请使用支持JavaScript的浏览器! Replication timing using transient hemimethylation_蚂蚁淘,【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
当前位置: > 首页 > 技术文章 >
Replication timing using transient hemimethylation
来自 : 蚂蚁淘

KatherineFriedman


Reference:

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


Rationaleofmethylationtimingapproach

TheE.colidammethylaseaddsmethylgroupstoadenineinthesequence5"GATC3"(MarinusandMorris,1973;GeierandModrich,1979).Thisenzymemethylatesbothunmethylatedandhemi-methylatedsites.InyeastDNA,methylatedbasesareundetectable,unliketheDNAofmosthighereukaryotes(Hattmanetal.,1978).However,thedammethylaseproteincanbeexpressedatmoderatelevelsinyeastwithfewobviouseffectsoncellviABIlityorgrowthrate(Brooksetal.,1983;HoekstraandMalone,1985).ExpressionoftheproteinresultsinmoderatelevelsofDNAmethylation.TheaccessibilityofdifferentGATCsitestomethylationisvariable,presumablyduetodifferencesinchromatinstructurethataffecttheaccessoftheenzymetoDNA(Gottschling,1992;SinghandKlar,1992;Wrightetal.,1992;KladdeandSimpson,1994).

E.coliDNA,thoughnormallyfullymethylated,undergoesatransientperiodofhemimethylationimmediatelyfollowingpassageofthereplicationfork(reviewedinBarrasandMarinus,1989).Thenewlysynthesizedstrandisnotinitiallymethylated,butacquiresmethylationafterdammethylasecontactsthenewlyreplicatedDNAsequence.Methylatedandhemi-methylatedsequencescanbedistinguishedbytheirdifferentialsensitivitytocleavagebyDpnI(GeierandModrich,1979).FullymethylatedDNAiscleavedatGATCsitesandhemi-methylatedorunmethylatedDNAisnot(Figure1).Therefore,thetimeatwhichE.colisequencesbecomeresistanttocleavagebyDpnIcorrelateswiththeirtimeofreplication.AsimilarlogiccanbeappliedtoreplicationofyeastDNAinastrainexpressingthedammethylase.Again,replicationforkpassageresultsinhemi-methylationoftheDNA,detectableasresistancetocleavagebyDpnI(Figure1).

cartoon illustration of rationale

Strainconstruction

InordertoconstructastraincontainingconstitutivelymethylatedDNA,theE.colidammethylasegeneanditsendogenouspromoterwereexcisedfromplasmidpMFH1(HoekstraandMalone,1985)withHindIIIandPvuIIandinsertedintotheHindIII-SmaIsitesofthepolylinkerinpRS305(SikorskiandHieter,1989),aLeu+yeastintegratingvector.Theresultingplasmid(pRS305-DAM)waspartiallydigestedwithClaIandthelinearizedplasmidwasusedtotransformyeaststrainRM14-3a(MATacdc7-1bar1ura3-52trp1-289leu2-3,112his6).CleavageoftransformantDNAwithDpnIwasassessedbySouthernhybridization,andthetransformantexhibitingthehighestlevelofDpnIcleavagewasselected(RM14-3aMeth5strain).

Protocolformethylationtiming

1.Grow~330mlofRM14-3aexpressingdammethylase(RM14-3aMeth5)toanOD660ofabout0.3(3.85x106cells/ml)at23oC.Arrestwith200nMalphafactorfor1-1.25doublingtimes,untilthepercentbuddedcellsstopsincreasing(usually?5%).(RM14-3aisbar1,sothealphafactorconcentrationrequiredislow).

2.Transfercellsto37oCandwaitforcultureitselftoreach37oC.AddPronase(20microgram/mlfinalconcentration--dissolvePronasein5mlminimalmedium).WeusePronasefromCalbiochem.

3.Holdat37oCfor~120minutes(until%buddedcellsreaches95-98%).Removetwosamplesfor0timepoint,thenswirltheflaskinicewaterforoneminuteandreturnto23oC.Iusuallycollectsampleseveryfiveminutesfor55minutes,thencollect65and80minutetimepoints.

4.Collectingsamples:Freeze8mlof0.1%Na-azide,0.2MEDTAin"50ml"Oakridge-typeplasticscrewcapcentrifugetubes(freezetheseslantedat-20oCtoincreasesurfacearea).Duringtheexperiment,mix20mlculturewitha1/20volumeof10%Na-azideina35mlCorextube(onice),immediatelytransfertothetubeoffrozenEDTA/azide,andshakethetubetochillthesampleandmeltthefrozenEDTA.Spinthecellsdowninachilledcentrifuge,washwith1mlcoldwaterinEppendorftubes,andfreezethepelletsat-20oC.

5.ExtracttheDNAbysmashandgrabmethod.

6.Restrictiondigest:

a)WeusuallyresUSPendeachDNApelletin20microlitersofTE,thendigesthalfofeachsamplewithEcoRI(thisallowsustoruntwogelsfromoneexperiment).Itypicallyuse1microliterofEcoRI(at20,000units/ml)foreachsample.After~6hours,theDNAisprecipitated.Oneofthe0minutetimesamplesissavedatthispointasacontrolforthesizeoftheEcoRIfragment(i.e.don"tcutoneofthesampleswithDpnI).Therestofthesamplesareresuspendedin50microliterswater,andanequalvolumeofadigestmixcontainingDpnI,restrictionbuffer,andBSAisadded.After?hours,theDNAisprecipitatedandloadedina0.7%agarosegel.
b)Afterblottingthegel,theblotissequentiallyhybridizedwithafragmentadjacenttoARS1(earlyreplicating),theR11(latereplicating)fragment,andanyotherfragmentsofinterest.

Figure2AandBshowatypicalresultforARS1andR11.

Quantitation

Theblotsarequantitatedbyphosphorimager.TheextentofDpnIcleavageisexpressedasthefractionoftotalsignalinthelanethatisattributabletothefull-lengthEcoRIfragment.ThisfractionshouldincreaseasthefragmentisreplicatedbecausetheDNAbecomestransientlyresistanttoDpnIcleavage.Thefractionofsignalinthefull-lengthEcoRIfragmentisthennormalizedtothelargestvalueandplottedversusthetimeafterreleasefromthecdc7block(seeFigure2C).


References

Barras,F.,andM.G.Marinus.1989.ThegreatGATC:DNAmethylationinE.coli.TrendsGenet.5:139-143.

Brooks,J.E.,R.M.Blumenthal,andT.R.Gingeras.1983.TheisolationandcharacterizationoftheEscherichiacoliDNAadeninemethylase(dam)gene.NucleicAcidsRes.11:837-851.

Geier,G.E.,andP.Modrich.1979.RecognitionsequenceofthedammethylaseofEscherichiacoliK12andmodeofcleavageofDpnIendonuclease.J.Biol.Chem.254:1408-1413.

Gottschling,D.E.1992.Telomere-proximalDNAinSaccharomycescerevisiaeisrefractorytomethyltransferaseactivityinvivo.Proc.Natl.Acad.Sci.USA89:4062-4065.

Hattman,S.,C.Kenny,L.Berger,andK.Pratt.1978.ComparativestudyofDNAmethylationinthreeunicellulareucaryotes.J.Bacteriol.135:1156-1157.

Hoekstra,M.F.,andR.E.Malone.1985.ExpressionoftheEscherichiacolidammethylaseinSaccharomycescerevisiae:Effectofinvivoadeninemethylationongeneticrecombinationandmutation.Mol.Cell.Biol.5:610-618.

Kladde,M.P.,andR.T.Simpson.1994.Positionednucleosomesinhibitdammethylationinvivo.Proc.Natl.Acad.Sci.Usa91:1361-1365.

Marinus,M.G.,andN.R.Morris.1973.IsolationofdeoxyribonucleicacidmethylasemutantsofEscherichiacoliK-12.J.Bacteriol.114:1143-1150.

Sikorski,R.S.,andP.Hieter.1989.AsystemofshuttlevectorsandyeasthoststrainsdesignedforefficientmanipulationofDNAinSaccharomycescerevisiae.Genetics122:19-27.

Singh,J.,andA.J.Klar.1992.ActivegenesinbuddingyeastdisplayenhancedinvivoaccessibilitytoforeignDNAmethylases:anovelinvivoprobeforchromatinstructureofyeast.GenesDev.6:186-196.

Wright,J.H.,D.E.Gottschling,andV.A.Zakian.1992.Saccharomycestelomeresassumeanon-nucleosomalchromatinstructure.GenesDev.6:197-210.

免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。