自噬与核糖体的关系

KellyandBedwell

TABLE2.PlasmidsusedPlasmidpDB1227pDB1226pDB0690pDB0691pDB0722pDB0723pDB0720pDB0721pDB0688pDB0689pYDF23

DescriptionPOP2-HA/YCplac33TPA1-HA/YCpLac22CTY775/lucCGACCTY775/lucUGACCTY775/lucCAACCTY775/lucUAACCTY775/lucUAGCCTY775/lucCAGCCTY775/lucCAAACTY775/lucUAAALEU2,CEN/ARStor1-1

ReferenceThisstudyThisstudy

Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Keelingetal.(2004)Reinkeetal.(2006)

cycloheximide,1mg/mLheparin).GradientswerecentrifugedinaBeckmanSW41rotorat35Krpmfor160minat4°C.Fractionswerecollected,andabsorbanceat254nmwasrecordedusinganISCOUA-5absorbancemonitor(Teledyne).

quencingusingprimersDB3023,DB3024,andDB3025.pDB1226wasmadebyfirstamplifyingtheTPA1ORF932bpupstreamstartand200bpdownstreamstopcodonwithprimersDB0873andDB0874andinsertingthisintothePstI–BamHIsitesofYCpLac22(GietzandSugino1988).ASalIrestrictionsitewasthenintroducedatthestopcodonusingsite-directedmutagenesisandprimersDB0876andDB0877.TheamplifiedfragmentwassubclonedbackintotheoriginalconstructusingtheKpnIandBamH1sites.Finally,a～250bpSalI–BamHIfragmentwithaSalIsitefollowedbyanHAtagand200bpoftheTPA13′UTRfollowedbyaBamHIsitewasamplifiedwithDB0874andDB3184andinsertedintotheSalI–BamH1sitesoftheconstruct.ForpDB1287,the5′UTRandORFofSCH9wasamplifiedandHA-taggedusingprimers3933and3934fromchromosomalDNAgivinga～3000bpfragmentwithHindIII–BamHIsites.ThiswasclonedintotheHindIII–BamHIsitesofYCplac33(GietzandSugino1988).The500bp3′UTRwasamplifiedusingprimers3935and3936tointroduceBamHIandSacIsites.Thiswasthenclonedintothelargerconstruct.Finally,plasmidpYDF23,whichcarriesthedominant-negativetor1-1allele,wasakindgiftfromDr.TedPowers.

Strains

TheSaccharomycescerevisiaestrainsusedinthisstudyaredescribedinTable1.StrainsYDB0665,YDB0657,andYDB0707werederivedfromstrainMHY501usingstandardgenetictechniques.StrainsYDB0681,YDB0680,YDB0682,YDB0688,YDB0695,YDB0683,YDB0687,YDB0689,andYDB0717werederivedfromstrainsMHY501,MHY659,orYDB0665byintegratingcarboxy-terminaltagsintotheappropriategenomiclocusasdescribed(Longtineetal.1998).YDB0686andYDB0679weremadeusingstandardge-netictechniquesstartingwithstrainYDB0646/pDB1227.StrainsYDB0708andYDB0709werederivedfromYH129/wandYH129/1,respectively.StrainsYDB0694andYDB0695werederivedfromYWO607andYWO612asdescribed(Longtineetal.1998).yJC750wasagiftfromDr.JeffColler.YJW615wasagiftfromDr.JonathanWeissmanandYH129/w,YH129/1,YWO607,andYWO612weregiftsfromDr.DieterWolf.

Dualluciferaseassays

Thedualluciferasereportersusedtomonitorreadthroughofstopcodonsinyeastweredescribedpreviously(Keelingetal.2004).Strainsharboringtheindicatedplasmidsweregrowntologinselec-tivesyntheticmediaandassayedforreadthrougheitherduringnu-trientexcessor6haftertheonsetofnitrogenstarvation.Thedualluciferaseassayswerecarriedoutaspreviouslydescribed(Grentzmannetal.1998;Keelingetal.2004).Thissystemmonitorsreadthroughofastopcodonbymeasuringfireflyluciferaseactivity,andallowsthenormalizationtothelevelofupstreamRenillalucif-eraseactivityexpressedinthesameopenreadingframe.Assayswerealsodonewithreporterthatcontainedasensecodoninplaceofthestopcodontodeterminemaximum(100%)readthrough.

Serialdilutionassays

CellsweregrownovernightinYPD.Ofnote,1A600unitofcellswasspundownandresuspendedin1mLsterilewater.Fivefoldserial

Plasmids

TheplasmidsusedinthisstudyaredescribedinTable2,whiletheoligonucleotidesusedareshowninTable3.pDB1227wasmadebyfirstamplifyingaSalI-POP2-HA-BamHIfrag-mentfromchromosomalDNAusingprimersDB2996andDB2997,andinsertingtheresult-ingfragmentintotheSalI–BamHIsitesofYCpLac33(GietzandSugino1988).AHindIII–SalIfragmentcorrespondingtothe500bpprecedingthestartcodonwasthenam-plifiedfromchromosomeDNAusingprimersDB2998andDB2999wasinsertedintotheHindIII–stly,afragmentcorre-spondingto248bpdistaltothestopcodonwasamplifiedusingprimersDB3000andDB3001fromchromosomeDNAandinsertedintotheBamH1–SacIsitesoftheconstruct.Thefinalconstructwasconfirmedbyse-

TABLE3.OligonucleotidesusedOligonucleotideDB0873DB0874DB0876DB0877DB2996DB2997DB2998DB2999DB3000DB3001DB3184

Sequence

5′-CCGGCTGCAGATCAAGAATGCTAATCAATTC-3′5′-CCGGGGATCCAGTTAAACTTATATTCATTC-3′5′-GGAAGATGAAGCGTCGACAATTAACCCGTC-3′5′-GACGGGTTAATTGTCGACGCTTCATCTTCC-3′5′-GGCCGTCGACATGCAATCTATGAATGTACA-3′5′-GGCCGGATCCTTATTATGCGTAATCCGGCACGTCGTAGGGATATTGGTCCCCATCAATACCGT-3′5′-GGCCAAGCTTGAAGAAAGAAGTTGAGAAGA-3′5′-GGCCGTCGACAATCCTTTTTGACCCTTTAT-3′5′-GGCCGGATCCTCATTTATGTACTATATGTA-3′5′-GGCCGAGCTCTCTTTCATTAATCGCCACCT-3′5′-GGCCGTCGACGTATCCCTACGACGTGCCGGATTACGCATAAACAATTAACCCGTCTTATTA-3′

908RNA,Vol.21,No.5