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CLONTECHInnovat iveTools t oAccelerat eDiscoveryYeast Protocols HandbookPT3024-1(PR13103)Published 14 March 2001Cat alog#:K1612-1MATCHMAKER Two-Hybrid Syst em 3(many)MATCHMAKER GAL4 LibrariesK1615-1MATCHMAKER Library Const ruct ion&Screening KitK1603-1MATCHMAKER One-Hybrid Syst emNL4000AAMATCHMAKER Random Pept ide Library5398-1GAL4 AD Monoclonal Ant ibody5399-1GAL4 DNA-BD Monoclonal Ant ibodyFOR RESEARCH USE ONLYYeast Protocols HandbookTable of ContentsI.Introduction 4II.Introduction to Yeast Promoters 5III.Culturing and Handling Yeast 10IV.Preparation of Yeast Protein Extracts 12A.General Informat ion 12B.Preparat ion of Yeast Cult ures for Prot ein Ext ract ion 12C.Preparat ion of Prot ein Ext ract s:Urea/SDS Met hod 13D.Preparat ion of Prot ein Ext ract s:TCA Met hod 15E.Troubleshoot ing 17V.Yeast Transformation Procedures 18A.General Informat ion 18B.Reag ent s and Mat erials Req uired 19C.Tips for a Successful Transformat ion 20D.Int eg rat ing Plasmids int o t he Yeast Genome 20E.Small-scale LiAc Yeast Transformat ion Procedure 20F.Troubleshoot ing Yeast Transformat ion 22VI.a-and|3-Galactosidase Assays 23A.General Informat ion 23B.In vivo Plat e Assay Using X-g al in t he Medium 25C.Colony-lift Filt er Assay 25D.Liq uid Cult ure Assay Using ONPG as Subst rat e 26E.Liq uid Cult ure Assay Using CPRG as Subst rat e 27F.Liq uid Cult ure Assay Using a Chemiluminescent Subst rat e 28G.a-Gal Quant it at ive Assay 32VII.Working with Yeast Plasmids 34A.General Informat ion 34B.Plasmid Isolat ion From Yeast 34C.Transforming E.co/wit h Yeast Plasmids 36VIII.Analysis of Yeast Plasmid Inserts by PCR 39A.General Informat ion 39B.Tips for Successful PCR of Yeast Plasmid Templat es 39IX.Additional Useful Protocols 42A.Yeast Colony Hybridizat ion 42B.Generat ing Yeast Plasmid Seg reg ant s 43C.Yeast Mat ing 44X.References 46XI.MATCHMAKER and Related Products 49APPENDICESA.Glossary of Technical Terms 50B.Yeast Genet ic Markers Used in t he MATCHMAKER Syst ems 52C.Media Recipes 53A.Yeast Media 53B.E.co li Media 56D.Solut ion Formulat ions 57E.Plasmid Informat ion 61F.Yeast Host St rain Informat ion 64CLONTECH Laboratories,IProtocol#PT3024-1Version#PR13103Yeast Protocols HandbookTable of Contents continuedList of TablesTable I.Yeast Promot er Const ruct s Used t o Reg ulat e Report er Gene Expression in MATCHMAKER Plasmids and Host St rains6Table II.Yeast Promot er Const ruct s in t he MATCHMAKER Cloning Vect ors9Table III.Comparison of p-g alact osidase Assays25Table IV.Select ed Yeast Genes and Their Associat ed Phenot ypes52Table V.MATCHMAKER Report er Genes and Their Phenot ypes52Table VI.MATCHMAKER Two-Hybrid Syst em Cloning Vect ors61Table VILMATCHMAKER Two-Hybrid Syst em Report er and Cont rol Plasmids62Table VIII.MATCHMAKER One-Hybrid Syst em Cloning,Report er&Cont rol Plasmids63Table IX.Yeast Report er St rains in t he MATCHMAKER One-and Two-Hybrid Syst ems64List of FiguresFig ure 1.Seq uence of GAL4 DNA-BD recog nit ion sit es in t he GAL1,GAL2,MEL1 UASs and t he UASG 17_mer6Fig ure 2.Urea/SDS prot ein ext ract ion met hod14Fig ure 3.TCA prot ein ext ract ion met hod16Notice to PurchaserPract ice of t he t wo-hybrid syst em is covered by U.S.Pat ent s#5,283,173 and#5,468,614 assig ned t o t he Research Foundat ion of t he St at e Universit y of New York.Purchase of any CLONTECH t wo-hybrid reag ent does not imply or convey a license t o pract ice t he t wo-hybrid syst em covered by t hese pat ent s.Commercial ent it ies purchasing t hese reag ent s must obt ain a license from t he Research Foundat ion of t he St at e Universit y of New York before using t hem.CLONTECH is req uired by it s licensing ag reement t o submit a report of all purchasers of t wo-hybrid reag ent s t o SUNY St ony Brook.Please cont act Barbara A.Sawit sky of SUNY St ony Brook for license informat ion(Tel:[phone];Fax:[phone]).All plasmids(except for pACT2,pAS2-1,pGADT7 and pGBKT7)are licensed from The Research Foundat ion of t he St at e Universit y of New York.pACT2,pAS2-1,and yeast st rains Y187 and Y190 are licensed from Baylor Universit y.pGBKT7 is a derivat ive of pODB-8 and is licensed from t he Universit e de Bordeaux.The pBridg e ThreeHybrid Vect or is t he propert y of t he Inst it ut Nat ional de la Sant e et de la Recherche Medicale(INSERM).pGADT7 and CG-1945 are t he propert y of CLONTECH Laborat ories,Inc.AH109 is t he propert y of CLONTECH Laborat ories,Inc.,and is a derivat ive of PJ69-2A which is t he propert y of t he Universit y of Wisconsin Research Foundat ion(WARF).The Polymerase Chain React ion(PCR)process is covered by pat ent s owned by Hoffman-LaRoche and F.Hoffmann-La Roche,Lt d.This product is int ended t o be used for research purposes only.It is not t o be used for drug or diag nost ic purposes nor is it int ended for human use.CLONTECH product s may not be resold,modified for resale,or used t o manufact ure commercial product s wit hout writ t en approval of CLONTECH.2001,CLONTECH Laborat ories,Inc.All rig ht s reserved.Protocol#PT3024-1Version#PRCLONTECH Laboratories,Inc.3Yeast Protocols HandbookI.IntroductionThe Yeast Prot ocols Handbook provides backg round informat ion and g eneral yeast prot ocols t hat complement our syst em-specific User Manuals.The prot ocols in t his Handbook have been opt imized wit h our yeast-based MATCHMAKER Two-Hybrid and One-Hybrid Syst ems,and MATCHMAKER Libraries.The Yeast Prot ocols Handbook is especially useful for researchers who wish t o use yeast as a vehicle for t heir molecular biolog y experiment s,but have lit t le or no prior experience working wit h yeast.For novice and experienced users alike,t he Yeast Prot ocols Handbook will help you obt ain t he best possible result s wit h your MATCHMAKER and ot her yeast-relat ed product s from CLONTECH.This Handbook includes:det ailed informat ion on cult uring and handling yeast informat ion on t he yeast promot ers used in t he MATCHMAKER Syst ems t wo prot ocols for preparing prot ein ext ract s from yeast q uant it at ive and q ualit at ive p-g alact osidase assays(for use wit h lacZyeast report er st rains)a simple,opt imized prot ocol for isolat ing plasmids from yeast PCR amplificat ion and yeast colony hybridizat ion prot ocols for t he rapid analysis of posit ive clones obt ained in a library screening a small-scale,lit hium acet at e yeast t ransformat ion prot ocol addit ional prot ocols for working wit h cert ain yeast plasmids and host st rainsThe special applicat ion of yeast t ransformat ion for one-and t wo-hybrid library screening is covered in det ail in each product-specific User Manual.The special applicat ion of yeast mat ing for library screening is covered in t he Pret ransformed MATCHMAKER Libraries User Manual.About our yeast-based productsThe MATCHMAKER GAL4 Two-Hybrid Syst ems(#K1604-1,K1605-1,K1612-1)and LexA Two-Hybrid Syst em(#K1609-1)are complet e kit s for ident ifying and invest ig at ing prot ein-prot ein int eract ions in vivo using t he yeast t wo-hybrid assay.The MATCHMAKER One-Hybrid Syst em(#K1603-1)provides t he basic t ools for ident ifying novel prot eins in vivo t hat bind t o a t arg et DNA seq uence such as a c/s-act ing reg ulat ory element.MATCHMAKER Two-Hybrid Syst ems are compat ible wit h our pBridg e Three-Hybrid Vect or(#6184-1)for t he invest ig at ion of t ert iary prot ein complexes.The MATCHMAKER Libraries are const ruct ed in vect ors t hat express insert s as fusions t o a t ranscript ional act ivat ion domain,and are t hus a convenient resource for researchers wishing t o screen a library using t he one-or t wo-hybrid assays.Pret ransformed MATCHMAKER Libraries provide an even g reat er level of convenience for t hose wishing t o perform a t wo-hybrid library screening wit hout using larg e-or library-scale yeast t ransformat ions.CLONTECH offers an ext ensive line of kit s and reag ent s t hat support and complement t he MATCHMAKER Syst ems and Libraries.The YEASTMAKER Yeast Transformat ion Kit(#K1606-1)includes all t he necessary reag ent s and prot ocols for efficient t ransformat ion using t he lit hium acet at e met hod.Also available from CLONTECH:a select ion of GAL4 DNA-binding domain(DNA-BD)and act ivat ion domain(AD)hybrid cloning vect ors;t he pGilda Vect or for use wit h LexA-based t wo-hybrid syst ems;monoclonal ant ibodies and seq uencing primers;and yeast media,including Minimal SD Base and many different formulat ions of Dropout(DO)Supplement.Finally,t he pHA-CMV and pMyc-CMV Vect or Set(#K6003-1)can be used t o confirm prot ein int eract ions in mammalian cells.For ordering informat ion on t hese product s,please see Chapt er XI of t his Handbook or t he CLONTECH Cat alog.CLONTECH Laboratories,IProtocol#PT3024-1Version#PR13103Yeast Protocols HandbookII.Introduction to Yeast PromotersYeast promot ers and ot her c/s-act ing reg ulat ory element s play a crucial role in yeast-based expression syst ems and t ranscript ional assays such as t he MATCHMAKER One-and Two-Hybrid Syst ems.Differences in t he promot er reg ion of report er g ene const ruct s can sig nificant ly affect t heir abilit y t o respond t o t he DNA-binding domain of specific t ranscript ional act ivat ors;promot er const ruct s also affect t he level of backg round(or leakiness)of g ene expression and t he level of induced expression.Furt hermore,differences in cloning vect or promot ers det ermine t he level of prot ein expression and,in some cases,confer t he abilit y t o be reg ulat ed by a nut rient(such as g alact ose in t he case of t he GAL1 promot er).This chapt er provides a brief int roduct ion t o several commonly used yeast promot ers and c/s-reg ulat ory element s.For furt her informat ion on t he reg ulat ion of g ene expression in yeast,we recommend t he Guide t o Yeast Genet ics and Mo lecular Bio lo gy by Gut hrie&Fink(1991;#V2010-1);Mo lecular Bio lo gy and Genet ic Engineering o f Yeast s,edit ed by Heslot&Gaillardin(1992);St arg ell&St ruhl(1996);and Pring le et al.(1997;#V2365-1).UAS and TATA regions are basic building blocks of yeast promotersThe init iat ion of g ene t ranscript ion in yeast,as in ot her org anisms,is achieved by several molecular mechanisms working in concert.All yeast st ruct ural g enes(i.e.,t hose t ranscribed by RNA polymerase II)are preceded by a reg ion cont aining a loosely conserved seq uence(TATA box)t hat det ermines t he t ranscript ion st art sit e and is also a primary det erminant of t he basal t ranscript ion level.Many g enes are also associat ed wit h c/s-act ing element s-DNA seq uences t o which t ranscript ion fact ors and ot her acs-act ing reg ulat ory prot eins bind and affect t ranscript ion levels.The t erm promot er usually refers t o bot h t he TATA box and t he associat ed c/s-reg ulat ory element s.This usag e is especially common when speaking of yeast g ene reg ulat ion because t he cis reg ulat ory element s are relat ively closely associat ed wit h t he TATA box(Yoccum,1987).This is in cont rast t o mult icellular eukaryot es,where cis-reg ulat ory element s(such as enhancers)can be found very far upst ream o r downst ream from t he promot ers t hey reg ulat e.In t his t ext,minimal promot er will refer specifically t o t he TATA reg ion,exclusive of ot her c/s-act ing element s.The minimal promot er(or TATA box)in yeast is t ypically approximat ely 25 bp upst ream of t he t ranscript ion st art sit e.Yeast TATA boxes are funct ionally similar t o prokaryot ic Pribnow boxes,but are not as t ig ht ly conserved.Furt hermore,some yeast t ranscript ion unit s are preceded by more t han one TATA box.The yeast H/S3 g ene,for example,is preceded by t wo different TATA boxes:TR,which is reg ulat ed,and TC,which is const it ut ive(Mahadevan&St ruhl,1990).Yeast TATA boxes can be moved t o a new locat ion,adjacent t o ot her c/s-reg ulat ory element s,and st ill ret ain t heir t ranscript ional funct ion.One t ype of c/s-act ing t ranscript ion element in yeast is upst ream act ivat ing seq uences(UAS),which are recog nized by specific t ranscript ional act ivat ors and enhance t ranscript ion from adjacent downst ream TATA reg ions.The enhancing funct ion of yeast UASs is g enerally independent of orient at ion;however,it is sensit ive t o dist ance effect s if moved more t han a few hundred base pairs from t he TATA reg ion.There may be mult iple copies of a UAS upst ream of a yeast coding reg ion.In addit ion,UASs can be eliminat ed or swit ched t o chang e t he reg ulat ion of t arg et g enes.UAS and TATA regions can be switched to create novel promotersThe mix and mat ch nat ure of yeast TATA boxes and UASs has been used t o g reat advant ag e in yeast t wo-hybrid syst ems t o creat e novel promot ers for t he report er g enes.(For g eneral references on yeast t wo-hybrid syst ems,see Chapt er X.)In most cases,t he lacZ,HIS3,ADE2 and LEU2 report er g enes are under cont rol of art ificial promot er const ruct s comprised of a TATA and UAS(or operat or)seq uence derived from anot her g ene(Table I).In some cases,t he TATA seq uence and t he UAS are derived from different g enes;indeed,t he LexA operat or is a c/s-act ing reg ulat ory element derived from E.co li.For GAL4-based syst ems,eit her a nat ive GAL UAS or a synt het ic UASG 17.mer consensus seq uence(Heslot&Gaillardin,1992)provides t he binding sit e for t he GAL4 DNA-BD.For LexA-based syst ems,mult iple copies of t he LexA operat or provide t he binding sit e for t he LexA prot ein.If you are put t ing t og et her your own one-or t wo-hybrid syst em,you must make sure t hat t he report er g enes promot er will be recog nized by t he DNA-BD moiet y encoded in your DNA-BD fusion vect or.Protocol#PT3024-1Version#PRCLONTECH Laboratories,Inc.5Yeast Protocols HandbookII.Introduction to Yeast Promoters continuedTABLE I.YEAST PROMOTER CONSTRUCTS USED TO REGULATE REPORTER GENE EXPRESSION IN MATCHMAKER PLASMIDS AND HOST STRAINSPlasmid or host strain3Reporter geneOrigin of UASUAS regulated byOrigin of TATA sequenceExpression levelb Induced(uninduced)CG-1945lacZUASg 17-mer(x3)CGAL4CYC1lowHIS3GAL1GAL4GAL1hig h(slig ht ly leaky)HF7clacZUASg 17-mer(x3)CGAL4CYC1lowHIS3GAL1GAL4GAL1hig h(t ig ht)Y190lacZGAL1GAL4GAL1hig hHIS3GAL1GAL4HIS3(TC+TR)hig h(leaky)Y187lacZ。GAL1GAL4GAL1hig hSFY526lacZGAL1GAL4GAL1hig hPJ69-2AHIS3GAL1GAL4GAL1hig h(t ig ht)ADE2GAL2GAL4GAL2hig h(t ig ht)AH109HIS3GAL1GAL4GAL1hig h(t ig ht)ADE2GAL2GAL4GAL2hig h(t ig ht)lacZMEL1GAL4MEL1lowEGY48LEU2LexA op(x6)LexALEU2hig hp8op-lacZlacZLexA op(x8)LexAGAL1ehig hpHISiHIS3(none),(n-a.)HIS3(TC+TR)n.a.f(leaky)pHISi-1HIS3(none)f(n.a.)HIS3(TC+TR)n.a.f(leaky)pLacZilacZ(none)(n-a.)CYC1n.a.f(t ig ht)a See Appendices E&F for references.b When induced by a posit ive t wo-hybrid int eract ion;leaky and t ig ht refer t o expression levels in t he absence of induct ion.c Conserved 17-bp palindromic seq uence t o which t he GAL4 prot ein binds(Gut hrie&Fink,1991).d Y187 probably cont ains t wo copies of t he lacZg ene,ju