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Per2An h www.sciencemag.org (this information is current as of February 6, 2008 ): http://www.sciencemag.org/cgi/content/full/291/5506/1040 including high-resolution figures, can be found in the onlineUpdated information and services, http://www.sciencemag.org/cgi/content/full/291/5506/1040#otherarticles, 9 of which can be accessed for free: cites 24 articlesThis article 296 article(s) on the ISI Web of Science. cited byThis article has been http://www.sciencemag.org/cgi/content/full/291/5506/1040#otherarticles 80 articles hosted by HighWire Press; see: cited byThis article has been http://www.sciencemag.org/cgi/collection/physiology: subject collectionsThis article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this articlepermission to reproduce of this article or about obtaining reprintsInformation about obtaining registered trademark of AAAS. is aScience2001 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience on February 6, 2008 www.sciencemag.orgDownloaded from threehumanhomologs,histhemostsimilartod).Inaddition,mutationsinthefly()andinthemouse(m)produceasimilarshortperiodphenotype.Inhumans(andotheranimals),shortperiodmutationsarepredictedtophase-advancecirca-dianrhythmsunderentrainedconditions().Furthemore,unlikemandm,thephaseresponsecurveforlightinductionofRNAismaximalatCT14()whenphasedelaysareelicitedbylight.Thisiscon-sistentwithapredominantlyphasedelayfunc-tionformPER2.Thus,aloss-of-functionmu-tationinhPER2could,theoretically,leadtoaphaseadvance.Thelocalizationofhonchromosome2qterwasconfirmedbyisolatingaBACclone(552H8,CITBhumanBAClibrary)containingthehgeneforuseinfluorescenceinsituhybridizationexperiments().ThisBACmappedtothetipofchromosome2q().Inaddition,weusedapolymorphisminhgenotypeK2174andperformedtwo-pointlink-agemappingwithninemarkersnotedprevious-ly().ArecombinationdemonstratedthegenetobedistaltomarkerD2S338.Thehaplotypeoftheremainingeightmarkerswasfullylinkedtohinthisfamily.Theindi-vidualsinbranch3wereconsideredtorepre-sentphenocopies,andmutationanalysisofwasperformed.comprises23exons(Fig.2A)).Asequencingerroroftheh(GenBankaccessionnumber,NM003894)wasidentified;thereportedcDNAhasamissingbaseatposition3652thatshiftsthereadingframe,predictingtranslationof69aminoacidsthatarenothomologoustootherPERproteinsbeforethestopcodon.Withthecorrectedse-quence,theregion3ofthatbaseencodes78aminoacidsthatare64%identicaltomPER2.Single-strandconformationpolymorphism(SSCP)analysis()ofaffectedandunaffectedindividualsrevealedacomplexbandingpatterninexon17.Sequencingofthisexonfromindi-vidualsinK2174revealedfourchanges.Threeofthefou

rchanges[basepair2087(bp2087)A/G,bp2114A/G,andbp2117A/G]occuratwobblepositionsand,therefore,preservedtheaminoacidsequence.However,thebasechangeatposition2106(AtoG)ofthehcDNApredictssubstitutionofaserineataminoacid662withaglycine(S662G)(Fig.2).Thischangewasnotfoundin92controls.TheS662GchangecosegregateswiththeASPSphenotypeinthisfamily,exceptforthebranchinwhichtheFASPS-associatedmarkeralleleswereunlinked(Fig.1).Fouradditionalat-riskindividualsinthefamilycarrythemutationbutdidnotmeetstrictaffectioncriteria,althoughtheydidshowastrongtendencyofearlysleep-wakepreference(Horne-Ostbergscores74.44)(ToestablishwhetherthismutationcausesFASPS,theS662Gmutationwasfunctionallycharacterized().TodeterminewhetherS662islocatedwithintheCKIbindingsiteofhPER2,CKI,myc-epitopetaggedmPER2,andtheindicatedtruncationmutantsofmPER2(Fig.3)wereexpressedinrabbitreticulocytelysatesandmPER2peptideswereimmunopre-cipitatedwithantibodiestomyc().CKIcoprecipitatedwithmPER2(1to763)butnotmPER2(1to554),thusdemonstratingthatthebindingsiteofmPER2islocatedbetweenresidues554and763,correspondingtoresidues556to771ofhPER2(Fig.3).Studiesof)mutantsin)andthemutantinthegoldenhamster()indicatethatmutationsaffectingthefunctionofCKIdisruptendogenouscirca-dianclockfunctionleadingtoalteredperiodlengthsorarrhythmicity.Inaddition,hPER2andmPER2aresubstratesofCKI.BecausetheS662GmutationislocatedwithintheCKIbindingregion,hPER2andmPER2fragmentsextendingfromaminoacids474to815and472to804,respectively,wereusedtoevaluatetheeffectofthemutationonPER2phosphorylation).TotestwhethertheS662Gmutationelim-inatesapotentialphosphorylationsite,reticulo-cytelysatescontainingS-labeledhPER2frag-mentswereincubatedwithalowconcentrationofCKI(0.25ng/l).Anelectrophoreticmo-bilityshiftwasobservedwhenwild-type(S662)butnotmutant(G662)fragmentsweretreatedwithCKI(Fig.4A).Asimilarresultwasobtainedwithwild-typeandmutantmPER2fragments().Phosphatasetreat-mentconfirmedthatthisshiftwasduetophosphorylation(Fig.4).Whentheexperi-mentwasrepeatedwithahigherconcentra-tionofCKI(6.5ng/l),mobilityshiftswereobservedforboththewild-typeandmutanthPER2fragments(Fig.4B).Thus,regardlessofthephosphorylationstatusofS662,otherresiduesinthepeptidecanbephosphorylatedwithexcesskinase.preferentiallyphosphorylatespeptideswithacidic[forexample,DDDD-X-X-S]or phosphorylatedresidues[forexample,S(P)-X-X-S]immediatelyupstreamofthetargetresi- due(whereDisaspartate,Sisserine,S(P)isaphosphoserine,Xisanyaminoacid,andtheunderlinedªSºisthetargetofthesubsequentphosphorylation)().AnalysisofthehPER2sequencerevealsfouradditionalserine Fig.2.)GenomicstructureofhThehgenecon-tains23exons(col-oredrectangles).Theinterveningintronsarenotdrawntoscale.Themutationinkindred2174(S662G)occursinexon17.TheÒÓaboveexon22showsthelocationofthesequenceerror(a1basepairdele-tion)inthehcDNAGenBankse-quence.()Thehmutationinkindred2174.DNAsequencesfromthehofcontrol(upper)andFASPS(lower)individualsareshown.Ana

rrowmarksadoublepeakatposition2106inthehsequenceobtainedfromanaffectedindividual.ThisAtoGtransition(sequenceshownisofinversecomplementarystrand)predictssubstitutionofahighlyconservedserineresidueataminoacidposition662byaglycine.AdoublepeakwasnotedwheneachDNAstrandwassequencedinbothdirections.()AminoacidsequenceofvariousPERhomologsfromtheregionharboringtheFASPSmutation().Theserineatposition662isreplacedbyaglycine(G)onthemutantallele.Fourasterisksmarkfoursubsequentconservedserineresidueseachwithtwointerveningaminoacids.www.sciencemag.orgSCIENCEVOL2919FEBRUARY2001 on February 6, 2008 www.sciencemag.org Downloaded from ReferencesandNotes1.C.R.Jonesetal.NatureMed.,1062(1999).2.J.C.Dunlap,,271(1999).3.D.R.Weaver,J.Biol.Rhythms,100(1998).4.S.M.Reppert,,1(1998).5.D.P.KingandJ.S.Takahashi,Annu.Rev.Neurosci.713(2000).6.M.J.Hamblen-Coyleetal.J.InsectBehav.,4177.K.Wager-Smith,S.A.Kay,NatureGenet.,,238.L.P.Shearmanetal.,1013(2000).9.IndividualswhodidnotmeeteithertheconservativeaffectedorunaffectedcriteriawereclassiÞedasun-known.VenousbloodsamplesweregatheredfromindividualsfromASPSfamilieswhowerelikelytocontributetolinkageinformation.ParticipantssignedaÒConsentofParticipationÓform,whichwasap-provedbytheInstitutionalReviewBoardforHumanResearchattheUniversityofUtahSchoolofMedi-cine.HighÐmolecularweightgenomicDNAwasiso-latedfromwhole-bloodlysates,andlymphoblastoidcelllinesweretransformedwithEpstein-Barrvirus,asdescribed(10.TheßuorescentlylabeledmarkerswereusedtoamplifygenomicDNAintotalreactionvolumesof20linaMJRPTC-200thermocycler(MJResearch,Watertown,MA).TheproductswerevisualizedonanAppliedBio-systemsmodel377andanalyzedbytheGenotyperpeak-callingsoftware.Pairwisetwo-pointlinkageanal-ysiswithMLINKoftheLINKAGEprogramwasused.Diseasepenetrancewassetat0.95,withoutagenderdifference,andthenormalandFASPSallelefrequenciesweresetat0.999and0.001,respectively.11.ManualgenotypingwascarriedoutafterPCRofDNAsampleswithappropriateprimers,aspreviouslyde-scribed(12.K.L.Tohetal.,datanotshown.13.MarkersD2S338,D2S2338,D2S2285,D2S2253,D2S125,D2S395,D2S140,D2S2986,andD2S2987(fromcentromeretotelomere)wereusedforgeno-typingandhaplotypeanalysis.14.C.Dibetal.,152(1996).15.U.Albrechtetal.,1055(1997).16.R.J.Konopka,S.Benzer,Proc.Natl.Acad.Sci.U.S.A.,2112(1971).17.B.Zhengetal.,169(1999).18.E.B.Klermanetal.Am.J.Physiol.,R271(1996).19.J.Duffyetal.,Sleep,S92(1999).20.M.J.Zylkaetal.,1103(1998).21.Humanlymphoblastculturesweretreatedwithchol-cimid(0.025mg/ml)at37¡Cfor1.5hours.Coloemidtreatedcultureswerepelletedat500atroomtem-peraturefor8min.Pelletswerethenresuspendedwith0.075MKCl(3mlperpellet)for15minatroomtemperature.CellswerethenÞxedin3:1MeOH:aceticacidandstoredat4¡C.Humanbacterialarti-Þcialchromosomes(BACs)werelabeledwithspec-trumorangeusinganicktranslationkitperthemanufacturersprotocol(Vysis,DownersGrove,IL).SlideswerepreparedbydroppingÞxedcellsontoglassslidesandwashingwithexcessÞxative.The

slideswerethenwashedinaceticacidfor35minatroomtemperatureandweredehydratedfor2mineachin70,85,andÞnally100%EtOH.Chromosomesweredenaturedin70%formamidein2SSCat74¡Cfor5min,andslidesweredehydratedagainasaboveexceptinice-coldEtOH.Twomicrogramsoflabeledprobewasblockedwith2gofhumanCot-1DNAinHybrisolVI(ONCOR,Gaithersburg,MD).Theprobemixturewasdenaturedat74¡Cfor5minandthenpre-annealedat37¡Cfor15min.Twelvemicrolitersofpre-annealedprobewasappliedperslide,acoverslipwasadded,andedgesweresealedwithrubbercement.Slideswerehybridizedinadarkened,humid-iÞedchamberfor16hoursat37¡C.Hybridizedslideswerethenwashedin0.4SSCcontaining0.1%Tween-20at74¡Cfor2min,andthen1minatroomtemperaturein2standardsalinecitrate(SSC).Slideswereallowedtodryinthedarkatroomtemperatureandwerestainedwith42-phenylindole(DAPI)(Vectorlabs,Burlingame,CA)forchromosomevisualization.22.Thehintron-exonboundariesweredeterminedinordertocarryoutthemutationalanalysis.Intron-exonboundariesofthehgenewereobtainedbyacombinationofdirectsequencingofhDNAandsequencingofPCRproductsfromgenomicDNAwithprimersdistributedalongtheentirecDNA.Intronicsequenceofatleast100basepairsßankingeachexonboundarywasobtained.Intronsizesweredetermineddirectlyfromgenomicsequenceoresti-matedbythesizeofPCRproductsampliÞedusingoligonucleotidesfromadjacentexons.AllsequencingreactionswerecarriedoutwithanAppliedBioSys-temsmodel377DNAsequencer(FosterCity,CA).23.SSCPwascarriedoutasdescribed().PCRproductswerediluted,denatured,andelectrophoresedthroughacrylamidegelsandvisualizedonx-rayÞlmatÐ80¡Cfor12to24hours.AberrantSSCPbandswerecutdirectlyfromthedriedgelsandsequencedasdescribed(24.ComplementaryDNAclonesencodingmwerePCRampliÞedfromthecorrespondingplasmidsandclonedintothepCS2MT(myc-epitopetagged)vectoraspreviouslydescribed).Site-directedmutagenesisoftheserineresi-dueatposition662ofhPER2andthehomologousserine(659)ofmPER2wereperformedtosubsti-tuteaglycineresidue.MutagenesiswascarriedoutwiththeQuikChangeSite-directedMutagenesisKit(Stratagene)usingtheprotocoloutlinedtherein.EcoRIÐXbaIfragmentsencodingaminoacids474to815ofhPER2(andthecorrespondingaminoacids472to804ofmPER2)werePCR-ampliÞedwithprimerscontainingEcoRIandXbaIsites,gel-puriÞedwiththeGENECLEANkit(BIO101,Vista,CA)andweredirectionallyclonedintotheEcoRIÐXbaIsitesofthepCS2MTvector.ExpressionfromanSP6promotergenerates6-myc-taggedpeptides.Aseriesof3mutationsofmwereconstructed(encodingaminoacids1to554,1to763,1to810,and1to904)foruseinmappingthebindingsiteforCKIaspreviouslydescribedform).Allcon-structswereconÞrmedbysequencing.25.E.Vielhaberetal.Mol.Cell.Biol.,4888(2000).26.B.Klossetal.,97(1998).27.P.L.Lowreyetal.,483(2000).28.TranscriptionandtranslationofhandminsertswereperformedinvitrointhepresenceofS-methioninewiththeTnTSP6CoupledReticulo-cyteLysateSystem(Promega)overaperiodof90minat30¡C.ThelabeledproductswereincubatedwithCKIinbuffercontainingphosphataseinhibitors[25mMTris.HCl,pH7.5,15%gl

ycerol,20mMNaF,170nMokadaicacid,2mMdithiotreitol(DTT),10-glycerolphosphate,and150MATP].Twen-ty-microliteraliquotswereremovedatselectedtimepointsandboiledwithSDSgel-loadingbuffer(0.1%bromophenolblue,50mMTrisHCl,pH6.8,0.1MDTT,2%SDS,10%glycerol)tostopthereaction.Attheendoftheexperiment,20-laliquotsweredi-gestedwith35unitsofcalfintestinalphosphataseinbuffer(50mMTrisHCl,pH7.9,10mMMgCl,0.1MNaCl,1mMDTT)for30minwhereindicated.Allproductswereanalyzedbyelectrophoresisin8%SDSÐpolyacrylamidegels(SDS-PAGE)withanacryl-amide:bis-acrylamideratioof120:1toenhancemo-bilityshifts.ThegelswereÞxedanddriedandthebandsvisualizedusingPhosphorImagerscreensscannedwithScannerControlSIsoftware(MolecularDynamics,Sunnyvale,CA).29.H.Flotowetal.J.Biol.Chem.,14264(1990).30.A.Cegielskaetal.J.Virol.,269(1994).31.N.Dumaz,D.M.Milne,D.W.Meek,FEBSLett.312(1999).32.K.Sakaguchietal.J.Biol.Chem.,9278(2000).33.J.L.Priceetal.,83(1998).34.G.A.Keesleretal.,951(2000).35.WebtextisavailableatOnlineatwww.36.L.J.Ptaceketal.,863(1994).37.Single-letterabbreviationsfortheaminoacidresiduesareasfollows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H,His;I,Ile;K,Lys;L,Leu;M,Met;N,Asn;P,Pro;Q,Gln;R,Arg;S,Ser;T,Thr;V,Val;W,Trp;andY,Tyr.38.Wethankthefamilieswhoparticipatedinthiswork,A.Meloni-EhrigandF.OrÞnofortechnicalhelp,andtoG.A.Keesler,E.Vielhaber,andV.Hillforhelpfuldiscus-sionsandreagents.SupportedbyNIHgrantsHL/HD59596(L.J.P.),CA71074(D.M.V.),andPublicHealthServiceresearchgrantM01-RR00064fromtheNationalCenterforResearchResources.L.J.P.isanInvestigatoroftheHowardHughesMedicalInstitute.15November2000;accepted18December2000Publishedonline11January2001;Includethisinformationwhencitingthispaper.TRP-PLIK,aBifunctionalProteinwithKinaseandIonChannelLorenW.Runnels,LixiaYue,DavidE.Clapham*Weclonedandcharacterizedaproteinkinaseandionchannel,TRP-PLIK.Aspartofthelongtransientreceptorpotentialchannelsubfamilyimplicatedincontrolofcelldivision,itisaproteinthatisbothanionchannelandaproteinkinase.TRP-PLIKphosphorylateditself,displayedawidetissuedistribution,and,whenexpressedinCHO-K1cells,constitutedanonselective,calcium-permeant,105-picosiemen,steeplyoutwardlyrectifyingconductance.ThezincÞngercontain--kinasedomainwasfunctional.Inactivationofthekinaseactivitybysite-directedmutagenesisandthechannelÕsdependenceonintracellularaden-osinetriphosphate(ATP)demonstratedthatthechannelÕskinaseactivityisessentialforchannelfunction.PhototransductioninphospholipaseC(PLC)±mediatedactivationoftransientreceptorpotential(TRP)chan-nels,leadingtomembranedepolarization().ThemammalianTRPchannelfamilymaybedividedbysequencesimilarityintoshort,long,andosm9±likesubfamilies[reviewedin()].Receptor-mediatedstimulationofPLCactivatesmanymembersoftheshortTRPchannels,andphysicalorchemicalstim-uliactivateisoformsoftheosm9±likeTRPchannel.LongTRPchannels(LTRPC),suchwww.sciencemag.orgSCIENCEVOL2919FEBRUARY2001 on February 6, 2008 www.sciencemag.org Downloaded fro