[PDF] Clone: The History of a Euphonious Scientific Term - NCBI

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MedicalHistory,2002,46:381-402"Clone":TheHistoryofaEuphoniousScientificTermURSULAMITTWOCH*"WhenIuseaword",HumptyDumptysaid..."itmeansjustwhatIchooseittomean."'Itisrareforascientifictermtobeadoptedbythepublic,butthishasclearlyhappenedtothewordcloneanditsderivative,cloning;andpublicreaction,dominatedbyapprehensionthatcloningmightbeappliedfortheproductionofhumanbabies,hasbeenconsistentlynegative.2Toestablishthescientificbasisfromwhichpublicopinionstemsisnoteasy.Indeed,thequestion:"Whatisaclone?",hasnounequivocalanswer.InarecentarticleinScience,AnneMcLarenwrotethat"Overtheyears,clonesandcloninghavemeantdifferentthingstodifferentpeople",3surelyanunsatisfactorystateofaffairsforascientificterm.Thepresentarticlewillfollowthedevelopmentofthedifferentusesofthewordclonefromwhenitwasfirstcoinedclosetoacenturyagotothepresent.Ishallarguethatthepopularidentificationofaclonewithanorganismthatisakintoacarboncopyoftheoriginalisarelicfromanearlierstageoftheevolutionoftheterm.Indeed,thesamepointwasmadebyLeeMSilverinarecentNatureessay.4Itwouldseemthat,followingtheacceptanceofMendelianinheritanceatthebeginningofthetwentiethcentury,itbecamewidelyassumedthatorganismsaretotallyspecifiedbythenucleargenotype.Thisresultedinbothscientistsandthemediaignoringotherdevelopmentaldifferencestowhichorganismsaresubjected,eitherforthesakeofsimplifyingthemessageorwithaviewtoincreasingitsimpactonthepublic.*ProfessorUrsulaMittwoch,DepartmentofBiology,GaltonLaboratory,UniversityCollegeLondon,4StephensonWay,LondonNW12HE,UK.u.mittwoch@ucl.ac.ukIamgreatlyindebtedtotheLibrariansoftheBritishLibrary,ofUniversityCollegeLondon,TheWellcomeLibrary,TheRoyalSocietyofMedicineandtheLinneanSocietyfortheirhelpwithlocatingreferences.IwishtothankDrDerekChadwick,DirectoroftheNovartisFoundation,andMsJaneDempster,formakingavailabletheoriginaltaperecordingsoftheCibaFoundationSymposiumon'Manandhisfuture',referredtoinnotes73and84,andtoProfessorStuartRosenandMrsMaryWykes(UCLPhoneticsandLinguisticDepartment)fortranscribingthem;alsoDrAnnBurgessforhelpfulcomments,andDrJonTurneyandtwoanonymousrefereesforconstructivecriticismofanearlierversionofthemanuscript.'LewisCarroll,Throughthelookingglass,andwhatAlicefoundthere,Basingstoke,MacmillanChildren'sBooks,reprinted1997fromtextof1897,p.113.2WellcomeTrust,Publicperspectivesonhumancloning:asocialresearchstudy,London,WellcomeTrust,1998,p.4.3AnneMcLaren,'Cloning:pathwaystoapluripotentfuture',Science,2000,288:1775-80.4LeeMSilver,'Whatareclones?',Nature,2001,412:21.381

UrsulaMittwochVegetativeReproduction:TheSearchforaScientificTermThestorybeginsattheturnofthetwentiethcentury,whenHerbertJWebberwasinchargeofthePlant-BreedingLaboratoryoftheUnitedStatesDepartmentofAgriculture.In1899,WebberhadtravelledtoLondoninordertoattendaconferenceonhybridization,organizedbytheRoyalHorticulturalSociety,andreportedthatworkonhybridizationasameansofsecuringvariationandimprovementshadonlyrecentlybegun,chieflyonoranges,pineapples,apples,pears,wheat,cornandcotton.5Theyear1900witnessedtherediscoveryofMendel'spaperbythreeEuropeanbotanists,6andinthefollowingyearanEnglishtranslationofthepaperwaspublished,withaforewordbyWilliamBateson.7Whilethenewscienceprovidedincreasedopportunitiesforplantbreeders,themajorityofgardenersandfruitproducersseekuniformityratherthanvariationintheirproduce,andthroughouthistorytheyendeavouredtoby-passtheparticipationofovulesandpolleninthepropagationofmanyfoodplants.FollowingthedisseminationofMendel'slaws,Webberwassearchingforascientifictermtodenotethoseplantsthatarepropagatedvegetativelybybuds,grafts,cuttings,suckers,runners,slips,bulbs,tubers,etc.Suchplants,hewrote,"arenotindividualsintheordinarysense,butaresimplytransplantedpartsofthesameindividual,andinheredityandinallbiologicalandphysiologicalsensessuchplantsarethesameindividual".8Webberemphasizedthathewaslookingforatermthatcouldbeusedandunderstoodbythegeneralpublic.Itshould,therefore,beshort,euphonious,phon-eticallyspelled,easilypronouncedanddifferentfromanyotherwordinordinaryuse,sothatitwouldnotsuggestanymeaningotherthantheonedesired.Hesuggestedthewordclon,fromtheGreekKAOV,meaningatwig,sprayorslip,suchasisbrokenoffforpropagation.Clon(pluralclons,adjectiveclona!)wastobeappliedtovarietiesofapples,pears,strawberries,etc.,thatarenotpropagatedfromseeds,todistinguishthemfromplantssuchaswheatandcornthataregrownfromseed.Thewordwastobepronouncedwithalongo,asinGreek.Subsequently,CharlesLouisPollard,ofSpringfield,Massachusetts,suggestedanimprovementinorthography.9HepointedoutthatoneofthedefiniteindicationsofquantityinEnglishwordswasinthefinale,asintone,bite,hateetc.;andeventhoughrecentAmericanusagehadsometimesdispensedwiththefinale,itseemedimportanttodistinguishthederivationfromtheGreekwordKAcovfromthatofKAovos(thelatterhavinggivenrisetotheEnglishadjectiveclonic).Ifwrittenclon,thewordwouldnaturallybepronouncedclon.'Hesuggestedthatclonebeadoptedasthecorrectform.Thenewspellingbecamewidelyaccepted.'HerbertJWebber,'WorkoftheUnited8HerbertJWebber,'NewhorticulturalStatesDepartmentofAgricultureonplantandagriculturalterms',Science,1903,18:hybridisation',J.r.hort.Soc.,1900,24:128-45,501-3.pp.128,131-4,138.'CharlesLouisPollard,'Onthespellingof6RobertOlby,OriginsofMendelism,2nded.,"clon"',Science,1905,22:87-8.ChicagoUniversityPress,1985,pp.109-37.'°Idem,"'Clon"versus"Clone"',Science,7GregorMendel,'Experimentsinplant1905,22:469.hybridisation.WithanintroductorynotebyW.Bateson',J.r.hort.Soc.,1901,26:1-32.382

"Clone"ClassicalDefinitionsThetermclonewasdefinedbyCraneandLawrenceas"thecollectivenameofalltheplantsasexuallyreproducedbydivision,grafting,etc.,fromone(seedling)individual"."Theauthorspointedoutthatthismethodofreproductionhadhadaprominentplaceinhorticultureforcenturies,andwasoftentheonlymeansofperpetuatingimportantvarieties.CDDarlingtondefinedcloneas"agroupoforganismsdescendedbymitosisfromacommonancestor".'2Whilethisdefinitionwouldincludeanimals,theexampleshediscussedwereconfinedtoplantsbearinganextrachromosome(trisomics),orwhichareheterozygousforchromosomalchanges.Manysuchplantsaresterile,andiftheydosetviableseed,theyfailtobreedtrue.Thedefinitionsgivenaboveemphasizethatoriginallyclonewasacollectivenoun,referringtoagroup,allofwhosemembersweredescendedbyvegetativereproductionfromacommon,sexuallyproduced,ancestor.ABStoutintroducedthetermramettodenotetheindependentmembersofaclone.'3Headdedthat"[t]hebotanicalstatusoftheentiregroupofplants(ramets)constitutingaclonispreciselythatoftheindividualplant...fromwhichtheclonwasderived".WBTurrillwrotethat"withmanyperennialspecieshundredsoframets(thephysiologicallyindependent'individuals'ofaclone)canbeproducedwithinayear".'4Hefurtherstatedthattheserametswereallofthesamegeneticconstitution,apartfromraresomaticmutations,sothatahighdegreeofuniformitycouldbeachieved.ThewordrametisincludedintheOxfordEnglishDictionary.'"Itisevidentthatvegetativepropagationplaysanimportantroleinensuringtheproductionofuniformcropsofmanyplantsofeconomicimportance,andthisfact,coupledwiththeeasewithwhichthewordcloneispronouncedandremembered,causedthetermtoenterintothevocabularyofawiderpublic.In1928,TheTimesprintedaReuters'reportstatingthattheRubberResearchInstituteoftheFederatedMalayStateshadissuedacommunique,whichincludedthestatementthat,inatappingofbuddingscarriedoutbytheInstitute,thehighest-yieldingclonehadlatexvesselsofmuchsmallerborethanthelowest-yieldingclone.Thiswasfollowedbyanexplanationinparentheses:"(Note-Cloneisthetermgiventoallbudgraftstakenfromaparticularparenttree)".'6Someyearslater,theEconomistselectedthefollowingitemfromachairman'sreport:"wealsohaveaninterestinNamoeTonganRubberEstatesinSumatra,whichhavebeenplantedwithselectedhighyieldingclones".'7"MBCraneandWJCLawrence,The'5TheOxfordEnglishDictionary,2nded.,geneticsofgardenplants,London,Macmillan,Oxford,ClarendonPress,1989.1934,p.213."6Reuters'Report,'Potentialrubbertree2CDDarlington,Recentadvancesincytology,yields',TheTimes,20July1928,p.20.2nded.,London,Churchill,1937,p.574."'Langen(Java)RubberEstates3ABStout,'Thecloninplantlife',J.NewCompany-Improvedresults-strongposition',Yorkbot.Garden,1929,30:25-37,p.33.Economist,26Jan.1935,120:212."WBTurrill,Britishplantlife,London,Collins,1948,p.128.383

UrsulaMittwochHowever,neithereconomicsuccessnorpopularinterestinfruitssuchasapples,strawberriesandbananasinspiredthemassmediatotakeaninterestinclones.Thishappenedonlyafterthewordwasappliedtoorganismsthatwereverydifferentfromthecropplantsofagricultureandhorticulture,andthedevelopmentofnewtechniquesofculturingthem.Ishallbrieflyreviewthestepsthatledtoconsecutivechangesinthemeaningoftheterm.LifeCyclesofProtozoa:ClonesandPureLinesDuringthecourseofthenineteenthcentury,techniquesweredevelopedthatallowedprotozoatobeculturedandaccuratelyobservedunderthemicroscope.In1888,EMaupaspublishedadetaileddescriptionofthemethodologyforculturingciliateprotozoa,includingthreespeciesofParamecium,specifyingglassware,andthedifferentfoodorganismsthatneededtobepresentinthedropsofwatercontainingthedifferentspeciesofprotozoa,bothcarnivoresandvegetarians.Eachisolatedindividualhadtobefollowedonadailybasis,andthesameappliedtoeachofitsoffspringandfuturegenerations.Itemergedthattheseanimalscouldmultiplybybinaryfissionforvaryingnumbersofgenerations,butthateventuallythelinewoulddieoutunlesssexualreproduction,intheformoffusionbetweentwoindividuals,tookplace.'8AsubsequentinvestigationbyHSJenningsonvariationandinheritanceinprotozoa,publishedin1929,dealtwiththequestionoftheextenttowhichvariationcanoccurbetweenindividualswithinalineofasexuallyproducedoffspring,andcontainedasubheading'Theoccurrenceandinheritanceofvariationwithinclones,withtheresultsofselection'.'9Thewordclonewasdefinedasfollows:"Alltheindividualsdescendedbyuniparentalreproductionfromasingleindividualconstituteaclone.Asingleclonehasattimesbeencalleda'pureline.'Aseriesofsuccessivegenerationsdescendedunderculturefromsomememberofacloneiscommonlyspokenofasaline;sometimesasaseries."20Selectionexperimentswereundertakeninordertodiscoverwhetherdiversebiotypes,i.e.hereditaryconstitutions,couldbeisolatedfromasingleclone.Theconceptofthepureline,offeredasasynonymforclone,wasoriginallyintroducedbyWJohannsen2'todescribeallindividualsoriginatingfromasingleself-fertilizingindividual:"MiteinerreinenLiniebezeichneichIndividuen,welchevoneinemeinzelnenselbst-befruchtendenIndividuumabstammen"("bypurelineIdesignateindividualsthataredescendedfromasingleself-fertilizingindividual").Theterm"selbst-befruchtenden",i.e."self-fertilizing",demonstratesthatinJohannsen'sterminology,theconceptofthepurelinewasappliedtoasexuallyreproducingorganism,suchasthegardenbean,Phaseolusvulgaris,andnottovegetativereproduction.18EMaupas,'Recherchesexperimentalessur20Ibid.,p.126.Iamultiplicationdesinfusoirescilies',Arch.Zool.21WJohannsen,UeberErblichkeitinexp.gen.,1888,6:165-277,pp.191,179-84,PopulationenundinreinenLinien.EinBeitragzur254-9.BeleuchtungschwebenderSelektionsfragen,Jena,'9HSJennings,'Geneticsoftheprotozoa',Fischer,1903,p.9.Bibl.Genet.,1929,5:105-330,p.232.384

"Clone"TheadmixtureoftheconceptsofcloneandpurelinewasacceptedbyRobertHarringtonin1975,whenheappliedthetermclonetoanhermaphroditespeciesoffishthatreproducesbyself-fertilization.Hewrotethat"Selfinginthewildhadalreadyreducedthefishwithineachofthethreelinesofourstocktogeneticuniformity,viz.isogenicityplushomozygosity.Theselinesarereferredtoashomo-zygousclones.Thisusageoftheterm'clone'avoidsrepeatedcircumlocutionandmeansagrouporlineofdescentcomposedofuniparental,isogenicindividuals".22Thetermclonehasalsobeenappliedtoall-femalespeciesoflizardsbelongingtothegenus,Cnemidophorus,whichreproducebydiploidparthenogenesisandaregeneticallyhomogeneous,althoughmanyspeciesareheterozygous.23Jennings'1929paperhadbeenprecededbyanearlieronepublishedin1916,inwhichhechallengedJohannsen'sclaimthatselectionwithinpurelineswasineffective.AccordingtoJennings,selectionofcharacteristicssuchasnumberandlengthofspinesontheshelloftheprotozoan,Difflugiacorona,waseffectiveevenwithintheprogenyofasingleindividual.Hedidnot,however,usethewordclone,butfamily,foralltheindividualsdescendedbyfissionfromoneoriginalparent.24CulturesofCellsfromMulticellularOrganismsInadditiontothestudyofprotozoa,thesecondhalfofthenineteenthcenturyalsowitnessedthedevelopmentofthescientificstudyofbacteriology.25Havingdiscoveredthenatureoffermentation,LouisPasteurdevisedspecialliquidmediafortheirculture,whileRobertKochdevelopedsolidmediafortheisolationofpurecultures.Intheearlytwentiethcentury,pathologistsadaptedthetechniquesforculturingmicro-organismsforthestudyofhumancellsandthoseofothermulticellularorganisms.26Asinbacteria,techniqueswereworkedouttoenablequantitativestudiestobemadeoftherateofgrowthofnormalandcancercellsinvitro.However,theproblemofobtainingpureculturesfromanoriginalinoculumcontainingmanycellsprovedtobeharderthaninmicro-organisms.27Amajordifficultythatpersistedforseveraldecadeswasthatculturesfailedtogrowunlesstheoriginalinoculumconsistedofalargenumberofcells.Theproblemwassolvedforthefirsttimein1948byKatherineSanfordandcollaboratorsattheUSANationalCancerInstitute.2822RWHarrington,'SexdeterminationandLondon,EdwardArnold,1929,vol.1,pp.1-14,differentiationamonguniparentalhomozygotesof212-23.thehermaphroditicfish,Rivulusmarmoratus26Fischer,Tissueculture:studiesin(Cyprinodontidae:Atheriniformes)',inRexperimentalmorphologyandgeneralphysiologyReinboth(ed.),Intersexualityintheanimaloftissuecellsinvitro,London,Williamkingdom,BerlinandNewYork,Springer,1975,Heinemann(MedicalBooks),1925,pp.19-34.p.249.27HenryHarris,Thecellsofthebody:a23CharlesJCole,'Unisexuallizards',Sci.Am.,historyofsomaticcellgenetics,ColdSpringJan.1984,250:84-90,118,p.85.HarborLaboratoryPress,1995,pp.31-56.2SJennings,'Heredity,variationandthe28KatherineKSanford,WiltonREarleandresultsofselectionintheuniparentalGwendolynDLikely,'ThegrowthinvitroofreproductionofDifflugiacorona',Genetics,1916,singleisolatedtissuecells',J.natl.CancerInst.,1:407-534,pp.419,532-3.1948,9:229-46.2SWWCTopleyandGSWilson,Theprinciplesofbacteriologyandimmunity,2vols,385

UrsulaMittwochTheirpublicationcarriedthefollowingintroduction:"Oneobstacletothemoreaccurateuseoftissueculture,particularlyincancerresearch,hasbeentheinabilitytogrowasingle,isolated,cellintoacloneorpureculture."29Thereasonforpreviousfailuresturnedouttobetheinadequacyofeventhebestculturemedia,whichneededextensivemodificationbylargenumbersofcellsbeforetheycouldbeutilized.Accordingly,theauthorsreducedthevolumeofmediumsurroundingindividualcellsbygrowingthemincapillarytubes,andalsousedconditionedmedium,inwhichcellshadbeengrownpreviously.30ThenewtechniqueresultedintheestablishmentofaclonecultureofstrainLsarcoma-producingcellsfrommiceoftheC3Hstrain.3'Suchculturesdidnot,however,alwaysretaintheirhomogeneity.Someyearslater,theauthorsreportedthattwosub-linesderivedfromaclonecellculturethathadoriginatedfromasinglecelldifferedinmorphologyaswellasintheirabilitytoinducesarcomaswheninjectedintomice.32TheodorePuckandPhilipMarcusdevisedasimplifiedtechniqueforproducingculturesofHeLa(humancervicalcarcinoma)culturesfromsinglecells.Insteadoflimitingtheamountofmediumavailabletoindividualcells,theyplatedthemoutontoaconfluentlayerofcellsthathadbeenkilledbyirradiation.33Inasubsequentpublication,Puckandhiscolleaguesgavethefollowingdefinitionofclone:"Acloneisapopulation,allofwhosemembersaredescendantsfromthesamesingleorganism".34Butcanasinglemammaliancellberegardedasanorganism?Andcouldthisdefinitionnotbeequallyappliedtoanycellculturederivedfromanindividualorganism?Thecellculturesobtainedbythesetechniqueswereprobablyalltransformedcellswithabnormalchromosomeconstitutions.Theestablishmentofcellculturesfromasingleeuploidcellwithnormalchromosomesprovedtobemuchmoredifficult.35CellCulturesfromPlantsTechniquesforgrowingculturesfromplantmaterialdevelopedalongsimilarlinestothosederivedfromanimalcells.Theyalsopresentedsimilarproblemswhenculturesweregrownfromtissuesofcellswhich,eventhoughtheywerederivedfromthesameplant,mayhavehaddifferentpotentialities.In1954atechniquewasdescribedforobtainingculturesoriginatingfromsinglecellsofcrowngallsonmarigold(Tageteserecta),andoftobacco(Nicotianatabacum)fromnormalstems.36Theauthorsreferredtotheseas"planttissueculturesofsinglecellorigin".This29Ibid.,p.229.productionwithHeLacellsintissueculture:the30Ibid.useofX-irradiatedcellstosupplyconditioning31Ibid.,p.245.factors',Proc.natl.AcadSci.USA,1955,41:32KatherineSSanford,GwendolynDLikely,432-7.WiltonREarle,'Thedevelopmentofvariations3TheodoreTPuck,PhilipIMarcus,StevenJintransplantabilityandmorphologywithinaCieciura,'Clonalgrowthofmammaliancellsincloneofmousefibroblaststransformedtovitro',J.exp.Med.,1956,103:273-84,p.274.sarcoma-producingcellsinvitro',J.natl.Cancer35Harris,op.cit.,note27above,p.51.Inst.1954,15:215-37,pp.228-9.36WHMuir,ACHildebrandt,AJRiker,3TheodoreTPuck,PhilipIMarcus,'Arapid'Planttissueculturesproducedfromsinglemethodforviablecelltitrationandcloneisolatedcells',Science,1954,119:877-8.386

"Clone"workwasfollowedbyamoreextensiveinvestigation,includingculturesfromadditionalplantspecies,grownfrombothcrowngallandnormaltissues.Thenewpublicationcontainedthefollowingstatement:37Manysinglecellclonesgrewwellindependently,butsomeeitherfailedtogrow(about10percent)orgrewpoorlyaftertransfertoagar.Theterm"clone"isusedinaccordancewithEarl's[sic](1955)38definition.Itseemsparticularlyappropriate,sincethesinglecellcamefromaheterogeneousmixtureandculturepropagationisentirelyvegetative.In1959LudwigBergmannusedthetermcloninginapublicationdescribingadifferenttechniqueforisolatingsingleplantcellsfortissueculture.39Duringthesameperiod,FrederickCampionStewardandcollaboratorsdevelopednewtechniquesforstudyingthegrowthofplantcellsfromtissuesthathadceasedcelldivision.Thisinvolved,first,theidentificationofmediacontainingthecorrectcompositionofgrowth-regulatingsubstances.40Byexposingsmallpiecesofcarrottissuetoaregulatedmixtureofgrowthmediumandair,phloemtissuewhichhadceasedtogrowresumedcelldivision,andeventuallyevenisolatedcellswouldgrowintoentireplants,includingflowersandseeds.4'Thederivativesofphloemexplantsderivedfromagivenplantwerereferredtoasaclone.42In1970,Stewardlookedbackontheyearsduringwhichithadbecomepossibletogrowentireplantsfromisolatedsomaticcells.Heconsideredthatallpartsofthecellswereinvolvedinthechangesoccurringwhenaquiescentstatereturnedtocellproliferation.Thisraisedthequestionwhethersimilarchangescouldbeinducedinthecellsofanimals.EvidencefromtheexperimentsofJohnGurdon(seebelow)seemedtoshowthatthenucleiofcertainanimalcellsretainedalltheirgeneticinformation,butthatthiswasreleasedonlyincontactwiththecytoplasmofanenucleatedegg.Perhapseventually,theequivalentoftheplanttechniquesheredescribedmayinducesomaticcellsofanimalstoembarkdirectlyuponanembryologicaldevelopment,and,perhaps,ifthisisdone,theymayevenbefurnishedinsomewaywiththeequivalentofaparentalbloodsupply.Suchstepspresent,however,obviouslygreaterordersofdifficulty,eveniftheydonotconstituteinsuperablebarrierstothefreeclonaldevelopmentofhigheranimalsfromsomaticcells.437WHMuir,ACHildebrandt,AJRiker,'Thepreparation,isolation,andgrowthincultureofsinglecellsfromhigherplants',Am.J.Bot.,1958,45:589-97.38WREarle,Communicationon'Purecellstrains',inDCHetherington(ed.),AnnualreporttoTissueCultureAssociation,1955,ascitedbyMuiretal.onp.593,note37.Extensiveenquirieshavefailedtolocateacopyofthereport.3LudwigBergmann,'Anewtechniqueforisolatingandcloningcellsofhigherplants',Nature,1959,Aug.22:648-9.'4EMShantz,FCSteward,MSSmith,RLWain,'Investigationsonthegrowthandmetabolismofplantcells.VI.Growthofpotatotubertissueinculture:thesynergisticactionofcoconutmilkandsomesyntheticgrowth-regulatingcompounds',Ann.Bot.,1955,19:49-58.4FrederickCampionSteward,'Thecontrolofgrowthinplantcells',Sci.Am.,Oct.,1963,209:104-13.4FrederickCampionSteward,Marion0Mapes,'Thetotipotencyofculturedcarrotcells:evidenceandinterpretationsfromsuccessivecyclesofgrowthfromphloemcells',J.Indianbot.Soc.,1963,42A,MaheshwariCommemorationvol.,237-46,pp.238-242.43FrederickCampionSteward,'Totipotency,variationandclonaldevelopmentofculturedcells',Endeavour,1970,29:117-24,p.124.387

UrsulaMittwochBeforetakingupthetopicofdevelopmentinanimals,twointerludesdealingwithnomenclaturebasedonimmunologicalsciencewillbeinserted.Interlude1:TheClonalSelectionTheoryofAcquiredImmunityTheincreasinglywidespreaduseofthewordcloneanditsderivativesfortheoffspringofsinglecellsincultureisreflectedinthetitleofFMacfarlaneBurnet'sinfluentialClonalselectiontheoryofacquiredimmunity,publishedin1959,whichdealtwiththedifficultproblemofhowcellscanmakespecificantibodiestothevastvarietyofdifferentpathogens.Itcouldbearguedthattheimpactofthetheorywasenhancedbythechoiceoftitle,whichimpliedscientificnoveltyandatthesametimewaseasilyremembered.Indeed,theauthorbeganbysayingthatinstrugglingtofindtitlesforlecturesorarticles,thefirstthatusuallycametomindwas"Abiologicalapproachto-,orThenaturalhistoryof(infectiousdisease,cancer,war)"etc."Theunderlyingagentofantibodyproductionwasnotjustacell,butacloneofcells.Moreover,theecologicalandevolutionaryimplicationsofclones,asopposedtosexuallyreproducingpopulations,seemedtorepresentasimplificationofsubjectmatter.Thisconceptcouldbeappliedequallytobacteria.Burnet'stheorywasbasedonthepremisethatantibody-producingcellsformedpartofamobilepopulationofmesenchymalcellsconstantlyundergoingphysiologicalandmutationalchanges.Heproposedthat,whensomaticmutationalchangesoccur,newclonesareinitiated.45Clonesthatproduceantibodyglobulinscorrespondingtoparticularantigenicdeterminantswillbeataselectiveadvantage,whichisexpressedbyenhancedproliferation.Thiswillresultinantibodiesbeingproducedtocombatthelargevarietyofantigensthatmaybeencountered.Interlude2:MonoclonalAntibodiesForpracticalpurposes,antiseraraisedbyinjectingforeignproteinsintoanimalstendtolacksufficientspecificityandreproducibility.Awayoutwouldbetosetupculturesfromindividualantibody-secretingcells,whichproduceonlyonespeciesofantibody,butsuchculturesfailtosurvive.In1975,GeorgesKohlerandCesarMilsteinovercamethisproblembyhybridizinglymphocytes,theprecursorsofantibody-producingplasmacells,withmalignantmyelomacells,whichcanbemaintainedpermanently.46Theauthorsfirstimmunizedmicewithsheep'sredbloodcellsandthenfused(hybridized)theirspleencellswithmyelomacells.Thehybridcellssecretedantibodiesagainstbothparents.Individualcellsthatproducedanti-bodiesagainsttheredbloodcellsweresetupinculture,andtheclonescouldbemaintainedinculture.Inordertobecome"monoclonalantibodies"inthe4FMacfarlaneBurnet,Theclonalselection4GeorgesKohler,CesarMilstein,theoryofacquiredimmunity,Cambridge'ContinuousculturesoffusedcellssecretingUniversityPress,1959,pp.1-2.antibodyofpredefinedspecificity',Nature,1975,45Ibid.,p.67.256:495-7.388

"Clone"immunologicalsense,however,theyneededtoundergoadditionalselectionpro-cedurestoridthemofdeterminantsderivedfromthemyelomaparentandtoexpresssolelyantibodiestoasingledeterminant.47Becauseoftheimmenseimpactofthetechniqueonclinicalmedicineandbiologicalresearch,CesarMilsteinandGeorgesKohlerwereawardedtheNobelPrize.Hencetheterm"monoclonalantibodies"isfamiliartomanyscientistsotherthanthosespecializinginimmunology.ExperimentswithEnucleatedFrogs'EggsWemustnowaddressthequestionofthedevelopmentalpotentialofanucleusofadifferentiatedanimalcell.Istheeffectofthenucleustodirectthedevelopmentofthephenotypeconfinedalongrestrictedlines,orcouldeventhenucleiofdifferentiatedcellsinitiatenormalembryonicdevelopment?Theideaoftransplantingsomaticnucleiintoeggshasexistedsincetheendofthenineteenthcentury,butitssuccessfulapplicationhadtowaituntiltechniquesofactivation,enucleationandthetransferofnucleusintotheegghadbecomeavailable.48Bythe1950ssufficientadvanceshadbeenmadetoenableRobertBriggsandThomasKingtoreportsuccessfultransplantationinthefrog,Ranapipiens.49Byremovingthenucleifromfrogs'eggsandreplacingthemwithnucleifromembryosattheblastulastage,theauthorssucceededinraisingfrogsuptoadulthood.Inanotherseriesofexperiments,KingandBriggsmadeserialtransplantationsintoenucleatedfrogs'eggs.Thisincludedisolatingnucleifromablastulaandinsertingthemsinglyintoenucleatedeggs,eachofwhichwasallowedtodevelopintoanewblastula.Theprocesswasthenrepeated,usingblastulaeobtainedfromthepreviousexperiments.Sinceitcouldbeassumedthatallblastulaesoformedcontainedidenticalnucleiderivedfromtheoriginalblastula,theywerereferredtoasa"nuclearclone".50Subsequently,theadjective"nuclear"tendedtobeomitted,andalltheoffspringofaparticularblastulawerereferredtoasaclone.Duringthe1960s,JohnGurdoncarriedouttransplantationexperimentsontheSouthAfricanclawedfrog,Xenopuslaevis,andinanarticleinScientificAmerican,concludedadescriptionofserialnucleartransplantationasfollows:"Theeffectisthesameasinthevegetativepropagationofplants,namelytheproductionofaclone:apopulationconsistingofmanyindividualsallhavinganidenticalsetofgenesintheirnuclei";whilethelegendtoanillustrationexplainingserialtransplantscontainedthestatement:"Thecells'geneticallyidenticalnucleiarethentransplantedintoenucleateeggs,givingrisetoaclone:apopulationcomprisedofgenetically47CesarMilstein,'Monoclonalantibodies,Sci.frogs'eggs',Proc.natl.AcadSci.USA,1952,38,Am.,Aug.1980,243:66-74.455-63.4'MarieADiBerardino,Genomicpotentialof5ThomasJKing,RobertBriggs,'Serialdifferentiatedcells,NewYork,Columbiatransplantationofembryonicnuclei',ColdSpringUniversityPress,1997,pp.28-53.HarborSymp.quant.Biol.,1956,21:271-90,49RBriggs,TJKing,'Transplantationofp.277.livingnucleifromblastulacellsintoenucleated389

UrsulaMittwochidenticalindividuals".51Theindividualsthemselveswerereferredtoas"transplantfrogs".Inalaterarticle,Gurdonspeculatedonthenatureofcytoplasmicmoleculesthatregulategeneactivity,buthestillmaintainedthatallfrogsobtainedbythetransplantationofnucleifromthesamedonorembryointomanyrecipienteggsconstitutedaclone,"becausetheyareallgeneticallyidenticaltwins";52andagain,thecomparisonismadewithvegetativereproductionaspractisedinplants.Thiscomparisonwasevidentlybasedontheconstancyofthenucleargenomeratherthanthephenotyperesultingfromtheinteractionofanycytoplasmicparticleswiththegenome.MitochondrialDNAThestrikingparallelismbetweenthebehaviourofchromosomesduringmeiosisandthetransmissionofpairedMendeliancharactersnotonlypersuadedgeneticiststhatthechromosomeswithinthenucleuswerethebearersofthehereditarymaterial,butalsogaverisetothesupplementaryideathatthecytoplasmwasdevoidofgeneticelements.Theoppositeview,emerginginthe1950sand1960s,thatsomeDNAwaspresentalsoinacytoplasmiccomponent,themitochondria,53eventhoughsupportedbyincreasinglystrongevidence,54wasatfirstvigorouslyrejected.5Therelativelymassivecytoplasmoffrogs'eggsappearedtobeparticularlyrichinmitochondrialDNA.Intwospecies,RanapipiensandXenopuslaevis,eggswerefoundtocontain300-500timesmoreDNAthandidsomaticcells,andtheevidencesuggestedthatmostofthisDNAwasofmitochondrialorigin.56Buteventhesefindings,aswellasothers,suggestingthatmitochondriacontainedafewdozengenes,5'didnotimpingeontheexpandinguseofthetermcloneanditsexclusiveconnectionwithnucleargenes.Mitochondriaarethepowerplantsofthecellandarenowknowntocontaingenesthatareessentialforenergygeneration.Inrecentyearsanincreasingnumberofmutationshavebeenidentifiedinhumanmitochondriathatgiverisetodegenerativediseases,particularlyofthebrainandofthemuscles.58Thesemutationsareinheritedsolelythroughthemother.Progressivelymalfunctioningmitochondriahavealsobeenassociatedwiththeagingprocess.Recentlyithasbeenestablishedthat51JBGurdon,'Transplantednucleiandcell56IgorBDawid,'Evidenceforthedifferentiation',Sci.Am.,Dec.1968,219:24-35,mitochondrialoriginoffrogeggcytoplasmicpp.27,28.DNX,Proc.Nat!.AcadSci.USA,1966,56:52Idem,'Eggcytoplasmandgenecontrolin269-76,p.269.development',TheCroonianLecture,1976,Proc.5DavidWilkie,'EarlyrecollectionsoffungalRSoc.Lond,B,1977,198:211-47,p.218.geneticsandthecytoplasmicinheritance5SylvanNass,MargitMKNass,controversy',inMNHallandPLinder(eds),'IntramitochondrialfiberswithDNATheearlydaysofyeastgenetics,PlainView,ColdcharacteristicsII.EnzymaticandotherhydrolyticSpringHarborLaboratoryPress,1993,treatments',J..CellBiol.,1963,19:613-29.pp.259-69,p.263.5DBRoodyn,DWilkie,Thebiogenesisof58DouglasCWallace,'MitochondrialDNAinmitochondria,London,Methuen,1968,pp.3-10.aginganddisease',Sci.Am.,Aug.1997,277:55DWilkie,Thecytoplasminheredity,40-7.London,Methuen,1964,pp.1-8.390

"Clone"mitochondrialDNAencodesthirteenpolypeptideswhichinteractwithalargenumberofpeptidesencodedbythenucleustoformthemitochondrialelectrontransportsystem.59Thissuggeststhatcompatibilitybetweenthetwogeneticsystemscouldbeimportant.Evenmammalianeggs,althoughverymuchsmallerthanthoseofamphibia,containabout100,000mitochondria,containing2x105copiesofthemitochondrialgenome,60comparedwithaboutathousandmitochondriainthecellsofsomaticcells.6'Thelargenumbermaybeinpreparationfortheenergeticdemandsofembryogenesis.Analternativeexplanationisthat,sincemitochondriadonotreplicateinearlyembryodevelopment,alargereservoirneedstobepresentintheoocyte.2Mitochondriacontroltissue-specificenergyproduction,andthereisinteractionbetweenthemitochondrialandnucleargenomes.6'Eventhoughquestionsremainregardingdetailedmechanisms-andnotwithstandingthefactthatthenumberofnuclearhumangenesexceedsthatpresentinmitochondriabyafactorofthreemagnitudes-therecannolongerbeanydoubtthatthequestion"Docytoplasmicgenesmatter?"mustbeansweredintheaffirmative.MoreScientificDefinitionsIn1972,LeonKasspublishedanarticleopposingdifferentreproductivetech-nologies,andinasectionon'Cloning,orasexualreproduction:stateoftheart',wentintosomedetailtodefinethetermclone.Theauthorbeganbysayingthat"Ingeneticterms,asexualreproductionisdistinguishedfromsexualreproduction...bytwocharacteristics:thenewindividualsare,first,derivedfromasingleparent,andsecond,geneticallyidenticaltothatparent".Furthermore,"Anunlimitednumberofidenticalindividuals,i.e.,aclone,allgeneratedasexuallyfromasingleparent,couldbeproducedbynucleartransplantation".64Thefirstcriterion,thatanindividualproducedbyasexualreproductionhasonlyasingleparentwouldseemtobequestionableinthecaseofindividualsproducedbynucleartransplantationintoanenucleatedeggfortworeasons:first,theeggcytoplasmcomesfromasecondindividual,andsecond,theeggisasexualcell,producedbymeiosis,ratherthanasomaticcell,producedbymitosis.Theinvolvementofthecytoplasmofaneggensuresthatthetechniquehasatbestsome,butnotall,thecharacteristicsofasexualreproduction.Thecorrectnessofthesecondcriterionisquestionablebecauseofthepresenceofmitochondrialgenesinthecytoplasm,whicharelikelytoaffectthefunctionofthenucleargenes.59PierreUBlier,FranceDufresne,RonaldS62Cummins,op.cit.,note60above,Burton,'Naturalselectionandtheevolutionofpp.172-3.mtDNA-encodedpeptides:evidencefor63Ibid.,pp.174-5.intergenomicco-adaptation',TrendsGenet.,2001,6LeonRKass,'Newbeginningsinlife',inM17:400-6.PHamilton(ed.),Thenewgeneticsandthefuture'JimCummins,'MitochondrialDNAinofman,GrandRapids,MI,WBEerdmans,mammalianreproduction',Rev.Reprod.,1998,3:1972,pp.15-63,pp.42-5.172-82.61GuyBrown,Theenergyoflife,London,HarperCollins,1999,p.100.391

UrsulaMittwochKassrejectedthenotionofthosewhowouldconfinethetermcloningtolarge-scalereplicationandusetheterm"nucleartransfer"forsingleinstancesofasexualreproduction:...hopingtherebynottobringtheopprobriumofthemassuseuponindividualcases....Whilethetermclonedoesimplyanaggregate,itisanaggregatewhichisformednothorizontally(duringonegeneration)butverticallyovertime(generationaftergeneration).Thuseventhefirstasexually-producedoffspringandhisprogenitortogetherformaclone,albeitasmalloneforthetimebeing."Nucleartransfer"isbutthenameofoneofseveralpossibletechniquesthatcouldgiverisetoacloneandthusdoesnotserveasagenerictermforthegeneticallyandhumanlysignificantfeaturesofasexualreproduction.Moreover,thedesiretoavoidforthesmall-scaleusetheoffensiveconnotationofmassproductionbegsthequestionofwhethertheopprobriumisnotequallyfittingandleadstothedevelopmentofeuphemism.Ishallthususe"cloning"assynonymouswith"asexualproduction,artificiallyinduced."65ThisparagraphwascitedbyRobertMcKinnellintheprefaceofhisbookCloning:nucleartransplantationinamphibia,publishedin1978,whereheexplainedthereasonforchoosingthistitle:"Biologistsgenerallyusetheword'cloning'torefertotheproductionofmultiplegeneticallyidenticalindividuals.Clonedamphibia,inthesenseofisogenicgroupsoffrogs,areproducedbynucleartransplantation,whichisthesubjectofthisbook.However,theword'cloning'isusedhereinamoregeneralsense,thatis,torefertooneormoreindividualsproducedbythemethod."'LikeKass,McKinnellappliedthetermcloningtotheproductionofasingleindividualoffspring.Inthesamebook,theword"clone"isgiventoagroupoffrogsthathavearisenbynucleartransplantationfromacommonblastulaandthereforearegeneticallyidentical,orisogenic."Theterms"isogenicgroups","syngeneticseries",and"nucleartransferclone"areusedsynonymouslyinthischapter.Thus"clone"isusedheretomeanagroup,inlieuofitsmeaningasamodeofasexualreproduction".67Inthefollowingyear,McKinnellpublishedanotherbook,addressedtothegeneralreader,68ofwhicharevisededitionappearedin1985.69Bythen,therehadbeensubstantialprogressinmammalianreproductivetechnology,includingin-vitroferti-lizationandimplantationofembryosintofostermothers.Afewmicewereclaimedtohavebeenbornasaresultofnucleartransplantation,70andthepossibilityofusingthetechnologyonhumanswasasubjectforspeculation,whichinturnincreasedtheuseofthewordscloneandcloningamongbothscientistsandthepublic.AccordingtoMcKinnell'sglossary,themeaningofclonehadnowbeenextendedtocomprisenotonlythecollective,butalsotheindividual:"Agroupofgeneticallyidentical65Ibid.,p.43.69Idem,Cloningoffrogs,mice,andother6RobertGMcKinnell,Cloning:nuclearanimals,rev.ed.ofCloning:abiologistreports,transplantationinamphibia,Minneapolis,Minneapolis,UniversityofMinnesotaPress,UniversityofMinnesotaPress,1978,pp.vii-viii.1985.67Ibid.,p.148.7KIllmensee,PCHoppe,'Nuclear68Idem,Cloning:abiologistreports,transplantationinMusmusculus:developmentalMinneapolis,UniversityofMinnesotaPress,potentialofnucleifrompreimplantation1979.embryos',Cell,1981,23:9-18.392

"Clone"organismsproducedwithoutsexualreproduction;anindividualproducedasexu-ally".7'Thepresenceoftheeggcytoplasmcontinuedtobeignored.Inadditiontothesetwodefinitions,McKinnellalsoconsideredthat"[t]hepro-ductionofmorethanoneembryofromseparatedcellsofasingleembryo,withouttheinterventionofsex,shouldberegardedasaformofcloning".72ThisuseofthetermseemstohavebeenforeshadowedbyJBSHaldane,73buttheclonesthatHaldaneenvisagedweretobeproducedbyaverydifferenttechnique.GeneCloningWiththeadventofrecombinantDNAtechnology,thetermcloneacquiredyetanothermeaningformolecularbiologists,whoextendeditsusetoasegmentofDNAthathadbeenmultipliedmanytimes.StanleyCohenandhisco-workersconstructedbacterialplasmidscarryingnewcombinationsofDNAsegments.ThesewereinsertedintoEscherichiacolibacteria,whichgaverisetocolonies.Theauthorswrote:"Asingleclonethathadbeenselectedforresistancetokanamycinandwhichwasfoundalsotocarryresistancetoneomycinandsulfonamide,butnottotetracyclin,chloramphenicol,orstreptomycinaftertransformationofE.colibyEcoRI-generatedDNAfragmentsofR6-5,wasexaminedfurther".74SubsequentlyitseemedasmallsteptoextendtheterminologyfromthebacterialclonetotheconstructofDNAthatwascontainedinit.InRecombinantDNA:ashortcourseJamesWatsonandhiscolleagueswrote:Throughoutthisbookwehavefrequentlyusedthewords"cloned","cloning,"and"clone";recentlytheyhavetakenonnewand,insomequarters,highlyemotivemeanings.Theword"clone"wasfirstusedtodescribeapopulationofcellsororganismsallderivedfromasinglecellororganismbyasexualmultiplicationinsuchawaythatalltheindividualsintheclonehavethesamegeneticconstitution.WhenitbecamepossiblebyrecombinantDNAtechniquestotakeageneoranyothersegmentofDNAandmultiplyitinbacteria,theterm"genecloning"seemedappropriate.75AccordingtoarecentdefinitioninNature,cloningis"[t]heprocessofgeneratingsufficientcopiesofaparticularpieceofDNAtoallowittobesequencedorstudiedinsomeotherway".76JBSHaldaneandAldousHuxleyInhisessayonscienceandthefuture,Haldaneconsideredvariouspossibilitiesbywhichprogressinbiologycouldcontributetothebettermentofhumankind.He71McKinnell,op.cit.,note69above,p.119.biologicallyfunctionalbacterialplastidsinvitro',72Ibid.,p.91.Proc.natl.AcadSci.USA,1973,70:3240-4,73JBSHaldane,'Biologicalpossibilitiesforp.3241.thehumanspeciesinthenexttenthousand5JamesDWatson,JohnToozeandDavidTyears',inGWolstenholme(ed.),ManandhisKurtz,RecombinantDNA:ashortcourse,Newfuture,London,J&AChurchill,1963,York,ScientificAmericanBooks,1983,p.206.pp.337-61.76DavidBaltimore,'Ourgenomeunveiled',7StanleyCohen,AnnieCYChang,HerbertNature,2001,409:814-16.WBoyer,RobertBHelling,'Constructionof393

UrsulaMittwochpredictedin-vitrofertilizationandcoinedthetermectogenesisfortheenvisageddevelopmentoftheembryoandfoetusinthelaboratory.TheproposedadvantagesofthesetechniqueswerethattheywouldcounteractthefallinthebirthratethatthreatenedvariousEuropeancountries,andalsothattheyenabledselectivebreedingfromasmallproportionofmenandwomenwithsuperiorachievements.77TheectogenesisthemewaselaboratedbyAldousHuxleyinhisfuturisticnovelBravenewworld,78thoughmainlyfortheproductionoflow-casteindividuals,whoweremass-producedinthemannerofFordmotorcars.InHuxley'snewworld,humanreproductionisconfinedtotest-tubes,inwhichthenutrientsaremodifiedtoproduceindividualsofdifferentclassestofittheirfuturestationsinlife.Thosedestinedtobelowerclass,Gammas,DeltasandEpsilons,whowouldbeneededtocarryoutmenialtasks,andwhoare,therefore,requiredinlargenumbers,aresubjectedtoatreatmentthatinducesthefertilizedeggtodivideseveraltimestoproducefromeighttoninety-sixidenticalindividuals.Huxleyreferredtothetechniqueas"Bokanovsky'sProcess",andtheprocessofdoingso"tobokanovskify".ItshouldbenotedthatthemethodofsocialengineeringdescribedbyHuxleyreliedmoreonmodifyingtheenvironmentofthedevelopingindividualsthantheirgeneticmake-up.Atthesametime,themassproductionofdesiredworkerswasfurtherpromotedbyrepeatedsubdivisionsoftheembryotobesubjectedtoaspecifictreatment,thusmaximizingtheuniformityoftheindividualstobeproduced.Thirtyyearslater,Haldanereturnedtothethemeofhowtoimprovethehumanracethroughadvancesinbiology.Anewpossibilitynowseemedonthehorizon:Wehaveknownhowtogrowmammaliancellsincultureforover50years.Humancells,notonlyfromembryos,childrenandcancers,butfromasixty-year-oldman,havebeengrownforyearsonend.Wedonotknowhowtoinducethemtoorganizethemselves.Butwemayfindoutatanymoment,aswehavealreadyfoundoutinsomecasesofplantcells.Itisextremelyhopefulthatsomehumancelllinescanbegrownonamediumofpreciselyknownchemicalcomposition.Perhapsthefirststepwillbetheproductionofaclonefromasinglefertilizedegg,asinBraveNewWorld.Butthiswouldbeoflittlesocialvalue.Theproductionofaclonefromcellsofpersonsofattestedabilitywouldbeaverydifferentmatter,andmightraisethepossibilitiesofhumanachievementdramatically.79Inthisquotation,Haldaneusesthetermclonetwice.Thefirst,inthereferencetoBravenewworld,relatestotheproductionofmultipleindividualsfromasinglefertilizedeggbyrepeatedsubdivisionsofanembryo,"Bokanovsky'sProcess".Torefertotheproductoftheprocessasaclonewouldappeartobeanoveluseoftheterm,which,asnotedabove,wassubsequentlyalsoadoptedbyMcKinnell.80Haldane,however,dismissesthisprocedureasoflittlesocialvalue.Thesecondreferenceistocellcultures.Manyofthesewouldnothaveoriginatedfromsinglecells,butthisfactmaynothaveseemedimportanttoHaldane,providedeachculturewasderivedfromasingleindividual.Sinceallsomaticcellsofanindividualcanbeassumedto77JBSHaldane,Daedalus:or,scienceandthe79Haldane,op.cit.,note73above,p.352.future,London,KeganPaul,Trench,Trubner,80McKinnell,op.cit.,note69above,p.91.8thimpression,1928,pp.63-8,352.78AldousHuxley,Bravenewworld,London,Chatto&Windus,1932.394

"Clone"havearisenbymitoticdivisionsfromtheoriginalfertilizedegg,anindividualoriginatingfromacellculturewouldbeaclonewithintheclassicaldefinitionoftheterm,i.e.apopulationofindividualsthataredescendedexclusivelyfromsomaticcells,withoutreferencetothenumberofcellsthatgaverisetothenewindividual.8'ContrarytothestatementbyGinaKolatathatHaldane,hookedonthemes-merizingstudiesbyBriggs,King,andlaterGurdon,speculatedontheresultsofthefrogstudiesatascientificsymposium,82thereisnomentionofthefrogstudiesinthesymposiumproceedings,83includingthepublisheddiscussiononethicalconsiderationswhichfollowsimmediatelyafterHaldane'scontributiontotheCibasymposium.'WhatHaldanedidenvisagewasthepossibilitythattechniquesmightbecomeavailabletoinducehumanculturedcellstodevelopintoadultindividuals,adevelopmentthatseemedalreadyfeasibleinplants.OnemaysurmisethathewasreferringtotheworkofFrederickCSteward,whosucceededingrowingentirecarrotplantsfromsomaticcellsinculture.85TherecanbenodoubtthatHaldanesimilarlyusedtheterm"toclone"fortheproductionofhumanbeingsfromsomaticcellsgrowninculture,andnotforembryosobtainedbynucleartransplantationintoenucleatedoocytes.Headdedthat:"Onthegeneralprinciplethatmenwillmakeallpossiblemistakesbeforechoosingtherightpath,weshallnodoubtclonethewrongpeople".86ThejuxtapositionofthewordscloneandcloningwiththemethodsofachievinghumanreproductionemployedbythetotalitarianregimedescribedinHuxley'snovelmayhavecontributedtoincreasingpublicanxietyaboutadvancesinhumanreproductivetechnologiesthroughoutthefollowingdecades.ArecentreportissuedjointlybytwoBritishauthoritiesstatesthat"ItisclearthatthetermcloningcarriesanautomaticstigmaformanybecauseofitsassociationwithimagerysuchasthatportrayedinBraveNewWorld",87eventhoughtheworddoesnotappearinthisbook.Indeed,publicfearandfascinationwiththedisastrouseffectscreatedbytheactivitiesofscientistsandtheirpredecessorsprecededthepublicationofthisnovelbymorethanathousandyears.GoetheandMaryShelleyTheoriginofGoethe'sballadoftheSorcerer'sapprentice,whoconvertedabroomintoaservanttofetchwaterfromthewell,butthencouldnotstophimeventhough81Darlington,op.cit.,note12above,p.574.developtothe8-cellstage,letonecelldevelop82Kolata,Clone:theroadtoDollyandanddeep-freezetheotherseven,apparentlyforthepathahead,London,AllenLane,Penguinthepurposeoftransplantation.Press,1997,p.61.85Steward,op.cit.,note41above.83Wolstenhohne,op.cit.,note73above.86Haldane,op.cit.,note73above,p.352.'4Discussion,'Ethicalconsiderations',in87HumanGeneticsAdvisoryCommissionandWolstenholme(ed.),op.cit.,note73above,HumanFertilisationandEmbryologyAuthority,pp.362-83.However,theoriginaltaperecordingCloningissuesinreproduction,scienceandrevealsthatoneparticipantdidaskaboutthemedicine,areport,London,DepartmentofTradefeasibilityofallowingafertilizedovumtoandIndustry,December1998,p.19.395

UrsulaMittwochtheincomingwaterthreatenedtofloodtheentirehouse,hasbeentracedtoatalebytheGreekwriterLucian,88wholivedinthesecondcenturyAD.Goethe'ssorcerer'sapprenticefollowedintheancienttraditionofmagicians,whocarriedouttheirworkbyutteringsecretincantationsoninanimateobjectslikebrooms,orasintheJewishmythoftheGolem,onapieceofclay.89MaryWollstonecraftShelleycreatedthearchetypalmadscientistinhernovel,Frankenstein,firstpublishedin1818.Thebookisnamedafteritsprinciplecharacter,aSwissstudentofanatomyandchemistry,whothoughtthathehaddiscoveredthesecretoflifeandwasthusabletobestowanimationonlifelessmatter.90Hethereuponsetouttocreateamanfrommaterialsobtainedfromthedissectingroomandtheslaughterhouse.Alas,theresultofhiseffortsturnedouttobeademon,whopursuedhiscreatorandfinallykilledhim.ThestorywasfurtherpopularizedbyasuccessfulHollywoodfilmin1931,91sincewhenthenameFrankensteinhasbecomeahouseholdword,albeitonethatisoftentransferredfromthemadscientisttothemonsterthathecreated.PublicInterest:AGlanceattheMediaTheexperimentsinnucleartransferinfrogswereoriginallyundertakentoelucidatetheproblemofcellandtissuedifferentiationbyprovidinginformationonwhetherthenucleusofanalreadydifferentiatedcellcouldsubstitutefortheoriginalnucleusderivedbythefusionofeggandsperm.Atfirst,interestinthisworkwasconfinedtospecialistsinthefield,butbeforelongitenteredthedomainofawiderpublic.Alreadyin1968,oneBritishnewspaperproclaimedthat"oneoftheofthemostextraordinaryofthepossibilitiesnowbeingexploredhasalreadybeenreportedundersuchheadlinesas'EinsteinfromCuttings'and'J'aimeMozartXXXIII'.Morescientificallyitisreferredtoas'cloningpeople'-thecreationofgeneticallyidenticalindividualsfrombodycells".92Theemergenceofavarietyofreproductivetechnologieswasundoubtedlyareasonforfurtherincreasingpublicinterest,whileanxietyabouttheirethicalimplicationsgaverisetoyetmorepublicity.Artificialinsemination,whichhadlongbeenusedinanimalhusbandry,cametobeappliedasameansofassistedreproductioninhumans,whilethemuchmorecomplextechniqueofin-vitrofertilizationwasdebatedlongbeforethefirst"test-tube"babywasbornin1978.9Inhiscriticalreviewof88LudwigGeiger(ed.),Goethe'sWerke,4th91JonTurney,Frankensteinsfootsteps:science,ed.,10vols,Berlin,GGrote,1890,vol.1,geneticsandpopularculture,NewHavenandpp.146-50."TheattributionofthepoemtoLondon,YaleUniversityPress,1998,LucianisbasedonStruwe,ZweiBalladenvonpp.29-34.Goethe,verglichenmitgriechischenQuellen,92GordonRattrayTaylor,'Carboncopyworaussiegeschopftsind,Konigsberg,1826".people',Observer,10March1968,ColourSuppl.,'9JosephDan,'Golem',inRJZWerblowsky,pp.6-12,p.9.GWigoder(eds),TheOxfordDictionaryofthe93RGEdwards,RuthEFowler,'HumanJewishReligion,NewYork,OxfordUniversityembryosinthelaboratory',Sci.Am.,Dec.1970,Press,1997,p.280.223:44-54.9MaryWollstonecraftShelley,Frankenstein,or,themodernPrometheus,3vols,London,Lackington,Hughes,Harding,etc.,1818.396

"Clone"reproductivetechnologies,includingin-vitrofertilizationandcloning,LeonKassregardedartificialinseminationasthebeginningofaslipperyslopethatwouldfinishwithectogenesis,"thefulllaboratorygrowthofababyfromspermtoterm"."94Ayearearlier,awell-publicizedarticlebyJamesWatsonhadappearedentitled'Movingtowardtheclonalman.Isthiswhatwewant?'.Watsonexplainedthattherapidprogressinworkingouttheconditionsfortest-tubeconceptionswouldbeforelongenableexperiments,similartothosethatproducedclonalfrogs,tobecarriedoutwithhumancells.Thegrowinguptoadulthoodofthesefirstclonalhumanscouldbeaverystartlingevent,afactalreadyappreciatedbymanymagazineeditors,oneofwhomcommissionedacoverwithmultiplecopiesofRingoStarr,anotherofwhomgaveusoverblownmultiplelikenessesofthecurrentsexgoddess,RaquelWelch.95Watson'sarticleattractedareply,castingdoubtonthepremisethatthetechniqueoftransplantinganucleusofahumancellintoanenucleatedeggwouldresultinafairlyexactcopyofthenucleardonor,since"thereisplentyofevidenceinothercreaturesthatthehostegg'scytoplasmwouldsubstantiallyaffecttheoutcome".96However,thepossibleeffectofthecytoplasmonthedevelopingembryoshasnot,sofar,beenamatterofpublicinterest.Inthelate1970s,abookappearedthatelectrifiedthemediaandcaughtscientistsunprepared.ItstitlewasInhisimage:thecloningofaman,anditdescribedthestory,whicheventuallyendedsuccessfully,ofhowtheauthor,DavidRorvik,becamethechosenintermediarytopavethewayforawealthymanwhowishedtosecureanheirbythenewprocessof"cloning".97Theofficialprovenanceofthebookwas"Geneticengineering".Couldthestorybetrue,orwasitahoax?Notwithstandingaten-pagebibliographythatwouldhavedonecredittoatextbook,Inhisimagebecameabest-seller.Intheend,followingalawsuit,thepublishers(butnottheauthor)admittedthatthestorywasuntrue.98Thosewhothoughtin1978thatcloningahumanbeingwas"impossible",werelikelytochangetheiropinionwithinthenexttwentyyears.ThebirthofDollythesheepwasannouncedin1997,albeitwithoutmentionofthewords"clone"or"cloning";butthesetermsweredisseminatedbythemediaevenbeforepublicationday,whentheissueofNatureproclaimed"Aflockofclones"onitsfrontcover.9FollowingthebirthofDollyanditshandlingbythemedia,cloningissueshavegeneratedwidepublicinterest,wherepromisesofunprecedentedbenefitsarecounteractedinthepublicmindbyalongtraditionoffearthattamperingwiththenaturalorderofthingswillleadtodisastrousconsequences.Thereislittledoubtthatsimplifiedterminologyhashelpedpeopletobecomefamiliarwithanadvance94Kass,op.cit.note64above,pp.42-3.98Kolata,op.cit.,note82above,pp.101-2.95JamesDWatson,'Movingtowardthe9IWilmut,AESchnieke,JMcWhir,AJclonalman.Isthiswhatwewant?',AtlanticKind,KHSCampbell,'ViableoffspringderivedMonthly,May1971:50-3,p.51.fromfetalandadultmammaliancells',Nature,9LionelJaffe,'Not-so-obsolescentmothers',1997,385:810-13.AtlanticMonthly,July1971:25-6.97DavidMRorvik,Inhisimage:thecloningofaman,London,HamishHamilton,1978.397

UrsulaMittwochinscience.Whetherithashelpedthepublicunderstandingofscience,orindeed,scienceitself,isanotherquestion.What'sinaName?Inordertokeeptrackoftheprogressivechangesinmeaningofthescientifictermclone,letusconsiderthesummaryprovidedinTable1.Thedifferentusagesfallintofourcategories:(1)theoriginal,orclassical,meaningoftheterm,i.e.thecollectiveofindividualsdescendedbyasexualreproductionfromasingleorganism;(2)cellculturesderivedfromasinglecell;(3)animalsproducedbythereplacementofthenucleusofanoocytebyadiploidnucleusofasomaticcell;(4)themultiplicationofgenesandotherDNAsequences.AsshowninthesecondsectionoftheTable,theuseofthetermclonewasfirstextendedfromgardenplantstounicellularprotozoadescendedbymitosisfromacommonancestor.Then,byequatingasexuallyproducedprotozoawiththepurelinesobtainedfromself-fertilizingfloweringplants,Jenningsobscuredtheboundarybetweenvegetativeandsexualreproduction,thelatterinvolvinggermcellsproducedbymeiosis.TheapplicationofthetermclonebySanfordandcolleaguestomammaliancellculturesoriginatingfromasingletissuecell-atechniquefirstsuccessfullyperformedin1948-furtherextendeditsusetoindividualcellsofmulticellularorganisms,whichwouldnaturallydividebymitosis,whereasthedefinitionbyPuckandhisfellowworkerscouldalsoapplytooffspringproducedbyself-fertilization.TheclonesofcompletecarrotplantsproducedbyStewardandMapesinthelate1950sareclosesttotheclassicaldefinition,apartfromthefactthatthecarrotswereproducedfromsinglesomaticcellsratherthanmacroscopicpiecesofplants.Alsoduringthe1950s,thetermclonebegantobeusedfortheoffspringproducedbyreplacingthenucleiofoocyteswithnucleiofsomaticcells(Table1,section3).Whilethecellcultureterminologycouldbeviewedasanextensionoftheclassicalusage,thenuclearreplacementtechniqueaddedanewdimension,sinceitwasappliedtooffspringoriginatingfromanegg,albeitanenucleatedone.Otherchangesfollowed.Whiletheuseofmanynucleifromthesameembryofornuclearreplacementledtothedefinitionofacloneasapopulationofgeneticallyidenticalindividuals,thisinturngaverisetotheideaofidenticaltwinsbeingclones,andthat,moreover,allclonesoriginateinasexual,orevenvegetative,reproduction.Eventuallythetermwasextendedfromthecollectivetotheindividualscomposingit.AsindicatedinthelastsectionoftheTable,withtheriseofmoleculargenetics,itspractitionersadoptedthetermcloningfortheirowntechniqueofmultiplyingsequencesofDNA.Today,thisusageiswidelyemployedbymoleculargeneticists,butitisthenuclearreplacementtechniquethathasgivenrisetoDollythathascaughttheinterestofthepublic.AmbiguousNomenclatureDefiningacloneasapopulationofgeneticallyidenticalindividualsisnolongeraccurate,sinceaninvestigationofthemitochondrialDNA(mtDNA)ofDollyand398

"Clone"Table1:Changesintheusageofthetermclone,resultinginfourcategoriesofmeaningYearDefinitionAuthor1903ThecollectionofplantspropagatedvegetativelyfromacommonWebberancestor.1934CollectivenameofalltheplantsasexuallyreproducedbyCraneandLawrence"division,grafting,etc.,fromone(seedling)ancestor.1937AgroupoforganismsdescendedbymitosisfromacommonDarlington"'ancestor.1929AllindividualprotozoadescendedbyuniparentalreproductionJennings"(i.e.binaryfission)fromasingleindividual.Inaddition,theconceptofclonewasequatedwiththatofapureline,i.e.thedescendantsofasingleself-fertilizingindividual.1948MammaliancellculturesoriginatingfromasingleisolatedtissueSanford,etal.vcell.1956"AcloneisapopulationofcellswhosemembersaredescendentPuck,etal.fromthesamesingleorganism".1958Culturesoriginatingfromsinglecellsoffloweringplants.Muir,etalvl1959ThewordcloningusedforthetechniqueofgrowingculturesBergmannv..fromisolatedplantcells.1963CompletecarrotplantsderivedfromaparticularstrainbyStewardandMapes"culturingindividualsomaticcells.1956SerialtransplantationofisolatednucleifromfrogblastulaeintoKingandBriggsxenucleatedoocytesgiverisetonuclearclones.1968Apopulationofgeneticallyidenticalindividuals.Gurdonx1972CloningissynonymouswithasexualreproductionartificiallyKassx"induced.Asingleparentandoffspringsoproducedformaclone.1975AllfrogsobtainedbytransplantationfromthesameembryoareGurdonxlidenticaltwins.Theiroriginisakintovegetativereproduction.1985Agroupofgeneticallyidenticalorganisms;anindividualMcKinnellx"vproducedasexually.1983AgeneorotherpieceofDNAmultipliedinbacteria.Watson"'HerbertJWebber,'Newhorticulturalandagriculturalterms',Science,1903,18:501-3.'MBCraneandWJCLawrence,Thegeneticsofgardenplants,London,Macmillan,1934,p.213.iCDDarlington,Recentadvancesincytology,2nded.,London,Churchill,1937,p.574."vHSJennings,'Geneticsoftheprotozoa',BibLGenet.,1929,5:105-330,p.232.vKatherineKSanford,WiltonREarleandGwendolynDLikely,'Thegrowthinvitroofsingleisolatedtissuecells',J.natl.CancerInst.,1948,9:229-46.ViTheodoreTPuck,PhilipIMarcus,StevenJCieciura,'Clonalgrowthofmammaliancellsinvitro',J.exp.Med.,1956,103:273-84,p.274."WHMuir,ACHildebrandt,AJRiker,'Thepreparation,isolation,andgrowthincultureofsinglecellsfromhigherplants',Am.J.Bot.,1958,45:589-97,p.593.ViiiLudwigBergmann,'Anewtechniqueforisolatingandcloningcellsofhigherplants,Nature,1959,Aug.22:648-9."'FrederickCampionSteward,Marion0Mapes,'Thetotipotencyofculturedcarrotcells:evidenceandinterpretationsfromsuccessivecyclesofgrowthfromphloemcells',J.Indianbot.Soc.,1963,42A,MaheshwariCommemorationvol.,237-46,pp.238-42.xThomasJKing,RobertBriggs,'Serialtransplantationofembryonicnuclei',ColdSpringHarborSymp.quant.Biol.,1956,21:271-90,p.277.XiJBGurdon,'Transplantednucleiandcelldifferentiation',SciAm.,Dec.1968,219:24-35,pp.27,28.""LeonRKass,'Newbeginningsinlife',inMPHamilton(ed.),Thenewgeneticsandthefutureofman,GrandRapids,MI,WBEerdmans,1972,pp.15-63,42-5.XiiiJBGurdon,'Eggcytoplasmandgenecontrolindevelopment',TheCroonianLecture,1976,Proc.PRSoc.Lond.,B,1977,198:211-47,p.218.XivRobertGMcKinnell,Cloningoffrogs,mice,andotheranimals,rev.ed.ofCloning:abiologistreports,Minneapolis,UniversityofMinnesotaPress,1985,p.119.x"JamesDWatson,JohnToozeandDavidTKurtz,RecombinantDNA:ashortcourse,NewYork,ScientificAmericanBooks,1983,p.206.399

UrsulaMittwochnineothersheepproducedbysomaticnucleartransfershowedthatthemtDNAwasderivedfromthecytoplasmoftheenucleatedoocytes,thusmakingthesheep"geneticchimeras".'°°Furthermore,thestatementbyGurdonandColman'0'thatthemtDNAofmammalianclonesisentirelymaternalinoriginissurelysignificant.Ifacloneoriginatesfromanucleardonorplusaneggcytoplasmofmaternal,i.e."mother"origin,theideathatitisderivedfromasingleparenthasevidentlybeenabandoned,whileitsorigininasexualreproductionisquestionable.Ambiguousnomenclaturewithregardtoeggsdevelopingintoembryosbyun-orthodoxpathwaysisnotconfinedtocloning.Halfacenturyago,AlanBoydenquestionedtheappropriatenessofreferringtoparthenogenesisasasexualre-productionandemphasizedthat"thereisnosuchthingasanasexualegg".'02Thehistoryofanegg'sdevelopmentisatleastasimportantasitssubsequentfate,andamodificationofsexualreproductionisnottobeconfusedwithasexualreproduction.Similarly,andapparentlyindependently,Imyselfpointedoutin1978that,sinceparthenogenesisinvolvestheproductionofanegg,whichplaysanessentialroleinthedevelopmentoftheembryo,theprocessshouldberegardedasincompletesexualreproduction.'03Ontheotherhand,thereasongivenbyRogerHughesforregardingparthenogenesisasnot"strictlyclonal"isthattheprocesscanproceedbydifferentroutesthatdonotnecessarilypreservethematernalgenotype.'04Whereasparthenogenesisremainedanissuethathasbeenlargelyconfinedtoscientists,thetopicofclonesandcloninghasbecomeofincreasingconcerntothepublic.Whilescientistsindifferentspecialtiesstillattachdifferentmeaningstotheterms,thepublicandpopularscientificjournalsidentifythemwiththetechniquethathasgivenrisetoDolly.l05Inordertoclarifytheterminologyofcloningaspotentiallyapplicabletohumans,ithasbeensubdividedintotwoapparentlydistinctmeanings:"reproductivecloning",definedas"whereanentireanimalisproducedfromasinglecellbyasexualreproduction",and"therapeuticcloning",involvingnuclearreplacementtechnologynotleadingtothecreationofidenticalindividuals.'06Thisevadesthequestionwhethertheprocessofembryosplittingthatleadstotheformationofidenticaltwinsisincludedinthefirstcategory,eventhoughthistechniquehasbeendescribedascloning.'07Ithasalsorecentlybeenpointedoutthattheidentificationof"nuclearreplacement"withcloningleadstoconfusion,sincereplacementbytwohaploidcellsMatthewJEvans,CaganGurer,JohnD105AndreaGraves,'Clonefarm',NewSci.,18Loike,IanWilmut,AngelikaESchnieke,EricAAug.2001:4-5.Schon,'MitochondrialDNAgenotypesinnuclear1HumanGeneticsAdvisoryCommissionandtransfer-derivedclonedsheep',Nat.Genet.,1999,HumanFertilisation&EmbryologyAuthority,23:90-3.Cloningissuesinreproduction,scienceand101JBGurdon,AlanColman,'Thefutureofmedicine.Aconsultationdocument,n.p.,1998,cloning',Nature,1999,402:743-6,p.743.pp.7-8.102AlanBoyden,'Isparthenogenesissexualor'07VasiliosTanos,JosephGShenker,asexualreproduction?',Nature,1950,166:820.'Reproductivehealthcarepoliciesaroundthe103UrsulaMittwoch,'Parthenogenesis',J.medworld.Ishumancloningjustified?',J.assist.Genet.1978,15:165-81,pp.165-6.ReprodGenet.,1998,15:1-9.1RogerNHughes,Afunctionalbiologyofclonalanimals,London,NewYork,ChapmanandHall,1989,p.8.400

"Clone"couldleadtobiparentalinheritance.'08AmoreaccuratetermtodescribetheDollytechniquewouldbe:somaticnuclearreplacementofanoocytenucleus.Themultitudeofmeaningsthathaveaccumulatedovertheyearsisinstarkcontrasttotheexpressedpurposeoftheoriginatorofthetermclon,whichwastodescribeanassemblageofcultivatedplantspropagatedvegetativelyratherthanbyseed,andwhichshouldnotsuggestanyothermeaning.'"Whydidthispurposefail?CoulditbethattheadditionaldesiderataputforwardbyWebber,thatthewordshouldbeshort,euphonious,phoneticallyspelledandeasilypronounced,unfitteditasascientificterm,whosecharacteristicshavebeendescribedasconstancyofmeaning,uglinessandemotionalneutrality?"0True,thelargemajorityofscientificterms,datingfromChaucertothetwentiethcentury,arepolysyllabic"'andoftennoteasytomemorize.Ontheotherhand,thewordgold,whichisphoneticallynotdissimilartoclone,hasexistedformorethanathousandyearsandstillreferstothesamechemicalelement.Wemustconcludethatthemultiplemeaningsofclonearenotduetopopularusage,buttothedesireofscientists,unhamperedbyanyregulatorybody,toextendthetermtoavarietyofnewlydiscoveredphenomenawithonlypartiallyoverlappingcharacteristics.Intheprocess,theoriginalmeaningofclonehasbeenalmostforgotten,eventhoughitshouldstillplayanimportantroleinthescienceoffruitgrowing.Forinstance,ediblebananasarealwaysseedless,andseveralhundredclonesarethoughttoexist,ofwhichmorethathalfaretriploid."2Itistobehopedthatthetermwillnotbeabandonedinitsappropriatesettingbecauseithasbeenpre-emptedfordifferentpurposesinthebiomedicalsciences.Thehistoryofthetermcloneillustratesthedangerofplacingconceptswithonlypartiallyoverlappingcharacteristicsunderoneumbrelladesignation,particularlyincommunicationsbetweenscientistsandthegeneralpublic.Inarecentessayaboutthegenome,HoraceFreelandJudsoncommentedontheconfusionengenderedbytheuseoftheterm"gene"inplaceof"allele",andwrotethat"Publicmisinformationislargelyandinoriginthefaultofscientiststhemselves"."3Sloppynomenclatureprovidesafertilesoilforthemediaandwritersofsciencefictiontoplayonpeople'sancientmistrustofscientists.Itishardlysurprisingthatthewordclone-whosemeaninghasrunthegamutfromplantsthataremerelytransplantedpartsofthesameindividual,tosheepandhumanswithverydifferentorigins-providedsuchanopportunity.Evidently,farmorevigilanceisneededonthepartofrefereesandeditorstoensureaccurateusageofscientificterms."'JacekZKubiak,MartinHJohnson,..'Ibid.,pp.47-63,88-111.'Humaninfertility,reproductivecloningand12JWPurseglove,Tropicalcrops:nucleartransfer:aconfusionofmeanings',monocotyledons2,Harlow,Longman,1972,Bioessays,2001,23:359-64.pp.351-5."'9Webber,op.cit.,note8above."'HoraceFreelandJudson,'Talkingabout"°THSavory,Thelanguageofscience,2ndthegenome',Nature,2001,409:769.ed.,London,AndreDeutsch,1967,p.112.401

UrsulaMittwochWhenterminologyshifts,specialists,likesomanyHumptyDumpties,willbeabletodiscerntheintendedmeaningcorrectly,butthegeneralpublic,justlikeAlice,willbeconfusedwhenconfrontedbyambiguousinformation."4Popularconfusionislikelytoperpetuatethelongtraditionofmistrustinggrandclaimsmadebyscientists."'EvenreadersofNature(2001,412:848)maybepuzzledbyarecent'Newsinbrief'pieceheaded'NochargeforJapan'sclonedmice',whichbegins:"Themouseclonesthatformedthebackboneoftheinternationalefforttomakesenseofthemousegenome...aretobemadeavailabletoresearchersfreeofcharge."Whenitisfurtherstatedthat21,000cloneshavebeengivenaway,itbecomesclearthattheinformationisnotaboutmicebutpiecesofDNA.402