M.K.Raghuraman

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Reference:

Friedman,K.L.,M.K.Raghuraman,W.L.Fangman,andB.J.Brewer(1995)AnalysisofthetemporalprogramofreplicationinitiationinyeastchromosomesJ.CellSci.Suppl.19:51-58.

Theprinciple:Anon-replicatingDNAcircleiscreatedinvivobysite-specificpop-outrecombination.Thisnon-replicatingfragmentisthenusedasaninternalcontroltomeasurethetwo-foldncreaseincopynumberofreplicatingchromosomalsequencesbetweenthestartandendofSphase.

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1.Growthecellsinminimalmediumwith1%Na-acetateor3%glycerolasthecarbonsource,aimingforabout20mlcellspertimepoint.(Raffinosehasnotworkedwellforme,soIavoidit.)

2.Arresttheculturewithalphafactor(200nMfinalconcentrationforbar1strains)whentheOD660isabout0.2-0.25.

3.Whenthecellshavearrested(morethan95%unbudded),remove20mlforthe"Uninduced"control,andadddrygalactose(2%w/vfinalconcentration)totheremainderoftheculture.

4.Allowtheinductiontoproceedfor2.5hr(R-KT-DIR)or4hr(R-Zeo-DIR),thenadddryglucose(2%w/vfinalconcentration).Thirtyminuteslater,shifttheculturetothe37^obath.

5.Oncetheculturehasreached37^oC(ittakes10-15min),addPronase(0.01-0.05mg/mlfinalconc)toreleasethecellsfromthealphafactorblock.[YoucanadddryPronase.IusuallydissolvethePronasein5mlofminimalmedium,justtoavoidhavingthedryPronasesticktothesidesoftheflask.]

6.Holdat37^oCfor90-120min(untilbuddedcellsaccumulate).Removeonesample(t=0),thenswirltheflaskinice-waterandreturnitto23^oC.Continuetocollectsamplesevery4min(untilt=80minormore).Iusuallytaketwo0-minsamples,andtakesamplesevery4minfromt=8throught=56,thentaket=64,72,and80minsamples--19samplesinall.

Theice-watertreatmentistoquick-chillthecultureto23^oC,andisusuallyfor~1min.Youcanempiricallydeterminehowlongittakestobringyourdesiredvolumeofmediumfrom37^oto23^oC.

Collectingsamples:Freeze8mlof0.1%NaN3,0.2MEDTAin"50ml"Oakridge-typeplastic,screw-cappedcentrifugetubes(frozenslantedtomaximizethesurfacearea).Duringtheexperiment,mix20mlofcellswith1/50volumeof10%NaN₃ina35mlCorextube(onice)andimmediatelytransferthemixtoatubeoffrozenEDTA/NaN₃.Alternatively,squirtthe10%azideonthefrozenazide/EDTAandimmediatelyaddthecellsample.VortexorshakethetubevigorouslytochillthesampleandbreakupthefrozenEDTA.Spindownthecellsinachilledcentrifuge,washwith1mlcoldwaterpersampleinEppendorftubesandfreezethepelletsat-20^oC.

7.ExtracttheDNA(smash-&-grabwithglassbeads);digest1/4to1/3ofitwiththeenzymeofchoicein40-60microliters(EcoRIworkswell,asitcutstheARS-lesscirclejustonce).Spliteachdigestintwoorthreetoruninduplicate(ortriplicate)gels.Ithelpstomakewide,well-separatedwells.Blotthegelsasusual.

8.HybridizesimultaneouslywithprobesfortheARS-lesscircle,anearlyMarker,alatemarker,andthesequenceofinterest.Quantitationcomesoutbestifthefragmentstobeprobedareinthe2kb-8kbrange.Sometimesthefragmentsjustdon"tworkout(i.e.,twofragmentsaretoocloseforcomfort),inwhichcaseyou"llhavetostriptheblotsandre-probe.Ifso,IusuallyincludetheARS-lesscircleprobeforeachhybridization.

9.Quantitation:weuseaMolecularDynamicsPhosphorImageroraPackardInstantImager.Thevaluesobtainedforthetwot=0samplesarecombinedandusedasthezerotimepoint(tobeusedfornormalizationofthelatersamples).

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Dothequantitationforeachgelandplotthemeanvaluesforeachtimepoint.

Here"satypicalplotshowingreplicationofARS1andR14(afragmentneartherightendofchromosomeV):

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Strains:R-KT-DIRandR-Zeo-DIRarebothderivedfromRM14-3a(MATa,cdc7,his6,leu2,trp1,ura3,bar1).TheARS-lesscassetteinR-KT-DIRisaKan^r-TRP1fragmentintegratedneartherightendofchromosomeV.Thet_1/2forexcisionofthiscassetteisabout1hr.TheARS-lesscassetteinR-Zeo-DIRcontainstheZeocin-resistancemarkerandaportionofthebacterialKanamycinresistancegene,andisintegratedatLEU2.Thet_1/2forexisionofthiscassetteisabout2hrat23degreesC.BothstrainshavetheZygosaccharomycesrouxii"R"recombinasegenebehindtheGALpromoterintegratedatLEU2.BothstrainsareLeu+.

Pronase:WeuseCalbiochem"sPronase(catalog#53702),whichseemsprettyclean--i.e.,platesspreadafterPronasetreatmentaren"tcontaminated--butIheardfromsomeonewho"dusedSigma"sPronaseandhadmassivegrowthofStreptomyces.