2infectionofmurinecells(15)andtransgenicmiceexpressinghumanCD4(16)andpro-videsarationalefortransgenicapproachestodevelopinganimalmodelsofHIVdisease.REFERENCESANDNOTES1.F.Cocchietal.,Science270,1811(1995).2.Y.Feng,C.C.Broder,P.E.Kennedy,E.A.Berger,ibid.272,872(1996).3.M.Samson,0.Labbe,C.Mollereau,G.Vassart,M.Parmentier,Biochemistry35,3362(1996);C.J.Raport,J.Gosling,V.L.Schweickart,P.W.Gray,I.F.Charo,J.Biol.Chem.271,17161(1996).4.H.Choeetal.,Cell85,1135(1996);B.J.Doranzetal.,ibid.,p.1149.5.T.Dragicetal.,Nature381,667(1996);H.Dengetal.,ibid.,p.661;G.Alkhatibetal.,Science272,1955(1996).6.S.Gartneretal.,Science233,215(1986).7.R.Atchisonetal.,unpublishedobservations.8.L.Boringetal.,J.Biol.Chem.271,7551(1996).9.WeclonedcDNAsencodinghumanormurineCCR5intotheexpressionvectorpcDNA3(Invitrogen)afterengineeringtheFLAGepitopeintotheNH2-terminusasdescribed(13).ExpressionofeachconstructwasdeterminedbyFACSwithanantibodytoFLAG(anti-FLAG)(BoehringerMannheim),andrelativeexpres-sionforeach(seebelow)wascalculatedastheper-centageofcellsexpressinghumanCCR5onthecellsurfacenormalizedtotheexpressionofhCCR5(de-finedas100%),withstandarderrorsofthemean.Themeanfluorescenceintensityofthepositivecellsfromanysinglesamplenevervariedfromtheaveragebymorethan30%inasingleexperiment.Therefore,neithertherelativenumberofpositivecellsnortheabsoluteexpressionlevelswithintransfectedcellsexplainsthedifferencesincoreceptoractivity.Chi-mericreceptorswerepreparedbytheoverlappoly-merasechainreaction(PCR)method(17).hCCR5(HHHH),humanCCR5(100%relativeexpression);mCCR5(MMMM),murineCCR5(126+49%);HMMM,NH2-terminusofhumanCCR5[aminoacids(aa)1to32]fusedtomurineCCR5(aa35to354)(77+22%);MHHH,NH2-terminusofmurineCCR5(aa1to34)fusedtohumanCCR5(aa33to352)(73+17%);MHMM,extracellularloop1andapor-tionoftransmembranedomain3ofhumanCCR5(aa86to118)replacingthecorrespondingsegmentofthemurinereceptor(aa88to120)(37+22%);MMHM,extracellularloop2andadjacentportionsofhumanCCR5(aa134to210)replacingthecorre-spondingregionofthemurinereceptor(aa136to212)(81+30%);MMHH,NH2-terminalhalfofmCCR5(aa1to162)fusedtotheCOOH-terminalhalfofhCCR5(aa161to352)(80+39%).10.I.F.Charoetal.,Proc.Natl.Acad.Sci.U.S.A.91,2752(1994).11.C.Franci,L.M.Wong,J.VanDamme,P.Proost,I.F.Charo,J.Immunol.154,6511(1995).12.WeclonedcDNAsencodinghumanCCR2Borchi-merasintotheexpressionvectorpCMV4(18)afterengineeringtheFLAGepitopeintotheNH2-terminusasdescribed(13).Expressionofeachconstruct(seebelow)wasdeterminedasdescribedearlier.Chimer-icreceptorswerepreparedbytheoverlapPCRmethod(17).5555,humanCCR5(100%relativeexpression);2222,humanCCR2B(87+2%);5222,NH2-terminusofCCR5(aa1to32)fusedtoCCR2B(aa45to360)(27+5%);2555,NH2-terminusofCCR2B(aa1to44)fusedtoCCR5(aa33to352)(108+17%);2255,CCR2B(aa1to136)fusedtoCCR5(aa124to352)(119+33%).13.F.S.Monteclaroand1.F.Charo,J.Biol.Chem.271,19084(1996);F.S.Monteclaroetal.,unpublishedobservations.14.J.Goslingetal.,unpublishedobservations.15.P.J.Maddonetal.,Cell47,333(1986).16.P.Loresetal.,AIDSRes.Hum.Retroviruses8,2063(1992).17.S.N.Ho,H.D.Hunt,R.M.Horton,J.K.Pullen,L.R.Pease,Gene77,51(1989).18.S.Andersson,D.L.Davis,H.Dahlback,H.Jornvall,D.W.Russell,J.Biol.Chem.264,8222(1989).19.M.A.Goldsmith,M.T.Warmerdam,R.E.Atchison,M.D.Miller,W.C.Greene,J.Virol.69,4112(1995).20.COS-7cellsweretransfectedwith2pLgofplasmidDNAperwellinasix-wellplateasdescribed(19).DNAsamplesconsistedofappropriatecombina-tionsof0.5p.gofahumanCD4expressionplasmid[pCD4Neo(19)]orplainvector,and1.5,gofachemokinereceptor-expressingplasmidorplainvector.About30hoursafteradditionofDNA,themediumineachwellwasreplacedwith1.0mlofmediumcontainingHIV-1Ba-L(-100to170ngofp24persample;source:NIHAIDSReagentRe-pository,passagedonprimaryhumanmacro-phages).About10hourslater,anadditional1.0mlofmediumwasaddedtoeachwell.After30hours,thecellswererecoveredfromthedishasdescribed(19)andanalyzedwithaFacScan(BectonDickin-son).StainingforintracytoplasmicHIV-1p24wascarriedoutwiththeFixandPermreagents(CaltagLaboratories),withamonoclonalantibodytop24(CoulterImmunology)andgoatanti-mousefluores-ceinisothiocyanate(FITC)-conjugatedsecondaryantibody(BectonDickinson).Cellswerefurtherstainedwithphycoerythrin(PE)-conjugatedanti-CD4(BectonDickinson).Appropriatecontrolsindi-catedthattheappearanceofdouble-positivecells(FITC+PE)wasdependentoncotransfectionwithbothCD4andhumanCCR5expressionplasmidsandonthepresenceofHIV-1Ba-L.21.H.Araiand1.F.Charo,J.Biol.Chem.271,21814(1996).22.WeacknowledgetheadviceofM.Warmerdam(transfection-infectionassay),E.Weider(FACSstud-ies),andL.Boring,H.Arai,andR.Speck(scientificinterpretation).WeappreciatetheassistanceofJ.CarrollandM.Cenicerosinthepreparationofthismanuscript.SupportedinpartbyNIHgrantHL52773(I.F.C.)andbyPfizer(M.A.G.).24September1996;accepted24October1996StatisticalLearningby8-Month-OldInfantsJennyR.Saffran,RichardN.Aslin,ElissaL.NewportLearnersrelyonacombinationofexperience-independentandexperience-dependentmechanismstoextractinformationfromtheenvironment.Languageacquisitioninvolvesbothtypesofmechanisms,butmosttheoristsemphasizetherelativeimportanceofexperience-independentmechanisms.Thepresentstudyshowsthatafundamentaltaskoflanguageacquisition,segmentationofwordsfromfluentspeech,canbeaccom-plishedby8-month-oldinfantsbasedsolelyonthestatisticalrelationshipsbetweenneighboringspeechsounds.Moreover,thiswordsegmentationwasbasedonstatisticallearningfromonly2minutesofexposure,suggestingthatinfantshaveaccesstoapowerfulmechanismforthecomputationofstatisticalpropertiesofthelanguageinput.Duringearlydevelopment,thespeedandaccuracywithwhichanorganismextractsenvironmentalinformationcanbeex-tremelyimportantforitssurvival.Somespecieshaveevolvedhighlyconstrainedneuralmechanismstoensurethatenviron-mentalinformationisproperlyinterpreted,evenintheabsenceofexperiencewiththeenvironment(1).Otherspecies
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