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Drosophila melanogaster

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MOLECULARANDCELLULARBIOLOGY,June1987,p.2134-2140Vol.7,No.60270-7306/87/062134-07$02.00/0Copyright©1987,AmericanSocietyforMicrobiologyDrosophilamelanogasterHomologsoftherafOncogeneGEORGEE.MARK,'*ROSSJ.MACINTYRE,2MARYE.DIGAN,3LINDAAMBROSIO,4ANDNORBERTPERRIMON4LaboratoryofHumanCarcinogenesis,NationalCancerInstitute,'andLaboratoryofMicrobialImmunity,NationalInstituteofAllergyandInfectiousDisease,3Bethesda,Maryland20892;SectionofGeneticsandDevelopment,CornellUniversity,Ithaca,NewYork148532;andDepartMentofGenetics,HowardHughesMedicalInstitute,HarvardMedicalSchool,Boston,Massachusetts021154Received3October1986/Accepted20February1987Amurinev-rafprobe,representingthekinasedomain,wasusedtoidentifytwouniquelociinDrosophilamelanogasterDNA.Themostcloselyrelatedtov-rafwasmappedbyinsituhybridizationtoposition2F5-6(Draf-1)ontheXchromosome,whereastheotherraf-relatedgene(Draf-2)wasfoundatposition43A2-5onchromosome2.ThenucleotideandaminoacidhomologiesofDraf-ltothekinasedomainofv-rafare61and65%,respectively.Thelargeamountofa3.2-kilobaseDraf-)transcriptdetectedineggsasamaternalmessagedecreasesduringembryonicdevelopment,andsignificantsteady-statelevelsareobservedthroughouttheremainderofmorphogenesis.WespeculatethattheDraf-1locusplaysanimportantroleinearlyembryogenesis.Acutelytransformingretroviruseshavebeenthesourceofthemajorityofoncogenicsequencesidentifiedtodate.Theseoncogeneswereacquiredbyrecombinationwith,andsubsequenttransductionof,cellularproto-oncogeneloci(reviewedinreference,2-4).Ofthefourmajoroncogeneclasses(srcfamily,rasfamily,nuclearoncogenes,andgrowthfactors),membersofthreeclasseshavebeenisolatedfromDrosophilamelanogaster.Threegenesrepresentingtherasfamily(28),fourgenesrepresentingthesrcfamily(10,11,19,33,34)andonerepresentingthec-mybproto-oncogene(15)havebeenidentifiedinD.melanogaster.Theconservationofproto-oncogenesequencesthroughoutthehundredsofmillionsofyearsofevolutionsinceinvertebratespeciationatteststotheessentialrolesplayedbythesegenesinnormalcellulargrowthanddifferentiation.Althoughtherelationshipbetweenseveraloftheseproto-oncogenesandrelatedsequencestoknownpolypeptideshasbeendemon-strated(exemplifiedbyplatelet-derivedgrowthfactorandsis;epidermalgrowthfactorreceptoranderbB;CSF-1receptorandfis;andinsulinreceptorandros-relatedse-quences),themechanismbywhichtheytransducetheirsignaltoeffectacellularresponseispresentlyobscure(3,13).Thelargestoncogenefamily,thesrcfamily,hasyieldedthegreatestnumberofDrosophilahomologs.Thusfarthesephylogeneticallyancienthomologshaveallbeenrepresenta-tivesofthosesrcfamilymembersencodingtyrosine-specificproteinkinases.WereportheretheisolationandpreliminarycharacterizationofaDrosophilagenethatcorrespondstotheproto-oncogenec-rafandtheidentificationofaraf-relatedDrosophilalocuswhichmayrepresenttherecentlyidentifiedraf-relatedproto-oncogenepkspresentontheshortarmofthehumanXchromosome(23).Thev-rafproductexhibitsserine-threonineproteinkinaseactivity(26),andc-rafshowssignificantprimaryandsecondaryaminoacidsequencehomologytoproteinkinaseC;thustheDrosophilahomologmightbeexpectedtosharethisspeci-*Correspondingauthor.ficity,andtheconservationofaminoacidsequencesob-servedissupportiveofthisexpectation.OurfindingextendsthenumberandvarietyofDrosophilaoncogene-relatedsequences.MATERIALSANDMETHODSHybridization,cloning,andDNAsequencing.AD.melanogastergenomiclibrarymadefromsize-selectedpar-tialEcoRI-digestedDNAligatedintoCharon4Awasob-tainedfromTipBenyajati(LittonBionetics,Frederick,Md.).Hybridizationofplaquelifts(1)wasperformedunderrelaxedstringencyconditions,asweretheSouthernblotanalyses.Nitrocellulosefilterswereprehybridizedat60°for2hin5xSCC(1xSSCis0.15mNaClplus0.015Msodiumcitrate)-5xDenhardtsolutionandthenhybridizedovernightat600Cin5xSSC-1xDenhardtsolution-10%dextransulfateandabout5x106cpmofnick-translatedprobeperml(30)representingthekinasedomainofv-raf(0.68-kilobase[kb]XhoI-to-SstIIfragment[21]).Thefilterswerewashedrepeatedlyin2xSSC-0.1%sodiumdodecylsulfateat55°CandexposedtoX-rayfilmovernightwithanintensifyingscreen.SubcloningofDNAfragmentsintopBR322wasdoneessentiallybythemethodofManiatisetal.(20).TheDNAsequencewasdeterminedbythemethodofMaxamandGilbert(24)fromuniquelylabeledrestrictionsiteterminiafterisolationbySeaplaque(FMCCorp.MarineColloidsDiv.,Rockland,Maine)agarosegelelectrophoresisasde-scribedpreviously(22).Insituhybridization.ThemethodofBonnerandPardue(5)wasusedforinsituhybridization,withmodificationstoaccommodatetheuseofbiotinylatedprobes.Biotinylatedprobesweresynthesizedbyusingbiotin-11dUTP(BethesdaResearchLaboratories,Inc.,Gaithersburg,Md.)andtheprimerextensionprocedureofFeinbergandVogelstein(8).Hybridizationconditionswere2xSSC,50%formamide,and10%dextransulfatefor24hat37°C.DevelopmentofthehybridwasperformedasdescribedintheENZODeteckKit.LocalizationonthepolytenechromosomewasdeterminedbyusingthealignmentofBridges'chromosomemaps,describedbyLefevre(16).2134TWOraf-RELATEDLOCIIND.MELANOGASTER2135lIIAtlIr4'%AAI^^>1^IAIIjAIAt4ki'ti-IIIflii111^^1lIkIAIIA23i-W-412.UeICASCEIFAXCHROMOSOMEELk1CIOIEI$IOiDIEIAC|DIE!,A6C|CI4h414142'443t4344i2RCHROMOSOME_r_p.:a->:'-Y* a . ..,,t,.ts[_:;t.r.*:>,,,,.4.3.>Rz_ka3SE^s;. l


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