpACT2.

• MatchmakerpB42ADLD-InsertScreeningAmplimers(CatNo.9108-1)areforamplifyinginsertsintheLexAsystemADcloningvectorpB42AD.

• MatchmakerDNA-BDVectorInsertScreeningAmplimers(CatNo.5417-1)areforconventionalPCRamplificationofinsertsintheGAL4DNA-BDcloningvectorspGBT9,pGBKT7,pAS2,andpAS2-1.

• MatchmakerLexADNA-BDInsertScreeningAmplimers(CatNo.9109-1)areforconventionalPCRamplificationofinsertsinpLexAandpGilda.

B. TipsForSuccessfulPCRofYeastPlasmidTemplates 1.Optimizationofthermalcyclingparameters Theoptimalcyclingparameterswillvarywithdifferenttemplates,primers,experimentalprotocols,

tubes,andthermalcyclers.RefertotheLD-InsertScreeningAmplimersUserManual,Ausubelet al.(1995),orRoux(1995)forsuggestionsonoptimizingPCRconditions.Insomecases,“touchdown”PCRmaybeneeded.WehavefoundthattouchdownPCRsignificantlyimprovesthespecificityofmanyPCRreactionsinawidevarietyofapplications(Donet al.,1991;Roux,1995). Briefly, touchdown PCR involves using an annealing/extension temperature that isseveraldegrees(typically3–10°C)higher thantheTmof theprimersduringthe initialPCRcycles(typically5–10).Theannealing/extensiontemperatureisthenreducedtotheprimerTmfortheremainingPCRcycles.Thechangecanbeperformedeitherinasinglesteporinincrementsoverseveralcycles;forexample,use72°Cforthefirstfivecycles,70°Cforthenext5cycles,and68°Cfortheremainingcycles.

2.Primerdesign PrimerdesignisthesinglelargestvariableinPCRapplicationsandthesinglemostcritical

factorindeterminingthesuccessorfailureofPCRreactions.Forbestresults,werecommendthatyouuseLD-Insert-ScreeningAmplimersfromClontech.However,ifyoudesignyourownprimers,besuretousesequencesflankingtheMCS.Always check and recheck your primer design before constructing or ordering primers.

LengthandG/Ccontent:Ingeneral,primersshouldhaveaTmofatleast70°Ctoachieveoptimalresultsinatwo-stepcyclingprogramwitha68°Cannealing/extensionstep.Therefore,whenever possible, primers should be at least 22 nucleotides (nt) long (25–30-mers arepreferred)andshouldhaveaGCcontentof45–60%.

VIII. AnalysisofYeastPlasmidInsertsbyPCR



3.Thermostablepolymerase TheAdvantage®2PolymeraseMixesaredesignedforLDPCR—i.e.,theycontainbothprimary

andproofreadingpolymerasestopermitamplificationofvirtuallyanyinsert,regardlessofsize.IfyoudonotuseanAdvantagePolymeraseMix,youwillneedtoprepareyourownpolymerasemixfromcommerciallyavailable,LDPCR-licensedDNApolymerases,suchasTaqorAmpliTaq®.WealsostronglyrecommendthatyouincludeTaqStartAntibodyinthepolymerasemix,forautomatichotstart(seeSectionB.4below).(TaqStartAntibodyispremixedintheAdvantage2PolymeraseMix.)

4.Useofantibody-mediatedorconventionalhotstart To minimize nonspecific amplification, we strongly recommend that you perform hot start

PCR.ThereareseveralmethodsavailableforhotstartPCR,includingthoseusingwaxbeads(Chou et al., 1992) or a manual hot start (D’Aquila et al., 1991). TaqStart Antibody(CatNo.639250,639251)providesanautomatichotstartwhenusedwithTaq orKlenTaqDNAPolymerase(Kellogget al.,1994).Antibody-mediatedhotstartwithTaqStartAntibodyismoreconvenientthanmanualhotstartorwax-bead-mediatedhotstart,andhasbeenproventobeatleastaseffectiveastheconventionalmethods.

5.Templatequality a. BecauseoftheexponentialnatureofPCRamplification,manyconventionalPCRapplications

suchasscreeningcDNAinsertsworkwellwithtemplatesofaverageorevenlowquality,includingplasmidDNAisolatedfromyeast.Use1-2µlofyeastplasmidDNApreparation(fromSectionVII.B)perPCR.

b. Besure touseasingle, well-isolatedyeastcolonywhen inoculating liquidcultures forpreparationofplasmidfromyeast(ChapterVII.B).

c. Iftheyeasttransformantcontainsmorethanoneplasmidinsertsequence,youmayseemultiplePCRbands.RestreaktheyeasttransformantontheappropriateSDmediumthatmaintainsselectiononthedesiredplasmid(s)butnotontheirinteractions(AppendixE).Theextragenerationsofgrowthwillallowsegregation(i.e.,loss)ofsomeoftheplasmids.Afterreconfirmingthepresenceofpositiveplasmidsusingaβ-galcolony-liftassay,repeattheplasmidisolationandPCRanalysisonwell-isolatedcolonies.Insomecases,itmaybenecessarytotransformE. coliwiththeyeastplasmidprep,andisolateplasmidfrom E. colitransformantstoensureahomogeneousplasmidpreparation(ChapterVII.C).

6.TipsforcharacterizingPCRproducts a. Electrophorese10µlsamplesofthePCRproductonanEtBr/0.8%agarosegeltoconfirmthat

thePCRworkedandtodetermineiftheplasmidprepcontainsmultiple(nonhomogeneous)plasmids.

b. Digest another 10-µl sample of each amplified insert with a frequent-cutter restrictionenzyme,suchasAluIorHae III,ina20-µlvolumereaction.RunthesesamplesonanEtBr/1.8%agarosegelinparallelwithDNAsizemarkersforcomparison.

7.GoodPCRpractices a. Preparereactionswithdedicatedpipettorsinadedicatedworkspace Due to the tremendousamplificationpowerofPCR,minuteamountsofcontaminating

DNAcanproducenonspecificamplification;insomeinstances,contaminantscancauseDNAbandsevenintheabsenceofaddedtemplateDNA.WerecommendthatyousetupyourPCRreactionsinadedicatedlabareaornoncirculatingcontainmenthoodandusededicatedpipettors,PCRpipette tipswithhydrophobicfilters,anddedicatedsolutions.Perform post-PCR analysis in a separate area with a separate set of pipettors.

b. Pipetting BecauseofthesmallvolumesusedinPCRexperimentsandthepotentialfortube-to-tube

variation,carefulpipettingtechniqueisextremelyimportant.Alwaysbesurethatnoextrasolutionisontheoutsideofthepipettetipbeforetransfer.Whenaddingsolutiontoatube,immersethetipintothereactionmixture,deliverthesolution,andrinsethepipettetipbypipettingupanddownseveraltimes.

VIII. AnalysisofYeastPlasmidInsertsbyPCRcontinued



VIII. AnalysisofYeastPlasmidInsertsbyPCRcontinued

c. UseaMasterMix Toreducetube-to-tubevariation,useamastermixwheneveryousetupmultiplePCR

reactions.Ifyouwish,includetheprimersinthemastermixalso.Ifyouaresettingupseveralsetsofparallelsamples,assemblemultiplemastermixes(e.g.,eachwithadifferentsetofprimers).The master mix should be thoroughly mixed before use (i.e., vortexed without bubbling).

d. Alwaysincludepositiveandnegativecontrols(i.e.,H2OinsteadofDNAtemplate).PositivecontrolsareprovidedwithallofClontech’sInsertScreeningAmplimerSets.



IX. AdditionalUsefulProtocols

A. YeastColonyHybridization

YeastcolonyhybridizationisanefficientwaytoscreenalargecollectionoflibrarytransformantsforthepresenceofanabundantcDNAinsert.Duplicatecoloniesbearingthesamelibraryplasmidcanthenbeeliminatedfromfurtheranalysis.WehavehadsuccesswiththismodificationoftheclassicprotocolofGrunsteinandHogness(1975;Kaiser,et al.,1994;Ausubelet al.,1994).Inthisprocedure,coloniesaredirectlyliftedontoanylonmembrane.β-glucuronidaseisusedtobreakcellwalls.

ReagentsandMaterialsRequired • AppropriateSDagarplatesthatwillkeepselectionontheplasmid(s)ofinterest(AppendixEor

thesystem-specificUserManual) • LabeledcDNAprobecomplementarytopreviouslyisolatedcDNAs

Note:Oligonucleotides,random-primedcDNAs,orPCR-generatedfragmentscanbeusedasprobes.OligonucleotideprobesmaybeadvantageousifthecDNAisamemberofaproteinfamilytoavoidinadvertentlyexcludingrelatedgenesthatarenotidenticaltothoseinitiallyobtained.

• 1Msorbitol/20mMEDTA/50mMDTT(preparefresh) • 1Msorbitol/20mMEDTA • 0.5MNaOH • 0.5MTris-HCl(pH7.5)/6XSSC(Ausubelet al.,1994) • 2XSSC(Ausubelet al.,1994) • 100,000units/mlβ-glucuronidase(typeHP-2crudesolutionfromHelixpomatia; SigmaCatNo.G-7017) • 82-mmcircularnylonmembrane,sterile • Whatman3MMpaper • 80°CvacuumovenorUVcross-linker • Additionalreagentsandequipmentforbacterialfilterhybridization(Ausubelet al.,1994)

1.Ifyouhavenotdonesoalready,collectthecoloniestobescreenedontoamasterplateinagridpatterntofacilitatefutureidentificationofthecolonies.Includeapositiveandnegativecontroloneachplate.Sincethiswillbeyourmasterplate,itisimportanttousetheappropriateSDagarmediumtomaintainselectiononallplasmids(includinganyreporterplasmid).Incubateplateat30°Cfor2–4daysuntilcoloniesappear.

2.Preparesorbitol/EDTA/DTTsolution. 3.Foreachplateofcoloniestobescreened,presoakaWhatman3MMpaperinthesorbitol/

EDTA/DTTsolution. 4.Using forceps, place a sterile, prelabeled, dry nylon membrane over the surface of the

plateofcoloniestobeassayed.Gentlyrubthemembranewiththesideoftheforcepstohelpcoloniesclingtothemembrane.

5.Pokeholesthroughthemembraneintotheagarinthreeormoreassymetriclocationstoorientthemembranetothemasterplate.

6.Whenthemembranehasbeenevenlywetted,carefullyliftitofftheagarplatewithforcepsandallowittoairdrybriefly(~5min).Placemembrane,colony-side-up,onapresoakedsheetofWhatman3MMpaper(fromStep3above)andincubatefor~30min.

7.Optional:Placemembranesat–70°Cfor5min,thenthawatroomtemperatureforoneormorecyclestofacilitatethedisruptionofthecellwalls.

8.Dilute the β-glucuronidase 1:500 in sorbitol/EDTA. Use 2 µl (of the 100,000 units/ml β−glucuronidasestock)permlofsorbitol/EDTAtogiveafinalconcentrationof200units/ml).Allow3–5mlofdilutedβ-glucuronidaseperfiltertobescreened.

9.Foreachmembranetobescreened,cutanotherpieceofWhatman3MMpapertofitinsidea100-mmpetridish.Placethepaperdiscinthedishcontainingthedilutedβ-glucuronidasetosaturatethepaper.Removeexcessliquid.

10.Carefullylayerthenylonmembrane,colonysideup,ontopoftheβ-glucuronidase-soakedfilter.Avoidtrappingairbubblesinbetweenthetwolayers.Coverthedish.Incubatethemembraneonthefilterforupto6hrat37°Cuntil>80%ofthecellslackacellwall.

Note:Theextentofcellwallremovalcanbedeterminedbyremovingasmallquantityofcellsfromthefiltertoadropofsorbitol/EDTAonamicroscopeslide,andobservingdirectlywithaphase-contrastmicroscopeat≥60Xmagnification.Cellslackingacellwallarenonrefractile.



11.PlacemembraneonWhatman3MMpapersaturatedwith0.5MNaOHfor8–10min. 12.Place membrane on Whatman 3 MM paper saturated with 0.5 M Tris-HCl (pH 7.5)/6X

SSCfor5min.Repeatstep12withasecondsheetofpresoakedWhatman3MMpaper. 13.PlacemembraneonWhatman3MMpapersaturatedwith2XSSC for5min.Thenplace

membraneondryWhatmanpapertoairdryfor10min. 14.Bakemembraneat80°Cfor90mininavacuumovenorUVcross-link. 15.Proceedasforbacterialfilterhybridization(Ausubelet al.,1994).

B. GeneratingYeastPlasmidSegregants

For some applications, it is useful to generate a segregant strain that has only a single type ofplasmid fromyeastcotransformantscontainingmore thanonekindofplasmid.Thereareseveralwaysthiscanbeaccomplished.Themostreliablebutalsomosttime-consumingwayistoisolatethe mixed plasmid DNA from yeast, use it to transform E. coli, isolate the desired plasmid from E. coli transformants,and transform thedesiredyeasthost strainwith the isolatedplasmidDNA.Alternatively,theyeastcotransformantstraincanbegrownforseveralgenerationsonSDmediumthatmaintainsselectiononthedesiredplasmidonly,asdescribedinSectionB.1below.Thesearchfor yeast segregants can be significantly accelerated if you are working with a cycloheximide-resistant yeast host strain and the unwanted plasmid confers sensitivity to cycloheximide,as described in Section B.2 below. Cycloheximide counterselection is an option with theMatchmakerTwo-HybridSystem2(CatNo.K1604-1),butcannotbeusedwiththehoststrainsprovidedwithPretransformedMatchmakerLibrariesortheoriginalMatchmakerSystem(CatNo.K1605-1).

1.Segregationbynaturallossofanunselectedplasmid a. Cultureindividualcotransformantcolonies(separately)in3mloftheappropriateSDliquid

selectionmediumfor1–2daysat30°Cwithshaking(230–250rpm).Themediummustmaintainselectionontheplasmidofinterest,butnotontheplasmidyouwishtolose.Undertheseconditions,theplasmidsthatarenotselectedforarelostatarateof10–20%pergeneration.RefertoAppendixEforinformationonyeastplasmidtransformation/selectionmarkers.

b. Spreadadilutedsampleofthisliquidcultureonagarplatesthatwillselectforthedesiredplasmid.

c. Incubatetheplateat30°Cfor2–3daysoruntilcoloniesappear. d. Usingsteriletoothpicksorpipettetips,transfer20–30individualcolonies(inanorderly

gridfashion)toappropriateSDselectionplatestoverifythattheyhavelosttheunwantedplasmidandretainedtheplasmidofinterest.

Note:StoretheyeastsegregantsontheappropriateSDselectionplateswrappedinParafilmat4°Cforuptotwoweeks.

IX. AdditionalUsefulProtocolscontinued



2.Cycloheximidecounterselectionofyeastsegregants Someyeasthoststrains,suchasCG-1945andY190,carrythecyhr2 mutantalleleandare

cycloheximideresistant(CyhR;C.Giroux,personalcommunication,forCG-1945,andHarperet al.,1993,forY190).Thewild-typeCYHs2 geneisdominanttothecyhr2 mutantallele.Thus,whentransformedwithaplasmidsuchaspAS2-1thatcontainsthewild-typeCYHs2gene,thehoststrainwillbecomesensitivetocycloheximide;thisholdstrueforaCyhRhoststraincotransformedwithaCYHs2-bearingplasmidandanotherplasmidthatdoesnotcarrytheCYHs2.gene.Therefore,onecaneffectivelyselectforyeastcellsthathavespontaneouslylosttheCYHs2-bearingplasmidwhileretainingtheotherplasmid,simplybyplatingthecotransformantsontheappropiateSDmediumcontainingcycloheximide.

Note:TheCYH2geneencodestheL29proteinoftheyeastribosome.Cycloheximide,adrugwhichblockspolypeptideelongationduring translation,prevents thegrowthof cells that contain thewild-typeCYH2 gene.CycloheximideresistanceresultsfromasingleaminoacidchangeintheCYH2protein.Cellscontainingboththesensitive(wild-type)andtheresistant(mutant)CYH2allelesfailtogrowonmediumcontainingcycloheximide.Therefore,thelossofaCYH2-containingplasmidcanbeselectedfordirectlyifthehostcarriestheresistantallelechromosomally(Guthrie&Fink[1991],pp306–307).

a. Fromeachoftherestreaked(CyhS)cotransformantsofinterest,pickacolony,1–3mmindiameter,andresuspenditin200µlofsterileH2O.Vortexthoroughlytodispersethecells.

Note:Donotpatchorstreakcellsfromthecolonyovertothecycloheximide-containingmedium.Cellstransferredinthiswayareattoohighadensityforthecycloheximideselectiontowork.

b. Spread100µlofthecellsuspensionontoanSD/–Leu/+cycloheximideplate.Alsospread100µlofa1:100dilution.

Note:Theconcentrationofcycloheximidetouseinthemediumdependsonthehoststrain.Forexample,use1.0µg/mlforCG-1945;10.0µg/mlforY190.

c. Incubatetheplateat30°CuntilindividualCyhRcoloniesappear.(Thisusuallytakes3–5days.) d. TransfertheCyhRcoloniestoappropriateSDselectionplatestoverifythattheyhavelost

theCYHs2-bearingplasmidandretainedtheplasmidofinterest.RefertoAppendixEforinformationonyeastplasmidtransformation/selectionmarkers.

Note:TheseyeastclonesarereferredtoasCyhRsegregants.StorethemontheappropriateSDselectionplateswrappedinParafilmat4°Cforuptotwoweeks.

C. YeastMating

Yeastmatingisaconvenientmethodofintroducingtwodifferentplasmidsintothesamehostcells,and, insomeapplications,canbeusedasaconvenientalternativetoyeastcotransformations(Bendixenet al.,1994;Harperet al.,1993;Finley&Brent,1994).SeeGuthrie&Fink(1991)orPringleet al. (1997) for informationon thebiologyof yeastmating.The following small-scaleprotocolworkswellforcreatingdiploidsbyyeastmating.IfyouwishtoscreenaPretransformedMatchmakerLibaryusingyeastmating,pleaserefertotheUserManualprovidedwiththoselibrariesforanoptimized,library-scalematingprotocol.

1.Preparationforyeastmating a. Ifyouhavenotdonesoalready,generateanappropriateyeaststraincontainingtheplasmid

ofinterest. b. Transform thechosenmatingpartnerseparatelywith theplasmidsyouwish to test in

combination with the plasmid of interest. Be sure to include transformations with theappropriatenegativeandpositivecontrolplasmids,ifapplicable.

c. SelectfortransformantsontheappropriateSDdropoutmedium. d. Foreachplasmidofinteresttobetested,setuppairwiseyeastmatingswithtransformants

containing control plasmids. Use either the standard procedure (Section C.2) or theprocedureadaptedformicrotiter(96-well)plates(SectionC.3).

2.Yeastmatingprocedure(standard) a. Pick one colony of each type to use in the mating. Use only large (2–3-mm), fresh

(<2-monthsold)coloniesfromtheworkingstockplates. b. Placebothcoloniesinone1.5-mlmicrocentrifugetubecontaining0.5mlofYPDmedium.

Vortextubestocompletelyresuspendthecells.




c. Incubateat30°Covernight(20–24hr)withshakingat200rpm. d. Spread100-µlaliquotsofthematingcultureontheappropriateSDminimalmedia.Use

doubledropouttoselectforbothplasmidsandtripledropouttoselectfordiploidsinwhichapositivetwo-hybridinteractionisoccurring.Proceedtostep4below.

3.Yeastmatingprocedure(microtiterplateversion) Ifyouhavemanyplasmidsofinteresttomatetoseveralcontrolstrains,itmaybemoreefficient

tosetupthematingsinseparatewellsofasterile,flat-bottommicrotiterplate.Inbetweensteps,keepplatecoveredwithasterilelid.

a. Aliquot160µlofYPDmediumtoeachwell. b. For each plasmid of interest to be tested, place a single transformant colony in a 1.5-ml

microcentrifugetubecontaining1mlofYPD.Vigorouslyvortexthetubetodispersethecells. c. Foreachtypeofcontrolplasmidtobeused,placeseveraltransformantcoloniesin3ml

ofYPDinasterile,10-mlconicaltube.Vigorouslyvortexthetubetodispersethecells. d. Aliquot20µlofthecellsuspensionfromStep3.bintoeachwellofaverticalcolumn.Use

aseparatecolumnforeachplasmidofinteresttobetested. e. Aliquot20µlofthecellsuspensionfromStep3.cintoeachwellofahorizontalrow.Use

aseparaterowforeachtypeofcontrolplasmid. f. Placeplateonarotatingplatformshakerandincubateat30°Cfor6–18hrat200rpm. Note:Donotrotateatahigherspeedorthemediumwillspilloutofthewells.

g. Spread 100 µl of each mating culture on 100-mm plates containing the appropriateSDminimalmediumandproceedtonextstep.

4.Incubateplatesat30°Cfor3–5daystoallowdiploidcellstoformvisiblecolonies. 5.ScoreforgrowthontheSDagarplates. 6.Confirmnutritionalandreporterphenotypesofdiploids Todetect(orreconfirm)protein-proteininteractions,assaythefreshdiploidcoloniesfromthe

SDselectionplates(Step4)aboveforβ-galactivityusingthecolony-liftfilterassay(SectionVI.C).Discardanyβ-gal-positivecoloniesthatcontainthecandidatelibraryplasmidalone.




X. References

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Bartel,P.L,Chien,C.-T.,Sternglanz,R.&Fields,S.(1993b)Eliminationoffalsepositivesthatariseinusingthetwo-hybridsystem.BioTechniques 14:920–924.

Brent,R.&Ptashne,M.(1985)AeukaryotictranscriptionalactivatorbearingtheDNAspecificityofaprokaryoticrepressor.Cell43:729–736.

Chien,C.T.,Bartel,P.L.,Sternglanz,R.&Fields,S.(1991)Thetwo-hybridsystem:Amethodtoidentifyandclonegenesforproteinsthatinteractwithaproteinofinterest.Proc. Nat. Acad. Sci. USA 88:9578–9582.

Fields,S.&Song,O.(1989)Anovelgeneticsystemtodetectprotein-proteininteractions.Nature340:245–247.

Fields,S. (1993)The two-hybrid system todetect protein-protein interactions.METHODS: A Companion to Meth. Enzymol.5:116–124.

Fields,S.&Sternglanz,R.(1994)Thetwo-hybridsystem:anassayforprotein-proteininteractions.Trends Genet.10:286–292.

Fritz,C.C.&Green,M.R.(1992)Fishingforpartners.Current Biol.2:403–405.

Golemis,E.A.,Gyuris,J.&Brent,R.(1996)Analysisofproteininteractions;andInteractiontrap/two-hybridsystemstoidentifyinteractingproteins.InCurrent Protocols in Molecular Biology (JohnWiley&Sons,Inc.),Chapters20.0and20.1.

Golemis,E.A.,Gyuris,J.&Brent,R.(1994)Interactiontrap/two-hybridsystemstoidentifyinteractingproteins.InCurrent Protocols in Molecular Biology(JohnWiley&Sons,Inc.),Ch.13.14.

Guarente,L.(1993)Strategiesfortheidentificationofinteractingproteins.Proc. Natl. Acad. Sci. USA90:1639–1641.

Gyuris,J.,Golemis,E.,Chertkov,H.&Brent,R.(1993)Cdi1,ahumanG1andSphaseproteinphosphatasethatassociateswithCdk2.Cell 75:791–803.

Luban,J.&Goff,S.P.(1995)Theyeasttwo-hybridsystemforstudyingprotein-proteininteractions.Curr. Opinion in Biotechnol.6:59–64.

Mendelsohn,A. R. & Brent, R. (1994) Biotechnology applications of interaction traps/two-hybrid systems. Curr. Opinion in Biotechnology 5:482–486.

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Alexandre,C.,Grueneberg,D.A.&Gilman,M.Z.(1993)StudyingHeterologousTranscriptionFactorsinYeast.METHODS: A Companion to Methods in Enzymology 5:147–155.

Ammerer,G.(1983)ExpressionofGenesinYeastUsingtheADCIPromoter.Methods in Enzymology101:192–201.

Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.,Struhl,K. (1994)Current Protocols inMolecular Biology (JohnWiley&Sons,Inc.)Vol.1,Chap.5.

Barnes,W.M.(1994)PCRamplificationofupto35-kbDNAwithhighfidelityandhighyieldfromλbacteriophagetemplates.Proc. Natl. Acad. Sci. USA 91:2216–2220.

Beier,D.R.&Young,E.T.(1982)CharacterizationofaregulatoryregionupstreamoftheADR2locusofS. cerevisiae.,Nature300:724–728.

Bendixen,C.,Gangloff,S.&Rothstein,R.(1994)Ayeastmating-selectionschemefordetectionofprotein-proteininteractions.Nucleic Acids Res.22:1778–1779.

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Breeden,L.&Nasmyth,K.(1985)RegulationoftheYeastHOGene.Cold Spring Harbor Symposium Quant. Biol.50:643–650.

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Campbell,K.S.,Buder,A.,Deuschle,U.(1995)Interactionofp56lckwithCD4intheyeasttwo-hybridsystem.Ann NY Acad Sci.766:89-92.

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Chien,C.T.,Bartel,P.L.,Sternglanz,R.&Fields,S.(1991)Thetwo-hybridsystem:Amethodtoidentifyandclonegenesforproteinsthatinteractwithaproteinofinterest.Proc. Nat. Acad. Sci. USA, 88:9578–9582.

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Chuang,S.-E.,Chen,A.-L.&Chao,C.-C.(1995)GrowthofE. coli atlowtemperaturedramaticallyincreasesthetransformationfrequencybyelectroporation.Nucleic Acids Res.23:1641.

D’Aquila,R.T.,Bechtel,L.J.,Videler,J.A.,Eron,J.J.,Gorczyca,P.&Kaplan,J.C.(1991)Maximizingsensitivityandspecificity



X. Referencescontinued

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Ebina,Y.,Takahara,Y., Kishi, F. & Nakazawa,A. (1983) LexA protein is a repressor of the colicin E1 gene. J. Biol. Chem.258:13258–13261.

Estojak,J.,Brent,R.&Golemis,E.A. (1995)Correlationof two-hybridaffinitydatawith in vitro measurements.Mol. Cell Biol.15:5820–5829.

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Flick,J.S.&Johnston,M.(1990)TwosystemsofglucoserepressionoftheGAL1promoterinSaccharomyces cerevisiae. Mol. Cell. Biol.10(9):4757–4769.

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Giniger,E.,Varnum,S.M.&Ptashne,M.(1985)SpecificDNAbindingGAL4,apositiveregulatoryproteinofyeast.Cell40:767–74.

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Kellogg,D.E.,Rybalkin,I.,Chen,S.,Mukhamedova,N.,Vlasik,T.,Seibert,P.&Chencik,A.(1994)TaqStartAntibody:HotstartPCRfacilitatedbyaneutrualizingmonoclonalantibodydirectedagainstTaqDNApolymerase.BioTechniques16:1134–1137.

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Ling,M.,Merante,F.&Robinson,B.H.(1995)ArapidandreliableDNApreparationmethodforscreeningalargenumberofyeastclonesbypolymerasechainreaction.Nucleic Acids Res.23:4924–4925.



X. Referencescontinued

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XI. MatchmakerandRelatedProducts

ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com

Generalreagentsforworkwithyeast • YeastmakerTMYeastTransformationSystem 630439 • YeastmakerTMCarrierDNA 630440 • YeastmakerTMYeastPlasmidIsolationKit 630441 • YPDMedium 630409 • YPDAgarMedium 630410 • MinimalSDBase(containsglucose) 630411 • MinimalSDAgarBase(containsglucose) 630412 • MinimalSDBase/Gal/Raf(containsgalactose&raffinose) 630420 • MinimalSDAgarBase/Gal/Raf(containsgalactose&raffinose) 630421 • Dropout(DO)SupplementsforusewithanySDBase many

GAL4-basedone-andtwo-hybridsystemsandrelatedproducts • MammalianMatchmakerTwo-HybridAssayKit 630301 • MatchmakerOne-HybridSystem 630302 • MatchmakerTwo-HybridSystem3 630303 • MatchmakerCo-IPKit 630449 • pCMV-Myc&pCMV-HAVectorSet 631604 • MatchmakercDNA&GenomicLibraries many • MatchmakerPretransformedLibraries many • MatchmakerADLD-InsertScreeningAmplimerSet 630433 • GAL4ADMonoclonalAntibody 630402 • GAL4DNA-BDMonoclonalAntibody 5399-1 • pGBKT7DNA-BDVector 630403 • pGADT7ADVector 630442 • pLP-GBKT7DNA-BDCreatorTMAcceptorVector 630406 • pLP-GADT7ADCreatorTMAcceptorVector 630405 • pBridgeTMThree-HybridVector 630404

Generalcloningreagents • QUICK-CloneTMcDNA many • GenomicDNA many • E. coliKC8ElectrocompetentCells 630435 • E. coliKC8ChemicallyCompetentCells 630434

Miscellaneousrelatedreagents • X-α-Gal 630407 • Luminescentβ-galDetectionKitII 631712 • Advantage®2PCRKit 639206 639207 • Advantage®2PolymeraseMix 639201 639202 • CHROMASPINTMTE-400Columns 636076


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3024-1 �0 Version No. PR13103

APPENDIXA.GlossaryofTechnicalTerms

Note: Many of these terms have other meanings in different contexts. For brevity, we have included only definitions relevant to this Yeast Protocols Handbook.

allele:Oneoftwoormoreformsthatcanexistatagivengeneticlocus(e.g.,his3-200 isamutantalleleandHIS3 isawild-typealleleatthehis3 locus).Instandardyeastnomenclature,mutantallelesarewritteninlowercaseitalics,whilewild-typeallelesarewritteninuppercaseitalics.

auxotroph:Astrainofyeastorothermicroorganisms thatwillproliferateonlywhen themedium issupplementedwithsomespecificnutrientnotnormallyrequiredbytheorganism.Forexample,Trp–yeaststrainsareauxotrophicfortryptophan(Trp);theyrequireTrpinthemedium.

cis-actingelement(orcis-actinglocus):ADNAsequencethataffectsthetranscriptionalactivityofgeneslocatedonthesameDNAmolecule,oftenviabindingofregulatoryproteinsorfactors.

confluent:Whenyeastorbacterialcoloniesgrowingonanagarplatearesonumerousthattheedgesofthecoloniestoucheachother.

clone:(a)Agroupofgeneticallyidenticalcellsorindividualsderivedbyasexualdivisionfromacommonancestor.(b)AheterologouscDNAfragmentinsertedintoavector;alsoreferstocopiesofthatoriginalcDNA.

colony:Avisiblecloneofcellsgrowingonsolidmedium.

diploid:Inyeast,acellhavingtwocompletechromosomesetsasaresultofmatingofhaploidaandαstrains.Acellcanalsobediploidforoneparticulargeneorseveralgenes,duetothepresenceofplasmids,orasaresultofgeneduplication.

dropout (DO)supplement:Amixtureofseveralaminoacidsandnucleosides thatmustbeaddedto minimal synthetic medium to support the growth of yeast strains that have defined nutritionalrequirements;typically,oneormorespecificnutrientsisleft(or“dropped”)outoftheDOsupplementsothattheresultingsyntheticdropout(SD)mediumwillonlysupportthegrowthofyeastthatareabletosynthesizethatnutrient.

gene:(a)Thefundamentalphysicalunitofheredity,recognizedthroughitsvariantalleles;(b)aDNAsequence that regulates and encodes a functional product, e.g., a polypeptide chain or an RNAmolecule.

geneticcomplementation:Theproductionofawild-typephenotypewhen(a)twodifferentmutationsarecombinedinadiploidcell;or(b)whenawild-typealleleonaplasmidisintroducedintoacellbearingadefectivechromosomalalleleviayeastmatingortransformation.

genome:TheentirecomplementofgeneticmaterialinacellexcludingautonomouslyreplicatingplasmidsandmitochondrialDNA.

genotype:Generally,alistofmutantallelesandexogenousgeneticelements.Wild-typeallelesaresometimeslistedaswellforclarityinaspecificexperimentalcontext.

haploid:Acellhavingonechromosomeset.Adiploidcellororganismcanalsobehaploidforagivengeneduetochromosomaldeletions.

hybridizationprobe:AdefinednucleicacidsegmentwhichcanbelabeledandusedtoidentifyspecificDNAclonesbearingthecomplementarysequenceviahybridization.

leakymutant:Amutantthatrepresentsapartialratherthanacompleteinactivationofthewild-typefunction;leakyphenotypescanresultfromamutationinthecodingregionorinthepromoterregion.Inyeastone-and two-hybridsystems,someof thehoststrainsare leaky forexpressionofcertainauxotrophicmarkers(forexample,HIS3expressioninY190).

matingtypes:Ageneticallyhaploidstateofunicellularorganismsthatcanreproducesexuallybycellularandnuclearfusiontoproduceadiploidorganism.InS. cerevisiae,therearetwomatingtypes,aandα,whichdifferonlyphysiologicallyandnotinphysicalform.

mutant:Anorganismorcellcarryingamutation.

mutantallele:Analleledifferingfromtheallelefoundinthestandardorwildtype.


Protocol No. PT3024-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR13103 �1

mutation:(a)Theprocessthatproducesageneorachromosomedifferingfromthewildtype.(b)TheDNAoraminoacidchangeresultingfromsuchaprocess.

operator:Inbacteria,aDNAregionthatactsabindingsiteforaspecificrepressorproteinandtherebyexertscontrolovertranscriptionoftheadjacentstructuralgeneoroperon.

operon:Inbacteria,asetofadjacentstructuralgenesthataretranscribedintoasinglemRNAmolecule,plustheadjacentregulatorygenesthataffecttranscriptionofthestructuralgenes.

PCR: Polymerase chain reaction; a process by which a defined segment of DNA is exponentiallyreplicatedin vitro bytheactionofathermostableDNApolymeraseduringrepeatedcyclesofheatingandcooling.

phenotype:Theobservablepropertiesofanorganismdeterminedbytheorganism’sgeneticconstitution(genotype)andtheeffectsoftheenvironment.

plasmid:Ageneticelementinbacteriaoryeastthatcanreplicateautonomouslyinthehostcell.Someplasmidscanalsobeinsertedintothehost’sgenomeindefinednaturalorexperimentalsituations,e.g.,viatransformationoflinearizedplasmidDNA.

promoter:ADNAsequencetowhichRNApolymerasecomplexbindsandinitiatestranscriptionofanadjacentstructuralgeneorgenecluster.Inyeast,thepromoteristypicallycomprisedofatleastoneTATAboxandothercloselyassociatedcis-regulatoryelements(e.g.,UASs).

prototroph:Astrainofyeastorothermicroorganismsthatwillproliferateevenifaparticularnutrientisnot supplied in themedium.Forexample,Trp– yeast strainsareprotototrophic forTrp; theycansynthesizetheirownTrpfromotherbiomoleculesanddonotrequireitinthemedium.Aprototrophictransformationmarkeror reportergenecanbeused tocomplement thecorrespondingauxotrophicalleleinanotherstrain.

segregation:Genetically,theproductionfromasinglecelloftwodaughtercellshavingdistinctgenotypesandphenotypesduetotheseparationoftwoallelesofagene.Inyeast,thiscanoccurduringsporulationorintransformantclonesasaresultoflossofaplasmid.

trans-actingelement:Agenethatcontrolstranscriptionalactivityofanothergenethroughadiffusablegeneproduct(protein)suchasarepressororactivator.

transformation:TheprocessofintroducingforeignDNAintoacell.

transformation markers: Genetic alleles whose phenotypes identify the presence of a plasmidintroducedintoacell;typically,suchmarkersaregenesthatcomplementanutritionalrequirementorconferresistancetoanantibiotic.

UAS:UpstreamActivatingSequence;yeastDNAsequencesthatcontroltheinitiationoftranscriptionofadjacentstructuralgenesviabindingofspecificregulatoryproteins.AnexampleisthebindingoftheyeastGAL4transcriptionalactivator(orDNA-BD)totheUASGoftheGAL1promoter.

wildtype:Thegenotypeorphenotypeofanorganismasitisfoundinnatureorinastandardlaboratorystrain.

APPENDIXA.GlossaryofTechnicalTermscontinued



APPENDIXB.YeastGeneticMarkersUsedintheMatchmakerSystems

table iv. selected yeast genes and their associated phenotypes

Allele

Wildtype Mutant Phenotypeofmutant

TRP1 trp1-901 Trp– Requirestryptophan(Trp)inthemediumtogrow,i.e.,isaTrp auxotroph

LEU2 leu2-3, 112 Leu– Requiresleucine(Leu)togrow,i.e.,isaLeuauxotroph

HIS3 his3-200 His– Requireshistidine(His)togrow,i.e.,isaHisauxotroph

URA3 ura3-52 Ura– Requiresuracil(Ura)togrow,i.e.,isaUraauxotroph

LYS2 lys2-801 Lys– Requireslysine(Lys)togrow,i.e.,isaLysauxotroph

ADE2 ade2-101 Ade– Requiresadenine(Ade)togrow;i.e.,isanAdeauxotroph;inaddition,confersapinkorredcolonycolortocoloniesgrowingon media low in adenine. The red pigment is apparently anoxidized, polymerized derivative of 5-aminoimidazole ribotidewhichaccumulatesinvacuoles(Smirnovet al.,1967;Weismanet al.,1987).

GAL4 gal4-542 Gal– Deficientinregulationofgalactose-metabolizinggenes(Flick&Johnston,1990;Johnstonet al.,1994)

(orgal4Δ)

GAL80 gal80-538 Gal– Deficient in regulation of galactose-metabolizing genes (GALgenesareconstitutivelyexpressed)

CYHs2 cyhr2 Cyhr Resistanttocycloheximide

table v. matchmaker reporter genes and their phenotypes

Reporter Gene Positive NegativeGene Description Phenotypea Phenotypea

lacZ Encodesβ-galactosidase LacZ+ LacZ– •Bluecolony •Whitecolony •β-galactivityabove •Undetectableorbackground background levelofβ-galactivity

HIS3 ConfersHisprototrophy His+ His– •GrowsonSD/–His •DoesnotgrowonSD/–Hisb

LEU2 ConfersLeuprototrophy Leu+ Leu–

•GrowsonSD/–Leu •DoesnotgrowonSD/–Leu

ADE2 ConfersAdeprototrophy Ade+ Ade–

•GrowsonSD/–Ade •DoesnotgrowonSD/–Ade •Pinkorredcolonycolor whengrownonmedium (suchasYPD)lowinAdeaRelativelevelsofbackgroundexpressionandreportergeneinductionaredependentonthepromoterconstructscontrollingthem.

SeeChapterIIforinformationonthepromoters.b 5–60mM3-ATmayberequiredtosuppressleakyHIS3 expressionincertainhoststrainsandtransformantsandtoobtainan

accurateHis–phenotype.



APPENDIXC.MediaRecipes

A.YEASTMEDIA

• YPDmedium YPDMedium(CatNo.630409)andYPDAgarMedium(CatNo.630410)areavailableinpowder

formfromClontech.OurYPDMediumisablendofpeptone,yeastextract,anddextroseinoptimalproportionsforgrowthofmoststrainsofSaccharomyces cerevisiae.SeeChapterXI fororderinginformation.IfyoupurchaseClontech’sYPDmedia,preparethemediumaccordingtotheinstructionsprovided.Alternatively,prepareyourownYPDmixtureasfollows:

20 g/L Difcopeptone 10 g/L Yeastextract 20 g/L Agar(forplatesonly) • [Optional]Foradenine-supplementedYPD(YPDA),add15mlofa0.2%adeninehemisulfate

solutionperliterofmedium(finalconcentrationis0.003%,inadditiontothetraceamountofAdethatisnaturallypresentinYPD).Adeninehemisulfatetoleratesautoclaving.

AddH2Oto950ml.AdjustthepHto6.5ifnecessary,thenautoclave.Allowmediumtocoolto~55°Candthenadddextrose(glucose)to2%(50mlofasterile40%stocksolution).Adjustthefinalvolumeto1Lifnecessary.

Note:Ifyouaddthesugarsolutionbeforeautoclaving,autoclaveat121°Cfor15min;autoclavingatahighertemperature,foralongerperiodoftime,orrepeatedlymaycausethesugarsolutiontodarkenandwilldecreasetheperformanceofthemedium.NotethatYPDfromClontechalreadycontainsglucose.

• [Optional]Forkanamycin-containingmedium,prepareYPDorYPDAasabove.Afterautoclavedmedium has cooled to 55°C, add 0.2–0.3 ml of 50 mg/ml kanamycin (final concentration10–15mg/L).

• SDmedium Minimal SD Base and Minimal SD Agar Base, either with dextrose (glucose), or galactose +

raffinose,areavailablefromClontechinpowderform.(SeeChapterXIfororderinginformation.)IfyoupurchaseClontech’sMinimalSDBase,prepareyourSD/Dropout(DO)mediumaccordingtothe instructionsprovided.Forexample, toprepareSD/–Leu/–Trpagar,youwillneed tocombineSDMinimalAgarBase(CatNo.630412)with–Leu/–TrpDOSupplement(CatNo.630412).

Alternatively,youcanpurchaseyeastnitrogenbasefromanothersupplier(e.g.,DifcoCatNo.0919-15-3)andprepareSD/DOmediumasfollows:

6.7 g Yeastnitrogenbasewithoutaminoacids 20 g Agar(forplatesonly) 850 ml H2O 100 ml oftheappropriatesterile10XDropoutSolution • AdjustthepHto5.8ifnecessary,andautoclave.Allowmediumtocoolto~55°Cbeforeadding

3-AT,cycloheximide,additionaladenine,orX-gal(seebelow). • Add the appropriate sterile carbon source, usually dextrose (glucose) to 2%, unless specified

otherwiseforyourapplication.Adjustthefinalvolumeto1Lifnecessary. Notes: • Ifyouaddthesugarsolutionbeforeautoclaving,autoclaveat121°Cfor15min;autoclavingatahighertemperature,for

alongerperiodoftime,orrepeatedlymaycausethesugarsolutiontodarkenandwilldecreasetheperformanceofthemedium.NotethatSDMinimalBasefromClontechalreadycontainsacarbonsource.

• Ifyoupurchasegalactoseseparately,itmustbehighlypurifiedandcontain<0.01%glucose.

• [Optional]For3-AT-containingmedium,addtheappropriateamountof1M3-ATstocksolutionandswirltomixwell.Theconcentrationof3-ATusedinthemediumdependsontheyeaststrainand,tosomeextent,onthepresenceoftransformingplasmid(s).Seeyoursystem-specificUserManualforfurtherinformation.

Notes: • 3-ATisheat-labileandwillbedestroyedifaddedtomediumhotterthan55°C. • 3-AT,acompetitiveinhibitoroftheyeastHIS3 protein(His3p),isusedtoinhibitlowlevelsofHis3pexpressedinaleaky

mannerinsomereporterstrains(Fields,1993;Durfeeet al.,1993).



APPENDIXC.MediaRecipescontinued

• [Optional] For cycloheximide-containing medium, add the appropriate amount of 1 mg/mlcycloheximide stock solution and swirl to mix well. The concentration of cycloheximide usedin themediumdependson theyeaststrain.Seeyoursystem-specificUserManual for furtherinformation.

Notes: • Cycloheximideisheat-labileandwillbedestroyedifaddedtomediumhotterthan55°C. • Cycloheximide-containing medium is used for selection of yeast strains, such asY190 and CG-1945, carrying the

cyhr2allele.

• [Optional]IfyouwishtoaddexcessadeninetoSDmedium,add15mlof0.2%adeninehemisulfatesolutionperliterofmedium.

• Pourplatesandallowmediumtohardenatroomtemperature.Storeplatesinverted,inaplasticsleeveat4°C.

• SD/Gal/Raf/X-galplates PrepareSDmediumasdescribedaboveexceptuse725mlofH2OanddonotadjustthepH.Autoclave,

andcoolto~55°C.Thenadd: Finalconcentration Toprepare1Lofmedium Galactose 2% 50mlof40%stock Raffinose 1% 25mlof40%stock 10XBUsalts 1X 100mlof10Xstock X-Gal 80mg/L 4mlof20mg/ml Pourplatesandallowmediumtohardenat roomtemperature.Storeplates inverted, inaplastic

sleeve,inthedark,at4°Cforuptotwomonths.Adjustfinalvolumeto1Lifnecessary. Notes: • Galactosemustbehighlypurifiedandcontain<0.01%glucose. • Ifthemediumistoohot(i.e.,>55°C)whenthesaltsolutionisadded,thesaltswillprecipitate.Also,X-Galisheatlabileand

willbedestroyedifaddedtohotmedium. • BUsaltsmustbeincludedinthemediumtoadjustthepHto~7,whichisclosertotheoptimalpHforβ-galactosidase

activity,andtoprovidethephosphatenecessaryfortheβ-galassaytowork. • Astheplatesage,saltcrystalswillforminthemedium.Thesedonotaffecttheperformanceofthemediumortheresults

oftheβ-galactosidaseassay. • If youareassaying forexpressionofa lacZ reportergene inasystem that requiresexpressionofaprotein froman

intact yeast GAL1 promoter (such as in the Matchmaker LexA Two-Hybrid System), you must use 2% galactose +1%raffinoseasthecarbonsourcesinsteadofglucose.IfyouarenotusingClontech’sSD/Gal/RafMinimalBase,besuretoobtainhigh-qualitygalactosethatisnotcontaminatedbyglucose.

StocksolutionsforusewithSDMedia• 1M3-AT(3-amino-1,2,4-triazole;SigmaCatNo.A-8056);prepareindeionizedH2Oandfiltersterilize.

Storeat4°C.Storeplatescontaining3-ATsleevedat4°Cforupto2months.

• 10XBUSalts Dissolvethefollowingcomponentsin1L(total)ofH2O: 70 g Na2HPO4•7H2O 30 g NaH2PO4

AdjusttopH7,thenautoclaveandstoreatroomtemperature.

•Carbonsources,filtersterilizedorautoclaved: Note: Autoclaveat121°Cfor15min;autoclavingatahighertemperature,foralongerperiodoftime,orrepeatedlymaycause

thesugarsolutiontodarkenandwilldecreasetheperformanceofthemedium.

•40%Dextrose(glucose)Storeat4°C. •40%Galactose(forLexATwo-HybridSystem;D(+)Galactose,e.g.,SigmaCatNo.G-0750)Storeat4°C. •40%Raffinose(forLexATwo-HybridSystem)Storeat4°C.

• 1mg/ml(1000X)CHX(Cycloheximide;SigmaCatNo.C-7698);prepareindeionizedH2O,filtersterilize,andaliquot.Storeat4°Cforupto2months.StoreplatescontainingCHXsleevedat4°Cforupto1month.

• 50mg/mlkan(kanamycin);prepareindeionizedH2O,filtersterilize,andaliquot.Storeat–20°Cforupto1month.Storeplatescontainingkansleevedat4°Cforupto1month.


Protocol No. PT3024-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR13103 ��


• X-gal (20 mg/ml in DMF) Dissolve 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside inN,N-dimethylformamide.Storeinthedarkat–20°C.

• 10XDropout(DO)Solution AcombinationofaMinimalSDBaseandaDOSupplementwillproduceasynthetic,definedminimal

mediumlackingoneormorespecificnutrients.Thespecificnutrientsomitteddependsontheselectionmediumdesired.ToprepareSD/–Leu/–Trpagar,forexample,combine–Leu/–TrpDOSupplement(Cat No. 630417) with Minimal SDAgar Base (Cat No. 630412). (With this naming convention[e.g.,SD/–Leu/–Trp], if anutrient isnot indicatedasmissing, it isassumed tobepresent in themedium.)ManyofthecommonlyusedDOSupplementscanbepurchasedfromClontech.InstructionsforpreparingthecorrespondingSD/DOmediumareprintedontheproducts’labels.

Alternatively,makeyourowndropout supplementby combining thenutrients listedbelowat theconcentrationsindicatedtopreparea10XDropoutSolution.A10XDropoutSolutioncontainsallbutoneormoreofthesenutrients.Notethatserine,asparticacid,andglutamicacidarenotincludedinthislistbecausetheymakethemediumtooacidicandbecauseyeastcansynthesizetheseaminoacidsendogenously.However,ifyouwishtoselectforyeaststrainAH109usingmediumthatlacksmethionine(i.e.,onSD/–Met),youshouldaddasparticacidtothe10XDOSolution—a10Xsolutionofasparticacidis1000mg/L.

10Xdropoutsupplementsmaybeautoclavedandstoredat4°Cforupto1year.

Nutrient 10XConcentration SigmaCat.No.

L-Adeninehemisulfatesalt 200 mg/L A-9126 L-ArginineHCl 200 mg/L A-5131 L-HistidineHClmonohydrate 200 mg/L H-9511 L-Isoleucine 300 mg/L I-7383 L-Leucine 1000 mg/L L-1512 L-LysineHCl 300 mg/L L-1262 L-Methionine 200 mg/L M-9625 L-Phenylalanine 500 mg/L P-5030 L-Threonine 2000 mg/L T-8625 L-Tryptophan 200 mg/L T-0254 L-Tyrosine 300 mg/L T-3754 L-Uracil 200 mg/L U-0750 L-Valine 1500 mg/L V-0500

Example:Tomakeoneliterof10X–Leu/–TrpDOSolution,combinethefollowing:

• 200mg adeninehemisulfate • 200mg arginineHCl • 200mg histidineHClmonohydrate • 300mg isoleucine • 300mg lysineHCl • 200mg methionine • 500mg phenylalanine • 2000mg threonine • 300mg tyrosine • 200mg uracil • 1500mg valine 1L Dissolvecomponentsin1LdeionizedH2O.Autoclave.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3024-1 �� Version No. PR13103

B. E. coliMEDIA

• Hanahan’sSOCMedium Finalconcentration ToPrepareOneLiter

Bactotryptone 2% 20 gYeastextract 0.5% 5 gNaCl 10 mM 10 mlof1MNaClKCl 2.5 mM 2.5 mlof1MKClMgCl2* 10 mM 10 mlof1MMgCl2•6H2OMgSO4* 10 mM 10 mlof1MMgSO4•7H2OGlucose* 20 mM 20 mlof1MglucoseDeionizedH2O to1 L

*BeforeaddingMgCl2,MgSO4,andglucosestocksolutions,separatelyfiltersterilizethemusinga0.2-µmfilter.

Addthebactotryptone,yeastextract,andNaClto900mlofdeionizedH2O;stirorshakeuntilsoluteshavedissolved.AddtheKCl.AdjustthepHto7with5NNaOH(~0.2ml).Adjustthevolumeto960mlwithdeionizedH2Oandautoclave.Justbeforeuse,addfilter-sterilizedMgCl2,MgSO4,andglucose.

• LBbroth Bacto-tryptone 10 g/L Bacto-yeastextract 5 g/L NaCl 5 g/L AdjustpHto7.0with5NNaOH.Autoclave.Storebrothat22°C.

• LB/ampagarplates PrepareLBbroth(Sambrooket al.,1989)asabove.Addagar(18g/L),autoclave,andcoolto

50°C.Addampicillinto50µg/ml.Pourplatesandstoreat4°C.

• M9 minimal medium for nutritional selection of E. coli transformants complemented by thewild-typeyeastgene.ForoptimalrecoveryofKC8andHB101transformants,adda1Xmixtureofaminoacids(i.e.,dropout[DO]supplement)lackingthespecificnutrientthatwillallowselectionofthedesiredplasmid.(ThesameDOsupplementsusedforyeastSDmediumcanbeusedtosupplementM9minimalmedium;seeAppendixC.AfordropoutrecipeorpurchasepremixedDOSupplementsfromClontech.)Inaddition,KC8requiresthiamine,andHB101requiresthiamineandproline,forgrowthonminimalmedium.

Prepare900mlofM9mediumasdirectedinSambrooket al.(1989).Toprepareagarplates,addagar(20g/L)priortoautoclaving.Afterautoclaving,allowmediumtocoolto55°C.Thenaddthefollowing:

• 1mlof50mg/mlampicillinstock • 1mlof1.0Mthiamine-HClstock • 100mlofanappropriatesterile10XDOstocksolution Inaddition,forHB101cellsonly: • 4mlofa10mg/mlstockofproline • StocksolutionsforusewithM9orLBmedia Ampicillin(50mg/mlinH2O).Storeat4°Cnolongerthan1month. Thiamine-HCl(1M,filter-sterilized) Proline(10mg/ml,filtersterilized) 10XDOstocksolution(AppendixC.A)




APPENDIXD.SolutionFormulations

A. ForPreparationofProteinExtracts

• ProteaseInhibitorSolution(concentrated) Always prepare solution fresh just before using. Place on ice to prechill. Typeofprotease(s) Toprepare688µl: inhibited: PepstatinA 0.1mg/ml 66µlofa1mg/mlstocksolution* Carboxylproteases (SigmaCatNo.P4265) inDMSO Leupeptin 0.03mM 2µlofa10.5mMstocksolution* Somethioland (SigmaCatNo.L2884) serineproteases Benzamidine 145 mM 500µlofa200mMstocksolution* Trypsin,plasmin, (SigmaCatNo.B6506) andthrombin Aprotinin 0.37mg/ml 120µlofa2.1mg/mlstocksolution* Someserine (SigmaCatNo.A6279) proteases *Storetheindividualstocksolutionsasdirectedonthelabelsandfollowlabelprecautions.

• PMSF(phenylmethyl-sulfonylfluoride)stocksolution[100X*] Dissolve0.1742gPMSF(SigmaCatNo.P7626)in10mlisopropanol.Wraptubeinfoiland

storeatroomtemperature.PMSFprimarilyinhibitsserineproteases. *Althoughthisisa100Xstocksolution,thefinalconcentrationofPMSFisgreaterthan1Xinsomemixtures,i.e.,

PMSFisusedinexcess.

Caution: PMSFishazardous.Weargloves.Handlewithcareandreadlabelprecautions.

• GlassBeads(425–600µm;SigmaCatNo.G-8772)

ForUrea/SDSProteinExtractionMethod: • Crackingbufferstocksolution Toprepare100ml: Urea 8 M 48g SDS 5%w/v 5g Tris-HCl[pH6.8] 40 mM 4mlofa1Mstocksolution EDTA* 0.1 mM 20µlofa0.5Mstocksolution Bromophenolblue 0.4 mg/ml 40mg DeionizedH2O Toafinalvolumeof100ml *EDTAprimarilyinhibitsmetalloproteases.

• Crackingbuffer(complete):Thefollowingrecipeissufficientforoneproteinextract.Scale- uprecipeasrequired. Prepare only the volume you needjust before use. BecausePMSFhasashorthalf-life(~7min)inaqueoussolutions,youmayneedtoadd additionalaliquotsofPMSFduringthecourseoftheprocedure.TheinitialexcessPMSFin theCrackingbufferquicklydegrades. Toprepare1.13mlofcompleteCrackingbuffer: Crackingbufferstocksolution 1ml(recipeabove) β-mercaptoethanol 10µl Proteaseinhibitorsolution 70µl,prechilled(recipeabove) PMSF 50µlof100Xstocksolution



APPENDIXD.SolutionFormulationscontinued

ForTCAProteinExtractionMethod:

• 20%w/vTCAinH2O(Storeat4°C;SeeSambrooket al.[1989]fortipsonpreparingTCA solutions.)

• TCABuffer Place on ice to prechill before use. Add the protease inhibitor solution and pMSF immediately prior to use. Toprepare10mlofTCAbuffer: Tris-HCl(pH8) 20 mM 200 µlofa1Mstocksolution Ammoniumacetate 50 mM 66.6 µlofa7.5Mstocksolution EDTA 2 mM 40 µlofa0.5Mstocksolution DeionizedH2O 9.7 ml Proteaseinhibitorsolution 50 µl/ml 500µl,prechilled(recipeabove) PMSF 100µlof100Xstocksolution

• SDS/glycerolstocksolution Toprepare12ml: SDS 7.3% w/v 3.5mlofa25%stocksolution Glycerol 29.1% v/v 3.5mlof100% Tris-base 83.3 mM 1.0mlofa1Mstocksolution,notpH-adjusted Bromophenolblue Spatulatip-full DeionizedH2O Toafinalvolumeof12ml

• Tris/EDTAsolution Toprepare10ml: Tris-base 200 mM 2.0mlof1Mstocksolution,notpH-adjusted EDTA 20 mM 0.4mlofa0.5Mstocksolution DeionizedH2O 7.6ml

• TCA-Laemmliloadingbuffer Prepare fresh just prior to use. Toprepare1ml: SDS/glycerolstocksolution 480 µl (Stocksolutionmayneedtobewarmedto 60°Ctoreliquefy) Tris/EDTAsolution 400 µl (Recipeabove) β-mercaptoethanol 50 µl PMSF 20 µl PMSFstocksolution(100X) Proteaseinhibitorsolution 20 µl Prechilled(recipeabove) DeionizedH2O 30 µl




B. ForTransformationofYeast

• HerringtestescarrierDNA(10mg/ml) Sonicated, herring testes carrier DNA in solution can be purchased separately

(CatNo.630440;seeChapterXIfororderinginformation),orcanbepreparedusingastandardmethod(Sambrooket al.,1989).Justpriortouse,denaturethecarrierDNAbyplacingitinaboilingwaterbathfor20minandimmediatelycoolingitonice.Useonlyhigh-qualitycarrierDNA;nickedcalfthymusDNAisnotrecommended.

• PEG/LiAcsolution(polyethyleneglycol/lithiumacetate) Prepare fresh just prior to use.

FinalConc. Toprepare10mlofsolution

PEG4000 40% 8mlof50%PEG TEbuffer 1X 1mlof10XTE LiAc 1X 1mlof10XLiAc

• Stocksolutions 50%PEG3350(Polyethyleneglycol,avg.mol.wt.=3,350;SigmaCatNo.P-3640)prepare

withsteriledeionizedH2O; ifnecessary,warmsolution to50°C tohelp thePEGgo intosolution.

100%DMSO(Dimethylsulfoxide;SigmaCatNo.D-8779) 10XTEbuffer:0.1MTris-HCl,10mMEDTA,pH7.5.Autoclave. 10XLiAc:1Mlithiumacetate(SigmaCatNo.L-6883)AdjusttopH7.5withdiluteaceticacid

andautoclave.

C. Forβ-galactosidaseFilterAssays

• Zbuffer

Na2HPO4•7H2O 16.1 g/L

NaH2PO4•H2O 5.50 g/L

KCl 0.75 g/L MgSO4•7H2O 0.246g/L AdjusttopH7.0andautoclave.Canbestoredatroomtemperatureforupto1year.

• X-galstocksolution

Dissolve 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-GAL; Cat No. 8060-1) inN,N-dimethylformamide(DMF)ataconcentrationof20mg/ml.Storeinthedarkat–20°C.

• Zbuffer/X-galsolution

100 ml Zbuffer 0.27 ml β-mercaptoethanol(β-ME;SigmaCatNo.M-6250) 1.67 ml X-galstocksolution

D. ForLiquidβ-galactosidaseAssayswithONPGasSubstrate

• Zbuffer(seeprecedingsectionforrecipe)

• Zbufferwithβ-mercaptoethanol To100mlofZbuffer,add0.27mlofβ-mercaptoethanol.

• ONPG(o-nitrophenylβ-D-galactopyranoside;SigmaCatNo.N-1127) 4mg/mlinZbuffer.AdjusttopH7.0andmixwell.

Notes: •ONPGrequires1–2hrtodissolve.

•Preparesolutionfreshbeforeeachuse.



E. ForLiquidβ-galactosidaseAssayswithCPRGasSubstrate

• Buffer1 Toprepare100mlofsolution HEPES 2.38g NaCl 0.9g L-Aspartate[hemi-Mgsalt;SigmaCatNo.A-9506] 0.065g BSA 1.0g Tween20 50.0µlDissolvetheabovecomponentsin75mlofdeionizedH2O.AdjustpHto7.25–7.30,thenbringvolume

to100ml.Filtersterilize.Storeat4°Cforupto3months. • Buffer2(20ml) Dissolve27.1mgofCPRGin20mlofBuffer1(finalconcentrationofCPRGis2.23mM).

Filtersterilize.Storeat4°Cinthedarkforupto3months.

F. Forα-GalQuantitativeAssays

• PNP-α-GalSolution 100mM(p-nitrophenylα-d-Galactopyranoside;SigmaCatNo.N0877)indeionizedH2O For10ml,dissolve30.1mgofPNP-α-Galin10mlofdeionizedH2O.Filtersterilize. Notes: •Preparesolutionfreshbeforeeachuse. •Keepthep-nitrophenylα-d-Galactopyranosidesolidanhydrous.Storeinadessicatorat–20°C.

• 10XStopSolution 1MNa2CO3indeionizedH2O(SigmaCatNo.S7795)

• 1XNaOAc 0.5Msodiumacetate,pH4.5(SigmaCatNo.S7545)

• AssayBuffer PrepareAssayBufferfresh,beforeeachuse,bycombining2volumes1XNaOAcBuffer

with1volumePNP-α-GalSolution[2:1(v/v)ratio].Mixwell.




APPENDIXE.PlasmidInformation

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APPENDIXE:PlasmidInformationcontinued

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.3

Hin

dIII

E

stoj

ake

t al.,

199

5

lexA

op(x

8),U

RA

3,a

mpr

(6.3

,2.1

,1.9

)(p

SH

18-3

4)

pB

42A

D-T

Le

xA2

H

SV

40la

rge

T-an

tigen

(87–

708)

–Trp

8.

5H

ind

III

Li&

Fie

lds,

199

3;

inp

B42

AD

,TR

P1,

am

pr

(3

.4,2

.1,1

.0,0

.9,0

.6,0

.5)

Chi

ene

t al.,

199

1

pC

L1

GA

L42

H&

2H

-2

wild

-typ

efu

ll-le

ngth

GA

L4

–Leu

~

15.3

H

ind

III

Fie

lds

&S

ong,

198

9

ge

nein

aY

Cp5

0de

rivat

ive,

(~

11.2

,2.8

,1.8

)

LE

U2,

am

pr

pG

AD

T7-

T

GA

L42

H-3

S

V40

larg

eT-

antig

en (8

4–70

8)

–Leu

10

.0

Xho

I/E

coR

IC

lont

ech

inp

GA

DT

7,L

EU

2,a

mpr

(8.0

,2.0

)

pG

BK

T7-

53

GA

L42

H-3

m

urin

ep5

3 (72

-390

)in

pGB

KT

7,

–Trp

8.

3B

amH

I/Eco

RI

Clo

ntec

h

T

RP

1,k

anr

(7.3

,1.0

)

pG

BK

T7-

Lam

G

AL4

2H

-3

Hum

anla

min

C(6

6-23

0)

–Trp

7.

9B

amH

I/Eco

RI

Clo

ntec

h

in

pG

BK

T7,

TR

P1,

kan

r

(7

.3,0

.57)

pL

AM

5’

GA

L42

H

Hum

anla

min

C(6

6–23

0)

–Trp

6.

0

Hin

dIII

B

arte

let a

l.,1

993a

inp

GB

T9,

TR

P1,

am

pr

(4

.7,0

.8,0

.6)

pL

AM

5’-1

G

AL4

2H

-2

Hum

anla

min

C(6

6–23

0)

–Trp

~

9.0

H

ind

III

Bar

tele

t al.,

199

3a

in

pA

S2-

1T

RP

1,a

mpr

(4.6

,2.2

,0.9

,0.8

5,0

.4)

pL

exA

-53

LexA

2H

m

urin

ep5

3 (72

–390

)in

pLex

A

–His

11

.1

Hin

dIII

Iw

abuc

hie

t al.,

199

3

HIS

3,a

mpr

(5.7

,5.2

,0.2

)

pL

exA

-Lam

Le

xA2

H

Hum

anla

min

C(6

6–23

0)

–His

10

.6

Hin

dIII

B

arte

let a

l.,1

993a

in

pLe

xA,H

IS3,

am

pr

(5

.2,4

.3,0

.9,0

.2)

pL

exA

-Po

sLe

xA2

H

LexA

/GA

L4 fu

sion

gen

e,

–His

~

13.5

H

ind

III

Gol

emis

et a

l.,1

994

HIS

3,a

mpr

(6

.0,4

.5,3

.0)

(pS

H17

-4)

pT

D1

GA

L42

H

SV

40la

rge

T-an

tigen

(84–

708)

–Leu

~

15.0

H

ind

III

Li&

Fie

lds,

199

3;

inp

GA

D3F

,LE

U2,

am

pr

(1

2,1

.3,1

.2,0

.5)

Chi

ene

t al.,

199

1

pT

D1-

1G

AL4

2H

-2

SV

40la

rge

T-an

tigen

(84–

708)

–Leu

~

10.0

H

ind

III

Li&

Fie

lds,

199

3;

inp

AC

T2,

LE

U2,

am

pr

(7

.3,1

.2,1

.0,0

.5)

pVA

3G

AL4

2H

m

urin

ep5

3(7

2–39

0)in

pG

BT

9–T

rp

6.4

H

ind

III

Iwab

uchi

et a

l.,1

993

TR

P1,

am

pr

(4

.6,1

.8)

pVA

3-1

GA

L42

H-2

m

urin

ep5

3(7

2–39

0)in

pA

S2-

1–T

rp

9.4

H

ind

III

Iwab

uchi

et a

l.,1

993

TR

P1,

am

pr

(4

.6,2

.2,1

.7,0

.9)

Chi

ene

t al.,

199

1

apB

42A

D-T

,pLA

M5’

,pLA

M5’

-1,p

LexA

-53,

pLe

xA-L

am,p

TD

1-1,

and

pV

A3-

1ar

ede

rivat

ives

oft

hep

lasm

ids

desc

ribed

inth

ein

dica

ted

refe

renc

es;p

lasm

ids

wer

em

odifi

eda

tClo

ntec

h.



APPENDIXE:PlasmidInformationcontinued

tab

le v

iii. m

at

ch

ma

ke

r o

ne-h

yb

rid

sy

st

em

cl

on

ing, r

ep

or

te

r &

co

nt

ro

l p

la

sm

ids

Dia

gn

ost

ic

S

elec

tio

no

n

Siz

eR

.E.S

ites

Vec

tora

Des

crip

tio

nb

SD

Med

ium

(k

b)

(kb

)R

efer

ence

p53

HIS

H

IS3

unde

rco

ntro

lof

–Ura

,–H

is

6.7

E

coR

I/X

ho Ic

Luo

et a

l., 1

996

p5

3bi

ndin

gsi

tes

inp

HIS

i,

(5

.7,1

.0)

H

IS3,

UR

A3 ,

am

pr

p53

BL

UE

la

cZu

nder

con

trol

of

–Ura

6.

4

Eco

RI/

Xho

Ic Lu

o et

al.,

199

6

p53

bind

ing

site

sin

pLa

cZi,

(6

.3,0

.1)

UR

A3 ,

am

pr

pG

AD

53m

m

urin

ep5

3 (72

–300

)fus

edto

–L

eu

7.6

E

coR

Id Lu

o et

al.,

199

6

GA

L4(7

68–8

81)A

D,L

EU

2,a

mpr

(7.6

)

pH

ISi

HIS

3 un

der

cont

rolo

f–U

ra,–

His

e 6.

8

Eco

RI/

Xho

IA

lexa

ndre

et a

l., 1

993

cl

oned

targ

ete

lem

ent,

(5

.7,1

.0)f

U

RA

3, a

mpr

pH

ISi-

1H

IS3

unde

rco

ntro

lof

–His

e 5.

4

Eco

RI/

Xho

IA

lexa

ndre

et a

l., 1

993

cl

oned

targ

ete

lem

ent,

(4

.4,1

.0)

am

pr

pL

acZ

ila

cZ u

nder

con

trol

of

–Ura

6.

9

Eco

RI/

Xho

ILu

o et

al.,

199

6

clon

edta

rget

ele

men

t,

(6.4

,0.0

4)f

U

RA

3, a

mpr

aIn

the

one-

hybr

ids

yste

m,a

put

ativ

ere

cogn

ition

seq

uenc

e(t

heta

rget

DN

As

eque

nce)

mus

tbe

clon

edin

toth

eM

CS

ofo

neo

fthe

rep

orte

rpl

asm

ids.

The

con

stru

ctis

then

use

dto

gen

erat

eth

ene

cess

ary

yeas

trep

orte

rst

rain

for

dete

ctin

gsp

ecifi

cD

NA

-pro

tein

inte

ract

ions

.b

Add

ition

alv

ecto

rin

form

atio

n,r

estr

ictio

nm

aps,

and

mul

tiple

clo

ning

site

(M

CS

)se

quen

ces

are

prov

ided

inth

eM

atch

mak

erO

ne-H

ybrid

Sys

tem

Use

rM

anua

l.c

p53H

ISa

ndp

53B

lue

are

notc

utb

yS

ma

I,w

hich

mak

esit

pos

sibl

eto

dis

tingu

ish

them

from

pH

ISia

ndp

LacZ

i,ea

cho

fwhi

chh

ave

asi

ngle

Sm

a Is

ite.

dpG

AD

53m

isn

otc

utb

yX

hoI.

e Le

aky

HIS

3 ex

pres

sion

inth

ese

plas

mid

spe

rmits

its

use

asa

sel

ecta

ble

mar

ker

onS

D/–

His

(w

ithou

t3-A

T).

f In

add

ition

,pH

ISia

ndp

LacZ

ihav

ea

sing

leS

ma

Isite

,whi

chm

akes

itp

ossi

ble

tod

istin

guis

hth

emfr

omp

53H

ISa

ndp

53B

lue,

whi

cha

ren

otc

utb

yS

ma

I.



APPENDIXF:YeastHostStrainInformation

ta

bl

e ix

. ye

as

t r

ep

or

te

r s

tr

ain

s in

th

e m

at

ch

ma

ke

r o

ne-a

nd t

wo-h

yb

rid

sy

st

em

s

Tran

sfo

rmat

ion

Str

ain

S

yste

m

Gen

oty

pea

Rep

ort

er(s

)b

Mar

kers

c R

efer

ence

s

SF

Y52

6G

AL4

2H

M

ATa,

ura

3-52

,his

3-20

0, a

de2-

101,

lys2

-801

,la

cZ

trp1

,leu

2H

arpe

ret

al.,

199

3

tr

p1-

901,

leu2

-3,1

12,c

anr ,

gal4

-542

,gal

80-5

38,

UR

A3

: : G

AL1

UA

S-G

AL1

TATA

-lacZ

HF

7c

GA

L42

H

MAT

a, u

ra3-

52,h

is3-

200,

ade

2-10

1,ly

s2-8

01,

HIS

3, la

cZ

trp1

,leu

2,

Fei

lotte

ret

al.,

199

4;

tr

p1-9

01, l

eu2-

3,1

12,g

al4-

542,

gal

80-5

38,

cyhr 2

C

.Giro

ux,p

erso

nalc

omm

unic

atio

n

LY

S2

: : G

AL1

UA

S-G

AL1

TATA

-HIS

3,

UR

A3

: : G

AL4

17-

mer

s(x3

)-CY

C1 T

ATA

-lacZ

Y18

7G

AL4

2H

-2,

MAT

α,u

ra3-

52,h

is3-

200,

ade

2-1

01,

lacZ

, ME

L1

trp1

,leu

2H

arpe

ret

al.,

199

3

GA

L42

H-3

,&

trp

1-90

1, le

u2-

3,1

12,g

al4Δ

,met

– ,g

al80

Δ,

PT

Lib

rarie

sd U

RA

3 : :

GA

L1U

AS-G

AL1

TATA

-lacZ

, ME

L1

CG

-194

5e G

AL4

2H

-2,

MAT

a,u

ra3-

52,h

is3-

200,

ade

2-10

1,ly

s2-8

01,

HIS

3, la

cZ

trp1

,leu

2,

Fei

lotte

ret

al.,

199

4;

tr

p1-9

01, l

eu2-

3,1

12,g

al4-

542,

gal

80-5

38,c

yhr 2

,

cyhr 2

C

.Giro

ux,p

erso

nalc

omm

unic

atio

n

LY

S2

: : G

AL1

UA

S-G

AL1

TATA

-HIS

3,

UR

A3

: : G

AL4

17-

mer

s(x3

)-CY

C1 T

ATA

-lacZ

Y19

0G

AL4

2H

-2

MAT

a,u

ra3-

52,h

is3-

200,

ade

2-10

1,ly

s2-8

01,

H

IS3,

lacZ

, ME

L1

trp1

,leu

2,

Har

per

et a

l.,1

993;

trp1

-901

, leu

2-3,

112

,gal

4Δ,g

al80

Δ, c

yhr 2

,

cyhr 2

F

lick

&J

ohns

ton,

199

0

LYS

2 : :

GA

L1U

AS-H

IS3 T

ATA

-HIS

3, M

EL1

U

RA

3 : :

GA

L1U

AS-G

AL1

TATA

-lacZ

EG

Y48

Le

xA2

H

MAT

α, u

ra3,

his

3, tr

p1, L

exA

op (

x6)-L

EU

2LE

U2

his3

, trp

1, u

ra3

Est

ojak

et a

l.,1

995

YM

4271

Le

xA2

H&

M

ATa,

ura

3-52

,his

3-20

0, ly

s2-8

01, a

de2-

101,

ade

5,

hi

s3, t

rp1

Liu

et a

l.,1

993

M

M1

H

trp1

-901

, leu

2-3,

112

, tyr

1-50

1,ga

l4Δ

,gal

80Δ

, ade

5 : :

his

G

PJ6

9-2A

f P

TL

ibra

ries

MAT

a, tr

p1-9

01, l

eu2-

3, 1

12, u

ra3-

52, h

is3-

200,

H

IS3,

AD

E2,

ME

L1

trp1

, ura

3, le

u2

Jam

ese

t al.,

199

6

ga

l4Δ

,gal

80Δ

, LY

S2

: : G

AL1

UA

S-G

AL1

TATA

-HIS

3,

G

AL2

UA

S-G

AL2

TATA

-AD

E2,

ME

L1

AH

109f

GA

L42

H-3

M

ATa,

trp1

-901

, leu

2-3,

112

, ura

3-52

, his

3-20

0,

HIS

3, A

DE

2, la

cZ,

trp1

, leu

2Ja

mes

et a

l.,1

996;

ga

l4Δ

,gal

80Δ

, LY

S2

: : G

AL1

UA

S-G

AL1

TATA

-HIS

3, M

EL1

M

EL1

Hol

tz,U

npub

lishe

d

G

AL2

UA

S-G

AL2

TATA

-AD

E2,

UR

A3:

:ME

L1U

AS-M

EL1

TATA

-lacZ

a T

hetr

p1, h

is3,

gal

4, a

nd g

al80

mut

atio

nsa

rea

llde

letio

ns;l

eu2–

3,1

12is

ad

oubl

em

utat

ion.

The

LY

S2

gene

isn

onfu

nctio

nali

nth

eH

F7c

and

CG

-194

5.S

eeC

hapt

erII

form

ore

info

rmat

ion

onth

epr

omot

ers

ofth

ere

port

erg

enes

.b

See

Tab

leV

for

mor

ein

form

atio

non

rep

orte

rge

nes

and

thei

rph

enot

ypes

.c

Gen

esth

ata

reu

sed

ass

elec

tion

mar

kers

inth

iss

yste

m.

d P

TL

ibra

ries

=P

retr

ansf

orm

edM

atch

mak

erL

ibra

ries.

eC

G-1

945

isa

der

ivat

ive

ofH

F7c

(F

eilo

tter

et a

l.,1

994)

.f

The

ade

2–10

1ge

neo

fthe

pre

curs

ors

trai

nw

asr

epla

ced

(by

reco

mbi

natio

n)w

ithth

eG

AL2

-AD

E2

repo

rter

con

stru

ct.



AmpliTaq®andGeneAmp®areregisteredtrademarksofHoffmann-LaRoche,Inc.,andF.HoffmannLaRoche,Ltd.

FalconTMisatrademarkofBectonDickinson&Co.

Galacton-StarTMandSapphireIITMaretrademarksofTropix,Inc.

GenBankTMisatrademarkoftheU.S.DepartmentofHealth&HumanServices.

Parafilm®isaregisteredtrademarkoftheAmericanCanCo.

Notes



Notes


Yeast Protocols Handbook Notes

yeast protocols handbook

Documents