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Selenoprotein W cDNAs From Five Species of Animals

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Selenoprotein W cDNAs From Five Species of AnimalsW,of,cDNAs,from,five,From,Five

    Selenoprotein W cDNAs From Five Species

    of Animals

BIOMEDICALANDENVIRONMENTALSCIENCES10,190.197(1997)

    ;SelenoproteinWcDNAsFromFiveSpeciesofAnimals

    ;P.D.WHANGER,S.C.VENDELAND,QPGU,M.A.BEILSTEIN,

    ;ANDL.W.REAM

    ;DepartmentofAgriculturalChemistry,OregonStateUniversity, ;Corvallis,Oregon97331,USA

    ;Thenucleotidesequencesoftheopenreadingfral’nesofcDNAsforselenoproteinWfrom

    ;skeletalmuscleofrat,mouse,sheep,rhesusmonkeyandhumanarereported.Theoretica1

    ;translationofthecodingsequencesindicatedhighlysimilarproteinsof88(mouseandrat)or

    ;87(human,monkeyandsheep)aminoacidsIn730f88positionsthespecifiedaminoacids

    ;areidenticalforal1fiveproteinsTGAencodingselenoeysteineisthe13thcooonofal1thecD.

    ;NAs.Themouse,ratandsheepopenreadingframesterminatewithTGAbutthehumanand

    ;rhesusmonkeycoOingregionsterminatewithTAATheencodedaminoacidsequencesarei-

    ;dentica1fortheratandmouseproteins.andforthehumanandmonkeyproteins.Thesimilar.

    ;ityofthecDNAscontinuesinthe3noncoOingregionsthroughtheputativeselenocysteinein.

    ;sertionsequence(SECIS)elementswhicharerequiredforcorrectinterpretationoftheseleno.

    ;cysteinecodon.TheregionbetweentheSECISelementsandthepolyadenylationsignals

    ;showedmuchlowersimilarity.TheclonedratgeneforselenoproteinWis5000bases1ong.

    ;withthe663basesofthecDNAinsixexons.Thetranscriptionstartsitewasidentifiedby

    ;nucleaseprotectionassaytobe16basesupstreamofthelongestcDNAclone.Acanonica1

    ;TATAboxoccurs150basesupstream.buttheassaydidnotindicatethepresenceof1onger

    ;mRNAs.

    ;INTRODUCTION

    ;Knowledgeofselenoproteinshasincreasedmarkedlywithinthelastseveral ;years.ThecDNAsencodinganumberofmammalianselenoproteinshavebee

nse.

    ;quenced.Theseincludethecellularglutathioneperoxidase(cGPX)fromhumans

    ;(Mullenbacheta1.,1987),rats(Hoeta1.,1988),mice(ChambersetnZ.. ;1986),andcattle(Mullenbacheta1.,1988),theplasmaGPXfromhumans(Taka

    ;hashieta1.,1990),phospholipidGPX(Schuckelteta1.,1991)),GPXfromin

    ;testinaltract(Chueta1.,1993),typesI(Berryeta1.,1991)),?(Daveyet

    ;nZ.,1995)and?

    (Croteaueta1.,1995)iodothyroninedeiodinases,selenoprotein ;Pfromratplasma(Hilleta1.,1991)andselenoproteinWfromratmuscle(Vende

    ;landeta1.,1995).Selenocysteineisencodedbythecodon,UGA,inallthesese

    ;lenoproteins.

    ;PublishedwiththeapprovalofOregonStateUniversityAgricuhuralExperimentStationastechnicalpaper

    ;number11028ThisresearchwassupportedbyPublicHealthServiceResearchGrantsnumbersDK38306

    ;and38341fromtheNationalInstituteofDiabetesandDigestiveandKidneyDiseases.

;Theresearchwith

    ;monkeysinwhichthetissueswereobtainedwassupportedinpartbyNIHgrantRR00163totheOregonRe.

    ;gionalPrimateResearchCenter,Beaverton,OR

    ;19O

    ;0895.3988/97

    ;CN11.2816

    ;Copyright?1997byCAPM

    ;

    ;SELEN0PR0TEINWcDNAs191

    ;AlthoughUGAisusuallyastopcodon.italsodirectstheincorporationofseleno

    ;cysteineintoselenoproteins(Stadtman,1990).AuniquetRNAservesasthesiteof

    ;selenocysteinesynthesisfromserineandrecognizestheUGAinthemRNA(Lein-

    ;felderela1.,1989).IneukaryoticmRNAs,interpretationofUGAasaselenocys

    ;teinecodonrequiresaSECISelement.whichformsaconservedstemloopstructurein

    ;the3untranslatedregionofthemRNA(Berryela1.,1993).Spacingbetween ;UGAcodonsandtheSECISelementdetermineswhetheraparticularUGAsp

ecifies

    ;selenocysteineorastop(Martinela1.,1996).

    ;Evidenceforalowmolecularweightproteincontainingselenium.nowcalled

     se

    ;lenoproteinW,waspresentedanumberofyearsago(Pedersenela1.,1972).Re

    ;centlythisselenoproteinwaspurifiedandcharacterized(Vendelandela1..1993)

    ;fromratskeletalmuscleandthecorrespondingcDNAsequencewasdetermined

    ;(Vendelandela1.,1995).ThiscommunicationpresentsthecDNAsequencesofthe

    ;openreadingframeforselenoproteinWfromskeleta1muscleofmouse,sheep,rhesus

    ;monkeyandhuman.ThesheepandrodentcDNAsshowthatUGAcanbothencode

    ;selenocysteineandserveasastopeodoninthesamemRNA.AlthoughUGAalsoen

    ;codesselenocysteineinprimates,TAAisthestopcodoninthesespecies. ;MATERIALSANDMETHODS

    ;Mouse,sheepandmonkeyskeletalmusclecDNAlibrarieswereconstructedin

    ;thelambdaExCellEeoRI/CIPvector(Pharmacia)aspreviouslydescribed(Vende

    ;1andela1.,1995).Ratsandmicewerefedadietwith1.0ppmseleniumfor10days

    ;beforecollectinglegmuscleforRN

    Aextraction.Musclefromasheepwascollected

    ;afterfeedingadietwith3.0ppmseleniumfor3.5months.Rhesusmonkeymuscle

    ;wassuppliedbytheOregonPrimateCenter,Beaverton,OR.Themonkeyswerefed

    ;astandarddietpriortotissuecollection.AhumanskeletalmusclecDNAlibrarywas

    ;purchasedfromClonteeh(PaloA1to,CA,USA).

    ;ProbeswerepreparedfromtheratselenoproteinWcDNAbvPCRandusedto ;screenmouse,sheep,rhesusmonkeyandhumancDNA1ibraries.Probeswere1abeled

    ;withdigoxigeninderivatizeddUTPincorporatedduringPCRusingstandardcondi.

    ;tions(DIGDNALabelingKit,BoehringerMannheimBiochimica).Thelibraries

    ;wereplatedatapproximately20,000pfuper150mmplate.Nylonmembranelifts

    ;fromtheplateswerehybridizedwiththedigoxigeninlabeledprobes. ;Probeswerede.

    digoxigeninantibodvusi;tectedbyalkalinephosphatasecoupledrabbitanti

    ngachemi.

    ;1uminescentsubstrate(CSPDkit,BoehringerMannheimBiochimica).Positive

    ;plaqueswerereplatedandisolatedasclones.Fourormoreindependentcloneswere

    ;isolatedfromeachlibrary.

    ;T’hecDNAinsertsinthelambdacloneswereexcisedinivoasinsertsinthe

    ;phagemidpExCel1.Theplasmidswereamplifiedandpurifiedflordyeprimersequenc-

    ;lngfromthevectorencodedSP6andT7sites.ThehumancDNAclonesinthevector

    ;lambdagtl0wereamplifiedbyPCRwithprimersflankingtheinsertandsequenced

    ;iromthesameprimers?Contirmingsequencewasobtainedfrominternalprimersde

    ;signedfortheindividualcDNAsbasedonthesequenceobtainedwiththeexterna1

    ;primers?Fourormoreclonesweresequencedfromeachlibrary.Oligonucleotidesvn

    ;thesisandsequencingwereperformedbytheCentralServicesLaboratoryoftheCen

    ;terforGeneResearchandBiotechnologyatOregonStateUniversity.Sequenceahaly

    ;seswereperformedwiththeGeneticsComputerGroup(GCG,Madison,WI)Se

    ;,?,}lj

    ;

    ;192WHANGERETAL

    ;quenceAnalysisSoftwarePackageversion7.2UNIX.

    ;TheratselenoproteinWgenewasidentifiedandisolatedfromagenomiclibrary

    ;inthephagevectorEMBLSP6/T7(ClontechLaboratories,Inc.)usingoligonu

    ;cleotideprobesderivedfromthepreviouslyisolatedrateDNA.Fragmentsofthegene

    ;ofapproximately2000bplengthwereamplifiedbyPCRwithprimersbasedoneDNA

    ;sequenceandtheSP6promoterencodedinthevector.Thegenomicfragmentswere

    ;sequencedfromtheendsbydyeterminaterreactionswiththePCRprimersandinter

    ;nallyusingprimersdesignedfromthenewlyrevealedsequence.Atotalof7088bp0f

    ;thegenomicclonewassequenced,whichextendedfrom2157bpupstreamofthecD

    ;NAsequencethroughthecDNApolyadenylationsite.

    ;Thetranscriptionstartsiteswereinvestigatedusingacommerciallyavailablekit

    ;(GuardianRnaseProtectionAssayKit,Clontech).TotalRNAwasisolatedfromrat

    ;skeletalmuscleandenrichedinpolyARNAbypassagethroughanoligodTc

    ellu

    ;losecolumn.AnantisenseRNAprobeforselenoproteinWmRNAwasmadefroma

    ;restrictionfragmentofthegeneextendingapproximately200basesupstreamfromthe

    ;5endofthecodingsequence.LivermRNAandmuscletRNAwereusedasnegative

    ;controls(ProdyandMerlie,1991).

    ;RESULTSANDDISCUSS10N

    ;ThenucleotidesequencesoftheopenreadingframesofselenoproteinWeDNAs

    ;forthefivespeciesaregiveninFig.1.ThecompletesequenceofthemouseeDN

Ais

    ;givenandthosebaseswhichdifferintheotherspeciesarelisted.Theblankareasin.

    ;dicatethesequenceintheotherspeciesarethesameasthemouse.InallspeciesTGA

    ;islocatedatcodingposition13.TGAisalsothestopcodonintherodentsandsheep.

    ;butTAAisthestopcodoninprimates.Thesheepissimilartotheprimatesinthat ;theopenreadingframeisonecodonshorterthantherodents.Thereisabout80%

    ;homologyofthenucleotidesequencesofthecodingregionbetweenthefivespecies.

    ;SelenoproteinWgenesandproteinsarehighlyconservedamongthespeciesex

    ;amined.ThecodingregionofthenucleotidesequencesofthefivecDNAsare80%i.

    ;denticalandtheirpredictedaminoacidsequencesare83%identical(Fig.2).The

    ;deducedaminoacidsequencesforselenoproteinWindicatethattheratandmousese

    ;quences,andthehumanandmonkeysequencesareidentica1.Thesheepandprimate

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