MARIETTA BIOL 309 - BIOL 309 NOTES

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Vol.4,No.10MOLECULARANDCELLULARBIOLOGY,Oct.1984,p.1%1-19690270-7306/84/101961-09$02.00/0Copyright©1984,AmericanSocietyforMicrobiologyMolecularCloningandCharacterizationofMutantandWild-TypeHuman13-ActinGenesJOHNLEAVITT,'*PETERGUNNING,2PATRICIAPORRECA,'SUN-YUNG,2tCHING-SHWUNLIN,1ANDLARRYKEDES2ArmandHammerCancerResearchCenter,LinusPaulingInstituteofScienceandMedicine,PaloAlto,California94306,1andMEDIGENProject,DepartmentofMedicine,StanfordUniversitySchoolofMedicineandVeteransAdministrationMedicalCenter,PaloAlto,California943042Received23April1984/Accepted19June1984Therearemorethan20P-actin-specificsequencesinthehumangenome,manyofwhicharepseudogenes.Tofacilitatetheisolationofpotentiallyfunctionalf-actingenes,weusedthenewmethodofB.Seed(NucleicAcidsRes.11:2427-2446,1983)forselectinggenomicclonesbyhomologousrecombination.AderivativeofthenVXminiplasmid,wAN7pl,wasconstructedbyinsertionofthe600-base-pair3'untranslatedregionofthel-actinmRNAexpressedinhumanfibroblasts.Fiveclonescontainingi-actinsequenceswereselectedfromanamplifiedhumanfetalgenelibrarybyhomologousrecombinationbetweenlibraryphageandtheminiplasmid.OneoftheseclonescontainedacompleteP-actingenewithacodingsequenceidenticaltothatdeterminedforthemRNAofhumanfibroblasts.ADNAfragmentconsistingofmostlyinterveningsequencesfromthisgenewasthenusedtoidentify13independentrecombinantcopiesoftheanalogousgenefromtwospeciallyconstructedgenelibraries,eachcontainingoneofthetwotypesofmutantl-actingenesfoundinalineofneoplastichumanfibroblasts.Theaminoacidandnucleotidesequencesencodedbytheunmutatedgenepredictthataguanine-to-adeninetransitionisresponsiblefortheglycine-to-asparticacidmutationatcodon244andwouldalsoresultinthelossofaHaeIIsite.DetectionofthisHaeIIIpolymorphismamongthefibroblast-derivedclonesverifiedtheidentityofthei-actingeneexpressedinhumanfibroblasts.Twoactinisoforms,Iandy,arecoexpressedindividingnonmusclecellsandundifferentiatedmyoblasts(26-28,30).Thesetwonearlyidenticalpolypeptidespolymerizeintomicrofilamentswhichformhighlypolymorphicaggregates(bundlesandcables)withinthesolubleandcytoskeletalportionsofthecytoplasm(5,21).Althoughthespecificstructuralandfunctionalrolesofcytoskeletalactinshavenotbeendetermined,itseemscertainthatpolymericactinisamajordeterminantofcytoarchitecture,cytoskeletalrear-rangement,movementoforganelles,andcellmotility.Electrophoreticvariantsof1-actinhavebeenfoundinneoplasticallytransformedhumanandmousefibroblasts(2,13-15,26).Thesevariantsappeartohavearisenasaconsequenceofsomaticmutationsinthestructural,-actingene(13,26).Twohumanvariantswereinducedorselectedinsuccessionaftermutagenesisinafibroblastcellculture(13,15),andtheirexpressionwasaccompaniedbyenhancedtumorigenicpotential(13).Thefirstofthetwomutationswaspresumablyinducedbythecarcinogen4-nitroquinolin-1-oxide(11)andhasbeendefinedasanexchangeofglycineforasparticacidatresidue244inthepolypeptide(26).OurknowledgeofthenucleotidesequenceforthemRNAde-rivedfromaP-actincDNAclone(23)andtheaminoacidexchangethatoccurred(26)predictsthataguanine-to-adeninetransitionhasoccurredinthesecondbaseofthecodon,whichinturnshouldresultinthelossofaHaeIIIrestrictionsitespanningthatcodon.Amutationatadifferentsite,superimposeduponthesamemutantgene,ispredictedbytheoccurrenceofasecondchargealterationofthemutant,-actinfoundinasubclonalderivative(HUT-14T)oftheoriginalmutantcelllineHUT-14(13).*Correspondingauthor.tPresentaddress:LinusPaulingInstituteofScienceandMedi-cine,PaloAlto,CA94306.Todefinebothmutationsandassesstheirroleinthechangingphenotypesofthehumanfibroblasttransformants,weclonedtheexpressedf-actingeneofhumanfibroblastsandcopiesofmutantandwild-typegenesfromtheHUT-14andHUT-14Tcelllines.Thecloningandidentificationofthesegeneswerecomplicatedbythepresenceofalargemulti-pseudogenesubfamilyfor,-actin(22).A600-base-pair(bp)sequencewhichisthe3'untranslatedregion(3'UTR)oftheP-actinmRNA,although0-actinspecificinsequence(22),didnotdistinguishtheexpressedgenefrommanyofthepseudogenesinhybridizationandduplexmeltingexperi-ments(22).Wethereforeturnedtoamethodofcloning(25)thatinvolveshomologousrecombinaitonbetweenXphagecontaining,B-actingenomicsequencesandthe3'UTRsequenceofthe3-actincDNAcarriedbyasmall7rVX-likeplasmid(25),7rAN7(H.Huang,personalcommunication).Thismethodpreferentiallyselected,byrecombination,thecloneswiththeleastdivergentsequence,i.e.,thefunctionalgeneandcloselyrelatedpseudogenes.MATERIALSANDMETHODSGeneralmethods.GrowthandtransformationofEsche-richiacoli,colonyhybridization(8),andpurificationofplasmidDNAweredoneasdescribedpreviously(3).Prepa-rationofCharon4AandXgtWESphagerecombinantDNA,agarosegels,andhybridizationblotsandtheconditionsusedforhybridizationwereasdescribedpreviously(22).Geno-micDNApreparationfrommammaliancells,DNAdigestionwithrestrictionenzymes,andhybridizationsonnitrocellu-loseblotswithdextransulfateweredoneasdescribedbyPonteetal.(24).Thehumancellstrainsweregrownandmaintainedaspreviouslydescribed(15).ConstructionoftheKD,HUT-14,andHUT-14Thumangenelibraries.PurifiedXCharon4A(1)vectorDNA(EcoRIarms),XgtWES.XB'(16)vectorDNA(full-lengthphage19611962LEAVITTETAL.genome),andpackagingextractspreparedfromE.coliBHB2688andBHB2690werepurchasedfromAmershamCorp.,ArlingtonHeights,Ill.TwosizeclassesoffullyorpartiallyEcoRI-digestedfragmentsfromgenomicDNA,2to14or10to23kilobases(kb),werepurifiedfrom0.5%agarosegels[SeakemHGT(P)]byadsorptiontoglasspow-der(31).TheEcoRIDNAfragments2to14kblongwereligatedtoAgtWESDNAarmsthatweregeneratedbyEcoRIandSacIdigestionofXgtWES.XB'DNA(16).TheEcoRIfragments(fullorpartialdigests)10or12to23kblongwereligatedintoACharon4AEcoRIarms.TheligationreactionmixtureconsistedofoneparthumaninsertDNAandthreepartsvectorDNA,66mMTris-hydrochloride(pH7.4),5mMMgC92,1mMATP,5mMdithiothreitol,100,ugofbovineserumalbumin(fraction5),andT4ligase(agiftfromR.Simoni,StanfordUniversity).Ligationreactions(13°C,overnight)werealwaystestedforcompletionbyagarosegelanalysisofsamplestakenatthebeginningandendofthereaction.A4-,usampleoftheligationreactionproductswasmixedwiththetwopackagingextracts,andphageassemblywasallowedtooccurfor2hatroomtemperature.Thepackagingreactionmixtureswerethendilutedwith0.5mlofphagedilutionbuffer(10mMTris-hydrochloride[pH7.4],10mMMgSO4,0.01%gelatin),followedimmediatelyby10,ulofchloroform,andstoredat4°C.PackagingtitersweredeterminedbyinfectionofE.coliLE392.Constructionofthe'nAN7,1miniplasmid.A600-bpEcoRI-BamHIfragmentofthecDNA(13-actin3'UTRsequence)insertinpHF,BA-3'UT(22)waspurifiedbygelelectrophoresisandadsorptiontoglasspowder(31)andthenligatedtotheEcoRI-BamHIlargefr


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