MakinglibraryDNAfromtheDNAwesendyou

Thelibraryisdistributedas18individualpoolsintheformofDNA.Youwillbesentaboutamicrogramofeach.TransformasuitableamountintoE.coli(useanykanamycin-andtetracycline-sensitivestrainsuitableformakingplasmidpreps).Selecttransformantswith40ug/mlkanamycinand/or3ug/mltetracycline(allowatleastanhourforexpressionfollowingtransformation).Obtain50,000coloniesforeachpool.ElutecoloniesfromplatesinLB;makea-70^oCstockofthiseluate.DiluteeluateintoLBplusantibiotictogiveaculturewithanalmostsaturateddensity.Growat37^oCforafewhours.MakeminiprepormidiprepDNA.

TransformingyeastwithDNAfromtheinsertionlibrary

OVERVIEW:MutagenizedDNAfromthelibraryisexcisedfromthebacterialvector.Itisthentransformedintoaura3strainofyeast.Thisprocedureisoutlinedinthisfigure.Thebeststrategyistoscreenafewthousandtransformantsfromeachpool.Useofacircle-zerostrainwillpreventrecoveryofinsertionsinthe2-micronplasmid.Screening30,000transformantsshouldgiveyou95%coverageoftheyeastgenome.

Tominimizedoubleintegrants,transformationsshouldcontainthelowestamountofDNApracticable.Wethereforerecommendthatapilotexperimentbeperformedtodeterminetransformationefficiencyofthestrain,andconditionsthenbescaledupasappropriate.ThepilotprotocolgivenbelowusesamodifiedversionofthemethodofChenetal.(1992)..Youshouldusewhatevertransformationprotocolworksbestinyourhands.

1. PlasmidDNAfrompoolsofthemTn-3xHA/GFP-mutagenizedgenomiclibraryisdigestedwithNotI.A2.1-kbbandfromthevectorshouldbeveryapparent,togetherwithabroadbandinthe8-kbregion,representinginserts.BecausesizedgenomicDNAwasusedtomakethelibrary,theinsertbandsarenotveryheterogeneousinsize.
2. A10-mlcultureoftheyeasthoststrainisgrowntoadensityof10⁷cells/ml(O.D.600of1).Useofsuchlogarithmically-dividingculturesincreasestransformationefficiency.
3. Cellsarepelletedandwashedoncewith5volumesofOneStepbuffer(0.2MLiAc,40%PEG4000,100mM[[beta]]-mercaptoethanol).Thiswashisespeciallyimportantwhenculturevolumesareincreased.
4. CellsareresUSPendedin1mlofOneStepbuffercontaining1mgofdenaturedsalmonspermDNA.100ulaliquotsofthissuspensionarethenaddedtotubescontainingfrom0.1to1ugofNotIdigestedplasmidDNA.
5. Tubesarevortexedtomixthecontentsthoroughly,thenincubatedat45^oCfor30minutes.
6. Cellsarepelletedandresuspendedin400ulofSC-ura.200ulisplatedontoSC-uramedium.Platesareincubatedat30^oCfor3to4days.

ScreeningforGFPfusions

Screeningforin-frameGFPfusionsinyeast

WehavenotdoneassaysofGFPactivityinyeast.

SeeNiedenthaletal(1996)fortheirmethods.

AnalyzingGFPfusionproteinlocalizationinyeast

WetestedmTn-3xHA/GFPbymutagenesisofBDF1,whichencodesachromatin-associatedprotein.Wegrewindividualbdf1::mTn-3xHA/GFPtransformantstoadensityof10⁷cells/mlinSC-ura.Thelastfourhoursofgrowthwereatroomtemperature,toallowformationoftheGFPchromophore.ThenweexaminedcellsdirectlyusingaLeitzmicroscopywithasystem13filter(thismaynotbeoptimal).In4of38transformants,wesawgreenfluorescenceofthenucleus.Fixationandspheroplastingofthecellsimprovedthesignal-to-noiseratio.

Identificationofthegenomicsiteoftransposoninsertion

Todeterminethesiteoftransposoninsertion,genomicDNAimediatelyadjacenttothetransposonsequencesmustberescued.WehavenotyetconstructedarescuevectorformTn-3XHA/GFP.Ifdemandforthelibraryishighwewillconstructone.Otherwise,wewillbehappytoprovidereagentsandinformationtoanotherlaboratorywhowishestoconstructit.InversePCRongenomicDNAcouldbeusedtorecoverthesiteofinsertion.Alternatively,CarlFriddlehasdevelopeda"vectorettePCR"rescueprotocolforlacZ-basedtransposons.IhavetranscribedCarl"sprotocolandmodifiedthesuggestedenzymesandprimers,tomakeitsuitableforvectorettePCRofthemTn-3xHA-basedtransposons.

UsingtheHATepitopetaggingfeatureofmTn-3xHA/GFP

Whentransposoninsertionhascreatedanin-framefusiontoGFPinthegeneofinterest,thetransposoncanbeexcizedtoleavea274bpinsertion(sequencegivenbelow)containingthe3xHAtag.Withthe5basepairduplicationcausedbytransposoninsertion,thisgivesanin-frame93-aminoacidinsertionintheprotein.ThepopouteventismediatedbycrerecombinaseandrequiresinductionoftheGAL1-10promoterongalactose.Ourstrainsgrowpoorlyongalactosebutgive80to100%popouts.

TheHAtripletagcanbedetectedbymousemonoclonalantibodies12CA5(Boehringer)orMMS101R(BAbCo,Richmond,California).Theseantibodyrecognisecross-reactingyeastproteinsofabout55kDor110kD,respectively,andcangiveaspottybackgroundonimmunofluorescence.Despitethisdrawback,the3xHAtaghasbeenusedextensivelyandsuccessfullyinyeast.Arabbitpolyclonalantiseraisalsoavailable(101c500;BabCo)butthiswaslessreactiveintheoneinstancewetried.Protocolsforyeastimmunofluorescencecanbefoundhere,orinMethodsinEnzymology194(1991).

1. TransformstrainwithpB227/GAL-cre,selectingonSC-leu.
2. ToderepresstheGALpromoter,inoculatetransformantsinto2mlsSC-ura-leuwith2%raffinoseascarbonsourceandgrowtosaturation.
3. Dilute1/100intoSC-leuwith2%galactoseascarbonsource(control:SC-leuwith2%glucoseascarbonsource).Growfor2days(somestrainsinducewithoutgrowing).
4. Ifgrown,dilute1/100.Spota10uldropontoanFOAplateandstreakitforsingles(non-quantitativeapproach!).OrplatedilutionsontoSCmediaandreplicatoidentifyura-colonies.Theinducedculturesshouldgive100xmoreUra-cellsthanthecontrol.
5. PCRprimersdesignedusingthesequencegivenbelowcanbeusedtodeterminepositionofthetag.TheIRelementsandpalindromicloxRregionshouldbeavoided.

N.B.Whentaggingessentialgenes,theoriginalstraintransformedshouldobviouslybediploid.Youcandissectthepopped-outversiontoseeifthetaggedgeneisfunctional.Onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype.

SequenceofHATtag(3xHA):

TRinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaAATttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

GenBankaccession

• mTn-3xHA/GFPisU54830

Antibioticsused: