[PDF] after deflagellation and during the cell cycle. gene Chlamydomonas

View - Download after deflagellation and during the cell cycle. gene Chlamydomonas

Previous PDF

Next PDF

1994, 14(8):5165. DOI: 10.1128/MCB.14.8.5165. Mol. Cell. Biol. J P Davies and A R Grossman

after deflagellation and during the cell cycle.geneChlamydomonas reinhardtii beta 2-tubulin Sequences controlling transcription of the

http://mcb.asm.org/content/14/8/5165

Updated information and services can be found at:

These include:

CONTENT ALERTS

more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to: on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

JOHNP.DAVIES*ANDARTHURR.GROSSMAN

MATERIALSANDMETHODS

5165 on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

5166DAVIESANDGROSSMAN

-170-160-150 -140-130

EcoRIKpnISall-60-50-40-30-20-10

IATAATATTSalI

ATTATAGCGAGCTACCAAAGCCATATTCAAACACCT

AsynchronousculturesweregrownonTris-acetate-phosphate(TAP)medium(18)orTAPmediumsupplementedwith50,ugofarginineperml.Gametesweremadefromcultures(5x106to8x106cellsperml)bywashingthecellstwicewithmediumV(36)andresuspendingtheminmediumVfor16to20h.ThegametesweredeflagellatedbypHshock(38).Synchronouscellculturesweremaintainedona12-h/12-hlight-darkcycleinTAPmedium(35).Cellgrowthwasmonitoredbycountingthecellsinahemocytometer.Cloning.ToexaminethefunctionofconservedsequenceswithinthetubB2promoter,wegeneratedaseriesoftubB2/arschimericgenescomposedofmutatedcopiesofthetubB2promoterfusedwiththearsreportergene.Mutationsina1.5-kbpEcoRIfragmentcontainingthetubB2promoterinpBluescriptKS+weregeneratedbyoligonucleotide-directedmutagenesis(2).TheoligonucleotideTAATGTC'TlTTGCAATAATATTATGGCTATl7F'AAACAGwasusedtochangetheGC-richregionlocatedbetweentheTATAboxandthetranscriptioninitiationsiteofthetubB2promoter(AGCCA-iGCCCCATT)toanAT-richregion(AGCCATAATATTATT)(Fig.1)(positions-12to-19).ThischangeablatestheGC-richregionandreducesthespacingbetweentheTATAboxandtranscriptioninitiationsiteby1nucleotide.TheplasmidcontainingtheentiretubB2promoterwiththismuta-tionwasrestrictedataKpnlsiteinthemultiplecloningsiteabout900bpupstreamofthetranscriptionstartsiteandatanXhoIsite65bpdownstreamofthetranscriptionstartsite.Thisfragment(about1kbp)wasfusedtoarstogeneratethenewplasmid,ptubB2AT/ars.TheoligonucleotidesTCTCGCAGCCCGCGGTACC'1TYTTGCTGGandCGGGGGGTCGAGGTACCATCGGTGTTGCATGwereusedtointroduceKpnIsites,singly,atpositions-144and-95relativetothetubB2transcriptioninitiationsite,andtheoligonucleotidesCGGCACGGAGCGTCGACGCAGCCCCGAAGGGandTGGCTATFlTAAACAGTCGACTGGCCCTGGAGCwereusedtointroduceSallsitesatpositions-64and-36,respectively(Fig.1).Togeneratethemutantchimericgenes,themu-tagenizedtubB2promoterswerecutonceattheintroducedrestrictionendonucleasesiteandonceattheXhoIsiteinthe5'-transcribedbutuntranslatedregionandfusedtothearsreportergene(12).Theseconstructsgeneratedaseriesofchimericgeneswithpromoterscontaining144,95,64,or36bpofthetubB2sequenceupstreamofthetranscriptionstartsite;theplasmidsharboringtheseconstructsweredesignatedptubB2AUS/ars,ptubB2AUS,3/ars,ptubB2AUS,3,2/ars,andptubB2AUS,3,2,1/ars,respectively.Diagramsofthesecon-structsarepresentedinFig.2.TheplasmiddesignationsarebasedonregionsdeletedfromtheoriginaltubB2promoter.TherearethreegroupsoftubboxmotifspresentinthetubB2promoter;thesearedesignated3, 2,and1(from5'to3').TheregionupstreamofthesemotifsisdesignatedUS(upstreamsequences).Intheplasmidname,anyregionnotedaftertheAsymbolhasbeendeletedfromthetubB2/arschimericgene.AconstructwiththetubB2sequencefromposition-64to-95

Construct

Number

1ptubB2/ars

2ptubB2AT/ars

3ptubB2AUS/ars5'tubB2ars

Us321GC

IAf e_00DL._.-Cells

Co-transformedTransformantsExpressingArs

7/26(27%)26/595(4.4%)

3/15(20%)38/751(5.1%)

ND

29/591(4.9%)

4ptubB2AUS,3/ars

5ptubB2AUS,3,2/ars

6ptubB2AUS,3,2,1/ars

7ars(nopromoter)

kbp-0.94/15(27%)40/690(5.8%) -0.2-0.100.10.2

0=L,I..-L_-le

ND19/1182(1.6%)

4/15(27%)13/1319(1.0%)

2/684(0.3%)_AbMOL.CELL.BIOL. on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

RESULTS

atposition-144,-95, -64,or-36relativetothetranscrip-tioninitiationsite(Fig.1)(seeMaterialsandMethods).Themutatedpromoterswerefusedtothearsreportergene,formingconstructsptubB2AUS/ars,ptubB2AUS,3/ars,ptubB2AUS,3,2/ars,andptubB2AUS,3,2,1/ars(Fig.2).Thechimericgeneswereintroducedintothecellwall-deficient(cwl5),arginineauxotrophic(arg2)C.reinhardtiistrainCC425viacotransformationwiththearg2gene(encod-ingargininosuccinatelyase)astheselectablemarker(14).EquimolaramountsofplasmidDNAcontainingtheselectablemarkerandthetubB2/arsconstructswereusedfortransforma-tion.Toselectfortransformants,cellswerespreadonsolidTAPmediumlackingarginine;transformantswereabletogrowintheabsenceofexogenouslyappliedarginine.Todeterminethefrequencyofcotransformation,randomlychosentransformantswereexaminedforthepresenceoftubB2/arsbyDNAgelblothybridizationwithradiolabeledarsDNA.Celllinescontainingarshybridizingsequencesthatwere

onceinternally.DigestedgenomicDNAwasfractionatedbyelectrophoresis,blottedontonitrocellulose,andhybridizedwithsequencesfromeitherthe5'orthe3'portionofars.IngenomicDNAcontaininganintactcopyofthechimericgene,DNAfragmentshybridizingwiththe5'and3'portionsofarswillbethesamesizesasthecorrespondingfragmentsfromthetubB2/arsplasmid.However,genomicDNAcontainingadis-VOL.14,1994 on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

5168DAVIESANDGROSSMAN

ruptedcopyofthechimericgenewillcontainfragmentsthatareeitherlargerorsmallerthanfragmentsfromtheplasmidDNA.Inall12celllinesanalyzedinthismanner,DNAfragmentsfromthe5'or3'endofthechimericgenewereeitherlargerorsmallerthanthepredictedsize.Thisindicatedthataportionofthechimericgenewaslostorrearrangedduringitsintegrationintothechromosome(datanotshown)andwouldaccountforthefindingthatthesecelllinesdonotexpressthechimericgene.ThenumberoftransformantsexpressingArswasdependentontheconstructintroduced.CotransformationwithptubB2/ars,ptubB2AT/ars,ptubB2AUS/ars,andptubB2AUS,3/arsre-sultedinahigherpercentageofArs-expressingtransformantsthandidcotransformationwithptubB2AUS,3,2/arsandptubB2AUS,3,2,1/ars(Fig.2)(5.8to4.4%comparedwith1.6and1.0%,respectively).BecauseptubB2AUS,3,2/arsandptubB2AUS,3,2,1/arswereapparentlycotransformedatthesamefrequencyastheotherconstructs,itmaybethatexpres-sionfromptubB2AUS,3,2/arsandptubB2AUS,3,2,1/arswasmoresensitivetothesiteofintegrationintothechromosomethanexpressionfromtheotherconstructs.Asacontrol,weintroducedthearsreportergenewithoutapromoterandanalyzedover600transformantsforArsexpres-sion.WefoundonlytwothatexhibitedArsactivityinTAPmedium;thelevelsofArsactivityinthesetwostrainsweremuchlowerthanthatobservedfortransformantsinwhichthechimericgenecontainedapromoter.ArsexpressioninthesecellsprobablyresultedfromintegrationofthearsgeneintothechromosomenearsequencesthatservedasapromoterorfromthegenerationofamutationcausingArstobeconstitutivelyexpressed.Whennoarsgenewasintroduced,only1in5,000transformantsexhibitedArsactivity(13).DNAgelblotanalysesofallstrainstestedconfirmedthattransformantsexpressingArsactivityinTAPmediumaftercotransformationwithatubB2/arsconstructcontainedatleastonecopyofthechimericgene.Specifically,31celllineswereexamined,and21hadonlyonecopyofthetubB2/arschimericgenewhile7hadtwocopiesand3hadmorethantwocopies.SomeoftheArs-expressingcelllineswerefurthercharacter-izedtodeterminewhethertheyreceivedtheentire5'portionofthechimericgene.Most(13of15)ofthecelllinesexpressingArsaftertransformationwithconstructscontaining900bpupstreamofthetranscriptionstartsite(ptubB2/ars,ptubB2AT/ars,ptubB2A2/ars,andptubB2A1/ars)containedatleast800bpoftheupstreamsequences,andthe otherscontainedatleast300bp.Most(11of12)ofthecellsexpressingArsaftertransformationwithchimericgeneshaving144bporfeweroftubB2promotersequences(ptubB2AUS/ars,ptubB2AUS,3/ars,ptubB2AUS,3,2/ars,andptubB2AUS,3,2,1/ars)containedtheentireportionofthetubB2promoterpresentintheoriginalchimericgene(datanotshown).RNAgelblotanalysesofArs-expressingstrainsconfirmedthatthechimericconstructsweretranscribed.Theuntrans-formedstrain,CC425,accumulatednoarsmRNAinvegetativecellsgrowinginTAPmedium(12)(Fig.3A,lane1),whiletransformantsexpressingArsactivityaccumulatedtranscriptsthathybridizedwiththearscDNA(Fig.3A,lanes2to7).HybridizationwithtubB2-specificsequencesdetectedboththeendogenoustubB2transcriptandthechimerictranscript(Fig.3B).Becausethereareonly65basesofthetubB2sequenceinthechimerictranscript,hybridizationwithtubB2wasmuchweakerthanwithars.However,longexposuresoftheblothybridizedwithtubB2DNAconfirmedthepresenceofthechimerictranscript(datanotshown).TodeterminewhetherthemutationsinthetubB2promoteraffecttranscriptionduringvegetativegrowth,wecomparedL.eqMr--

L.

CMd:<.

11)esr4

"cc3Xt4S-'EA uB..Br..H..lfubB21ars_-ars

1234567

B tubB21ars_- tubB2_ C 23S_-

18S0tubR2

12345 67

_t~~~~~~~~~~~~~~~~I

FTY__-

1234567

chimerictranscriptaccumulationlevelsinseveraltransfor-mantsexpressingeachconstruct.Sincetransformantscontain-ingmultiplecopiesofthechimericgenemayalsobeexpressingmorethanonegene,wecomparedtranscriptlevelsonlyamongtransformantscontainingasinglecopyoftubB2/ars.Thedifferenceintranscriptaccumulationwithintransformantsexpressingthesameconstructwasasgreatasthedifferenceamongtransformantsexpressingdifferentconstructs.Thus,withinthelimitsofourassay,wecoulddetectnocis-actingelementbetweenbp-900and-36(relativetothetranscrip-tioninitiationsite)thatgreatlyenhancesorrepressestran-scriptionoftubB2duringasynchronous,vegetativegrowth.SincethelevelofthechimericmRNAthataccumulatedintheseconstructsisapproximately10%ofthatobservedfortheendogenoustubB2transcript,itispossiblethatweeliminatedanenhancerelementduringconstructionofptubB2/ars(theenhancerelementcouldbemorethan900bpupstreamorwithinthecodingor3'untranslatedregionofthetubB2gene).Identificationofsequencesnecessaryforinducedtranscrip-tionfollowingdeflagellation.TotestwhethertheGC-richsequencesandtubboxmotifs arenecessaryforincreasedtranscriptionfollowingdeflagellation,weperformedRNAgelMOL.CELL.BIOL.

J,

0.=r- on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

SEQUENCESCONTROLLINGTUBULINTRANSCRIPTION

tubB2Iars_. B

0306012003060120

tabB2_-. C

0306012003060120030120

23S-_-Eu

18S

DptubB2AUS.3,2.:'arsp1ub2AUS.i3,2,Ulars

1}306012003{60120

.-s..t>'X.w...... u.

03060120030601200306012014.4)601211

FIG.4.RNAgelblotanalysesof transformantsexpressingchi-mericgenes(designationsareasinFig.2)afterdeflagellation.GameticcellsweredeflagellatedbypHshock,andRNAwasisolated0,30,60,and120minafterdeflagellation.Afterseparationbyelectrophoresis,theRNAwastransferredtonitrocellulosefiltersandhybridizedwitharscDNA(A)andtubB2-specificsequences(B).TheRNAblotsinpanelsAandBwereexposedtofilmfor16to20h.(C)Ethidium-stainedgel,witheachlanecontainingapproximately5jigofRNA.TheRNAblotsinpanelDwerehybridizedwiththearscDNAandexposedtofilmfor3to5days.ThemoreslowlymigratingbandspresentinptubB2AUS,3,2/arshavenotbeenidentified.Numbersoftransformantsexamined:ptubB2/ars,9;ptubB2AT/ars,5;ptubB2AUS/ISars,11;ptubB2AUS,3/ars,5;ptubB2AUS,3,2/ars,8;ptubB2AUS,3,2,1/

4t ......Wlm--

O..:.:::

00W.-W!"'I"':-,

s..

A66-.",-dommikk

mmpqwl.'Icpw on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

5170DAVIESANDGROSSMAN

A tiiifl22 (.'0IIsTFICI

NLliIIhL'

A I~-

LIfi\L1K3.I/a!1l-,

pIul).\lS.3.1;L1 khp-Ii()II.?(111},1).I ptuhtit)12A\US.3/ars trfhbB2-_ I)

34)6)124))612)4)34()(12))3))36)124)

I~~- ~~~~~~~~~~:..:::_-!T_

FIG.5.RNAgelblotanalysesoftransformantsexpressingchimericgenesafterdeflagellation.(A)Schematicdrawingsofthe tubB2/ars

23S_

180-4)306020

:.1{- -4bMOL.CELL.BIOL. _.(IC

I1)[LihI32).Mk'S."/IItF.,

I)LIhIi_2%-'!.r-.

L)ptLI1)i2.\/JI-\

M&A-I.R....... on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

WehavemeasuredtranscriptaccumulationfromconstructswithvariousmodifiedtubB2promotersfusedtothearsre-portergeneintransformedC.reinhardtii.Toourknowledge,thisisthefirstdetailedpromoteranalysisusingthisorganism.AccumulationofthetubB2transcript,aswellastranscriptsofothertubulin-encodinggenesinthisorganism,increasesfol-lowingdeflagellation,duringwhichtimethereisrapidflagellarregeneration.Thea-and,-tubulingenesofC.reinhardtiihavesimilarpromotersequences;thereisaGC-richelementbe-tweentheTATAboxandthetranscriptioninitiationsiteandmultiplecopiesofa10-bpconservedsequence(thetubboxmotif)(6).WehaveexaminedtheeffectofchangingtheGC-richsequenceordeletingthetubboxsequencesonthetranscriptionofchimericgenesinasynchronousandsynchro-nousvegetativecellsaswellasindeflagellatedgameticcells.Wehavereachedseveralconclusionsbasedontheworkpresentedhere.AlteringtheGC-richsequencedownstreamoftheTATAboxinthetubB2promotertoanAT-richsequencedoesnotgreatlyaffecttheexpressionoftubB2/arsinanasynchronouscellculture.AsimilarGC-richsequenceispresentinanumberofthegenesofC.reinhardtii,includinggenesencodingtheothertubulinsubunits(6),thesmallsubunitofribulose-1,5-bisphosphatecarboxylase/oxygenase(17),a70-kDaheatshockprotein(24),aradialspokeprotein(Rsp3)(37),achlorophylla/b-bindingprotein(19),andaproteinwithhomologytothe,BsubunitofaGprotein(31).Thepresenceofthissequenceinsomanygenesledinvestiga-torstospeculatethattheGC-richregionwasimportantfortranscription.ItwaspreviouslyreportedbyBandziulisandRosenbaum(4)thatinterruptingtheGC-richsequenceinthetubAlpromoterwitha12-bpsegmentofDNAseverelyinhibitedtranscriptionofatubAl/catchimericgeneinXenopusoocytes.OurresultssuggestthattheGC-richregionisnotimportantforconstitutivetranscriptionofthetubB2geneinvegetativecultures.ThereareanumberofdifferencesbetweenourstudiesandthoseofBandziulisandRosenbaum(4).WeanalyzedexpressionfromthetubB2promoterfusedtothearsreportergeneinC.reinhardtii,whileBandziulisandRosen-baum(4)workedwiththetubAlpromoterfusedtothecatreportergeneandexaminedexpressioninXenopusoocytes.Perhapsmoreimportantly,wechangedtheGC-richsequencesbyoligonucleotide-directedmutagenesisandreducedthenum-berofbasesbetweentheTATAboxandthetranscriptioninitiationsitebyonlyone.BandziulisandRosenbaum(4)introduceda12-bpsegmentofDNAintothemiddleoftheGC-richsequence;thissubstantialchangeinthedistancebetweentheTATAboxandthetranscriptionstartsitemaysignificantlyaffecttranscription(25).Ourresultsalsoindicate thatinasynchronous,vegetativeculturesthetubB2promotercontainingonly35bpupstreamofthetranscriptioninitiationsiteissufficienttodriveconstitutivetranscriptionofthechimericgenewithanefficiencysimilartothatofthepromotercontaining900bp.Sincealltubboxmotifs

areupstreamof-35,thesesequencesdonotappeartoserveasenhancersorrepressorsoftranscriptionduringvegetativegrowth.InadditiontoexaminingexpressionofthetubB2geneinvegetativecultures,weexploredtheroleoftheGC-richregionandthetubboxesongeneexpressionfollowingdeflagellationofgameticcells.WhentheGC-richsequenceswereconvertedtosequencesenrichedforAandT,therewasnogreateffectontheinducedtranscriptaccumulationofthetubB2/arschimericgenefollowingdeflagellation.Chimericgenescontainingdele-tionsoftubboxsequenceswerealsotestedfortheirabilitytoVOL.14,1994 on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

5172DAVIESANDGROSSMAN

A

FIG.6.RNAgelblotanalysisoftransformantsexpressingchimericgenes(designationsareasinFig.2)duringsynchronouscellgrowth.CellsweregrowninTAPmediumona12-hlight/12-hdarkcycle.(A)Averagecelldensityduringthecellcycleoffiveculturesthathadstartingdensitiesofapproximately106cellsperml.Thecelldensitiesweredeterminedbycountingthecellsinahemocytometer.Bars,standarderrorsofthemeans.Lightanddarkperiodsareindicated.RNAwasisolated2,6,10,14, 18,and22hfromthestartofthelightperiod.RNAwasseparatedbyelectrophoresisinagarosegels,trans-ferredontonitrocellulosefilters,andhybridizedwitharscDNA(B)ortubB2-specificsequences(C).(D)Ethidium-stainedgelswitheachlanecontainingapproximately10,ugofRNA.Numbersoftransformantsexamined:ptubB2/ars,four;ptubB2AUS,3/ars,two;ptubB2AUS,3,2/ars,one;ptubB2AUS,3,2,1/ars,three.RNAaccumulationlevelsinalltransformantsexpressingthesameconstructweresimilar.

HoursIntoCellCvcle

BCC425

-floursinto2610141822cellcycle ....::.:.:.:..... .............;s tubB21ars:: lHoursinto2610141822 cellcycle..;...

MMbB2D~~~~ ~~ ~~ ~~ ~~~~~~..ptulhl/lars

261014 1822ptthlIbB2AUlS.,3/ars

2610141822

2610141822

.:.pti61)LS2,2,I.'rs

26)I(14IX22

261014 18222610141822

floursintI261014182226101418222610 1418 2cellcvcle201014182221610141822 23S_
18S_

inducetranscriptaccumulationfollowingdeflagellation.Theinclusionoftwoorthreegroupsof tubboxsequencesinthe

4 E -D 4] Qw0 09 V3 2

LightDark

LightDark

001020IMOL.CELL.BIOL. on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

noraconstructinwhichtubboxgroup2wasmaintainedatitsoriginalpositioninthetubB2promoterbutwiththesequencesoftubboxgroup1changed(ptubB2XlAUS,3/ars)couldinduceelevatedtranscriptionfollowingdeflagellation.Becausetwoconstructswithdifferentsetsoftubboxes(ptubB2AUS,3/arsandptubB2A/Vars,containingfourandfivetubboxmotifs,respectively)showedincreasedtranscriptaccumulationfollow-ingdeflagellationwhilethreedifferentconstructswithonlyonetubboxgroup(ptubB2AUS,3,2/ars,ptubB2AUS,3,1/ars,andptubB2XlAUS,3/ars,containingtwotubboxsequencemotifs)didnot,wespeculatethataminimalnumberof tubboxsequencesarerequiredforincreasedtranscriptionfromtubu-lingenepromotersfollowingdeflagellation.Theconstructinwhichonlytubboxgroup2wasdeleted(ptubB2A2/ars)hasfivetubboxsequencemotifsbutcouldnotsupportinducedtranscription.Inthisconstructtheadjacenttubboxmotifs(fromgroups1and3)arefartherapartthanintheotherconstructsandmaybeonseparatesidesoftheDNAhelix.Therefore,wesuggestthatthedistancebetweenadjacenttubboxmotifs,andperhapstheirorientationwithrespecttoeachother,iscriticalforinducedtranscription.Alltubgeneshavemultiplecopiesoftubboxes,andtheirtranscriptionmayberegulatedbyamechanismsimilartotheoneregulatingtranscriptionoftubB2.However,thersp3andrsp6genes,whosetranscriptsincreasefollowingdeflagellation,haveonlyoneandtwocopiesofthetubboxsequencemotif,respectively.Ourdatasuggestthatthesesequencesarenotsufficienttosupportinducedtranscriptionfollowingdeflagel-lation.Thus,othersequencemotifsmaybeinvolvedinregu-latingtheaccumulationoftranscriptsfromthesegenes.Accumulationofboththea-andthe,-tubulintranscriptsisobservedfollowingdeflagellationofC.reinhardtii(7,23,32)andafterdeciliationofTetrahymenapyniformis(33).Duringthistimelargequantitiesoftubulinarerequiredfortheregenerationofflagellaorcilia.Thecorrelationoftheaccu-mulationoftubulinmRNAandtheamountoftubulinre-quiredbythecellraisesthepossibilityofautoregulatorycontroloftubulinsynthesis.Inanimalcellsthestabilityof,B-tubulintranscriptsismaintainedbyanautoregulatorysysteminwhichthelevelofunpolymerizedtubulinsubunitswithinthecellaltersthestabilityofP-tubulinmRNA(10).Whenthelevelofunpolymerizedtubulinsubunitsishigh,the1-tubulintran-scriptisunstable.Conversely,whentheconcentrationofunpolymerizedtubulinislow,thetranscriptisstable.Thus,accordingtothemodel,whentubulinisrapidlypolymerizingintomicrotubulesorflagella,theconcentrationofunpolymer-izedtubulinwithinthecellfallsand,-tubulinmRNAisstabilized.Oncethesestructuresarecompleted,theconcen-trationofunpolymerizedtubulinwithinthecellincreasesandthe3-tubulintranscriptsbecomeunstable.InC.reinhardtii,itappearsthattubB2mRNAaccumulationfollowingdeflagella-tioniscontrolledprimarilybytranscription,sincedeletingsequencesinthepromoterpreventstheincreasedaccumula-tionoftranscripts.Furthermore,itisunlikelythatthelevelofunpolymerizedtubulinmediatesthetranscriptionalregulationoftubB2followingdeflagellation,sinceinducedaccumulationoftubB2mRNAcontinuesevenwhenflagellargrowthisinhibitedbyaddingcolchicineorwithholdingCa2+ions(9).Undertheseconditionstheconcentrationofunpolymerizedtubulinwithinthecellshouldbehigh.Theaccumulationoftranscriptsfromboththea-andthe,-tubulingenesinC.reinhardtiiisalsoregulatedoverthecourseofthecellcycle(1).Thisregulationmaybeneededtocoordinatetubulinsynthesiswiththeformationofthemitoticspindleapparatusandtheassemblyofnewflagella.Cellcycle-regulatedexpressionoftubulinhasnotbeenobservedinmanyorganisms.Physarumpolycephalumincreasesaccumula-tionofthea-and,B-tubulintranscriptsabout40-foldpriortomitosis(8,30),whileTetrahymenathermophila(22)andHeLacells(5)increasetubulinmRNAaccumulationonly2-fold.InAspergillusnidulans,thereisnochangeintheleveloftubmRNAduringcelldivision(15).OurresultsdemonstratethattubB2larstranscriptsaccumu-lateanddeclineearlierinthecyclethanthetubB2mRNA.Itisunclearwhatiscausingthistimingdifference,althoughthereareanumberofpossibleexplanations.TranscriptionofthechimericgeneduringthecellcyclemaybeidenticaltothatoftheendogenoustubB2gene,butthestabilityoftheendoge-noustubB2transcriptmaybelowearlyinthecycle(e.g.,2and6h)andincreaseatlatertimes(e.g.,14and18h).Asmentionedpreviously,thestabilityof1-tubulintranscriptsinmammaliancellsiscontrolledbythepoolofunpolymerizedtubulinwithin thecell(10),aneffectmediatedbythepresenceofthefirstfouraminoacidsattheNterminusofthe1-tubulinpolypeptide(10,16,39).ThetubB2geneofC.reinhardtiiencodesthesamefourN-terminalaminoacids,suggestingthattheremaybesomecontroloftubB2mRNAstabilitybythepoolofunpolymerizedtubulinsubunitsundercertaincondi-tions.Thesefouraminoacidsarenotencodedbythetranscriptofthechimericgene,andifcellcyclecontroloftubB2mRNAaccumulationinvolvesthissequence,thestabilityofthechi-merictranscriptwillbedifferentfromthatoftheendogenoustubB2transcript.Theneteffectmaybetoshiftthetimeofmaximaltranscriptaccumulation.AnalternativepossibilityisthatsomesequencesinvolvedincontrollingthetimingoftranscriptionofthetubB2genewerelostduringconstructionofthetubB2larschimericgene.Sucharesultwasobtainedwithsequencescontrollingthecellcycle-regulatedtranscriptionoftheSaccharomycescerevisiaehistoneH2AandH2Bgenes.Thesegenesareregulatedbybothpositive-andnegative-controlelements.Whenonlythenegative-controlelementwasplacedupstreamofaconstitutivelytranscribedreportergene,transcriptsaccumulatedinacellcycle-regulatedmanner.How-ever,maximalaccumulationofthereportertranscriptoccurredearlierinthecellcyclethantheaccumulationoftheendoge-nousH2Btranscript(27).InC.reinhardtii,bothduringcelldivisionandfollowingdeflagellation,newflagellaaresynthesizedwiththeconcomi-tantincreaseintheexpressionofthetubgenes.OurresultssuggestthatdeletingtubboxsequencesalterstheregulatedexpressionoftubB2bothduringcelldivisionandfollowingdeflagellation.DuringthecellcyclethetubB2larschimericgenescontainingtheupstreamsequencesandallsevenofthetubboxmotifs(ptubB2/ars)oronlyfourtubboxmotifs(ptubB2AUS,3/ars)areregulatedinacellcycle-dependentmanner,butchimericgeneswithtwo(ptubB2AUS,3,2/ars)orno(ptubB2AUS,3,2,1/ars)tubboxsequencesshowlittlevari-ationinexpression.SincefollowingdeflagellationptubB2/arsisregulatedsimilarlytotheendogenoustubB2butitstimingofexpressionduringthecellcycleisdifferent,atleastsomeofthesignalsresponsibleforelevatedtubB2mRNAaccumulationfollowingdeflagellationaredifferentfromthosethatstimulatetubB2mRNAaccumulationduringthecellcycle.Becausemultiplecopiesofthetubboxsequencemotifappeartoberequiredforinducedtranscriptionfollowingdeflagellation,itislikelythatsomemultimericformofatranscriptionfactormaybindtothesesequencestoenhancetranscription.Thesignificanceoftheoverlappingtubboxesisnotclear,althoughtheymayfacilitatethebindingoftranscrip-tionfactorsandmayoptimizeprotein-proteininteractionsthatarerequiredforenhancedtranscription.VOL.14,1994 on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

5174DAVIESANDGROSSMAN

ACKNOWLEDGMENTS

REFERENCES

1.Ares,M.,andS.H.Howell.1982.Cellcyclestage-specificaccu-mulationofmRNAsencodingtubulinandotherpolypeptidesinChlamydomonas.Proc.Natl.Acad.Sci.USA79:5577-5581.2.Ausubel,F.M.,R.Brent,R.E.Kingston,D.D.Moore,J.G.Seidman,J.A.Smith,andK.Struhl.1988.Currentprotocolsinmolecularbiology.JohnWileyandSons,NewYork.3.Baker,E.J.,J.A.Schloss,andJ.L.Rosenbaum.1984.RapidchangesintubulinRNAsynthesisandstabilityinducedbydeflag-ellationinChlamydomonas.J.CellBiol.99:2074-2081.4.Bandziulis,R.J.,andJ.L.Rosenbaum.1988.Novelcontrolelementsinthealpha-1tubulingenepromoterfromChlamydo-monasreinhardtii.Mol.Gen.Genet.214:204-212.5.Bravo,R.,andJ.E.Celis.1980.Asearchfordifferentialpolypep-tidesynthesisthroughoutthecellcycleofHeLacells.CellBiol.84:795-802.6.Brunke,K.,J.G.Anthony,E.J.Sternberg,andD.P.Weeks.1984.RepeatedconsensussequenceandpseudopromotersinthefourcoordinatelyregulatedtubulingenesofChlamydomonasrein-hardtii.Mol.Cell.Biol.4:1115-1124.7.Brunke,K.J.,E.E.Young,B.U.Buchbinder,andD.P.Weeks.1982.CoordinateregulationofthefourtubulingenesofChlamy-domonasreinhardtii.NucleicAcidsRes.10:1295-1310.8.Burland,T.G.,K.Gull,T.Schedl,R.S.Boston,andW.F.Dove.1983.Celltype-dependentexpressionoftubulinsinPhysarum.J.CellBiol.97:1852-1859.9.Cheshire,J.L.,andL.R.Keller.1991.UncouplingofChlamydo-monasflagellargeneexpressionandoutgrowthfromflagellarexcisionbymanipulationofCa2+.J.CellBiol.115:1651-1659.10.Cleveland,D.W.1988.AutoregulatedinstabilityoftubulinmRNAs:anoveleukaryoticregulatorymechanism.TrendsBio-chem.13:339-343.11.Curry,A.M.,B.D.Williams,andJ.L.Rosenbaum.1992.Sequenceanalysisrevealshomologybetweentwoproteinsoftheflagellarradialspoke.Mol.Cell.Biol.12:3967-3977.12.Davies,J.P.,D.P.Weeks,andA. R.Grossman.1992.Expressionofthearylsulfatasegenefromthe12-tubulinpromoterinChlamy-domonasreinhardtii.NucleicAcidsRes.20:2959-2965.13.Davies,J.P.,F.Yildiz,andA.R.Grossman.1994.MutantsofChlamydomonaswithaberrantresponsestosulfurdeprivation.PlantCell6:53-63.14.Debuchy,R.,S.Purton,andJ.-D.Rochaix.1989.Theargininosuc-cinatelyasegeneofChlamydomonasreinhardtii:animportanttoolfornucleartransformationandforcorrelatingthegeneticandmolecularmapsoftheARG7locus.EMBOJ.8:2803-2809.15.Doshi,P.,C.A.Bossie,J.H.Doonan,G.S.May,andN.R.Morris.1991.Twoa-tubulingenesofAspergillusnidulansencodediver-gentproteins.Mol.Gen.Genet.225:129-141.16.Gay,D.A.,T.J.Yen,J.T. Y.Lau,andD.W.Cleveland.1987.Sequencesthatconferr-tubulinautoregulationthroughmodu-latedmRNAstabilityresidewithinexon1ofa,-tubulinmRNA.Cell50:671-679.17.Goldschmidt-Clermont,M.,andM.Rahire.1986.Sequence,evolutionanddifferentialexpressionofthetwogenesencodingvariantsmallsubunitsofribulosebisphosphatecarboxylase/oxyge-naseinChlamydomonasreinhardtii.J.Mol.Biol.191:421-432.18.Gorman,D.S.,andR.P.Levine.1966.Cytochromefandplastocyanin:theirsequenceinthephotosyntheticelectrontrans-portchain.Proc.Natl.Acad.Sci.USA54:1665-1669.19.Imbault,P.,C.Wittemer,U.Johanningmeier,J.D.Jacobs,andS.H.Howell.1988.StructureoftheChlamydomonasreinhardtiicabII-1geneencodingachlorophylla/bbindingprotein.Gene73:397-407.20.Keller,L.R.,J.R.Schloss,C.D.Silflow,andJ.L.Rosenbaum.1984.TranscriptionofaandXtubulingenesinvitroinisolatedChlamydomonasreinhardtiinuclei.J.CellBiol.98:1138-1143.21.Kindle,K.L.1990.High-frequencynucleartransformationofChlamydomonasreinhardtii.Proc.Natl.Acad.Sci.USA87:1228-1232.22.McGrath,K.E.,S.M.Yu,D.P.Heruth,A. A.Kelly,andM.A.Gorovsky.1994.Regulationandevolutionofthesinglealpha-tubulingeneoftheciliateTetrahymenathermophila.CellMotil.Cytoskeleton27:272-283.23.Minami,S.A.,P.S.Collis,E. E.Young,andD.P.Weeks.1981.TubulininductioninC.reinhardtii:requirementfortubulinmRNAsynthesis.Cell24:89-95.24.Muller,F.W.,G.L.Igloi,andC.F.Beck.1992.Structureofageneencodingheat-shockproteinHSP70fromtheunicellularalgaChlamydomonasreinhardtii.Gene111:165-173.25.Nagawa,F.,andG.R.Fink.1985.Therelationshipbetweenthe"TATA"sequenceandtranscriptioninitiationsitesattheHIS4geneofSaccharomycescerevisiae.Proc.Natl.Acad.Sci.USA82:8557-8561.26.Nicholl,D.S.T.,J.A.Schloss,andP.C.L.John.1988.TubulingeneexpressionintheChlamydomonasreinhardtiicellcycle:eliminationofenvironmentallyinducedartifactsandthemeasure-mentoftubulinmRNAlevels.J.CellSci.89:397-403.27.Osley,M.A.,J.Gould,S.Kim,M.Kane,andL.Hereford.1986.Identificationofsequencesinayeasthistonepromoterinvolvedinperiodictranscription.Cell45:537-544.28.Remillard,S.P.,andG.B.Witman.1982.Synthesis,transport,andutilizationofspecificflagellarproteinsduringflagellarregenera-tioninChlamydomonas.J.CellBiol.93:615-631.29.Sanger,F.,S.Nicklen,andA.R.Coulson.1977.DNAsequencingwithchain-terminatinginhibitors.Proc.Natl.Acad.Sci.USA74:5463-5467.30.Schedl,T.,T.G.Burland,K.Gull,andW.F.Dove.1984.CellcycleregulationoftubulinRNAlevel,tubulinproteinsynthesis,andassemblyofmicrotubulesinPhysarum.J.CellBiol.99:155-165.31.Schloss,J.A.1990.AChlamydomonasgeneencodesaGprotein,Bsubunit-likepolypeptide.Mol.Gen.Genet.221:443-452.32.Silflow,C.D.,andJ.L.Rosenbaum.1981.Multiplea-andl-tubulingenesinChlamydomonasandregulationoftubulinmRNAlevelsafterdeflagellation.Cell24:81-88.33.Soares,H.,L.Galego,R.G6ias,andC.Rodrigues-Pousada.1993.ThemechanismsoftubulinmessengerregulationduringTetrahy-menapyniformisreciliation.J.Biol.Chem.268:16623-16630.34.Weeks,D.P.,andP.S.Collis.1976.InductionofmicrotubuleproteinsynthesisinChlamydomonasreinhardtiiduringflagellarregeneration.Cell9:15-27.35.Weeks,D.P.,andP.S.Collis.1979.InductionandsynthesisoftubulinduringthecellcycleandlifecycleofChlamydomonasreinhardtii.Dev.Biol.69:400-407.36.Weeks,D.P.,P.S.Collis,andM.A.Gealt.1977.ControlofinductionoftubulinsynthesisinChlamydomonasreinhardtii.Na-ture(London)268:667-668.37.Williams,B.D.,M.A.Velleca,A.M.Curry,andJ.L.Rosenbaum.1989.MolecularcloningandsequenceanalysisoftheChlamydo-monasgenecodingforradialspokeprotein3:flagellarmutationpf-14isanochreallele.J.CellBiol.109:235-245.38.Witman,G.B.,K.Carlson,J.Berliner,andJ.L.Rosenbaum.1972.Chlamydomonasflagella.I.Isolationandelectrophoreticanalysisofmicrotubules,matrix,membranesandmastigonemes.J.CellBiol.54:507-539.39.Yen,T.J.,P.S.Machlin,andD.W.Cleveland.1988.Autoregu-latedinstabilityoffI-tubulinmRNAsbyrecognitionofthenascentaminoterminusoff-tubulin.Nature(London)334:580-585.MOL.CELL.BIOL. on March 1, 2013 by PENN STATE UNIVhttp://mcb.asm.org/Downloaded from

quotesdbs_dbs27.pdfusesText_33

[PDF] bêta 2-microglobuline - Prêts Étudiants

[PDF] Beta Bloquants Ifsigonesse Free Fr - Chirurgie

[PDF] Bêta hCG plasmatiques - Santé Et Remise En Forme

[PDF] beta marine - Hardy Engineering - Gas-Oil

[PDF] beta telecoms sur les marches africains, utilisation pour la - France

[PDF] BETA-BLOQUANTS - Musculation

[PDF] Beta-Glucan aus Hafer - Ernährungs

[PDF] BETA-Horn von

[PDF] Bêtabloquants - Chirurgie

[PDF] Bêtabloquants : jusqu`où en augmenter les doses, pourquoi ne pas - Musculation

[PDF] Bêtabloquants, grossesse et allaitement

[PDF] BETADINE 250 mg, ovule Polyvidone iodée - Chirurgie

[PDF] betaillere fixe 1 niveau / bovins betaillere fixe 1 niveau / bovins

[PDF] betaillere fixe 1 niveau betaillere fixe 1 niveau remorque / bovins

[PDF] Bétaillères - France