VectorsystemsforcloningdifferentsizesofDNAfragments

Cell-basedDNAcloninghasbeenusedwidelyasatoolforproducingquantitiesofpureDNAforphysicalcharacterizationandfunctionalstudiesofindividualgenes,geneclustersorotherDNAsequencesofinterest.However,thesizeofdifferentDNAsequencesofinterestcanvaryenormously(e.g.humangenesizesareknowntovarybetween0.1kband2.5Mb).Thefirstcell-basedcloningsystemstobedevelopedcouldcloneonlyrathersmallDNAfragments.Recently,however,therehavebeenrapiddevelopmentsincloningsystemsthatpermitcloningofverylargeDNAfragments.

Inordertoadaptnaturalplasmidmoleculesascloningvectors,severalmodificationsarenormallymade:

• Insertionofamultiplecloningsitepolylinker.Thisisashort(~30bp)syntheticsequencewhichcontainsuniquerestrictionsitesforavarietyofcommonrestrictionnucleases(pre-existingrestrictionsitesfortheseenzymeswillbedeletedfromtheplasmidifnecessarytoensurethepresenceofuniquecloningsites).
• Insertionofanantibioticresistancegene.Thehostcellsthatareusedmustnaturallybesensitivetotheantibioticinquestionsothatanyvectormoleculewhichtransformsahostcellcanconferantibioticresistance.Byplatingtransformedcellsonamediumcontainingtheantibiotic,onlythosecellsthathavebeentransformedbyvectormoleculessurvive.
• Insertionofaselectionsystemforscreeningforrecombinants.TypicallythisinvolvesarrangingforthemultiplecloningsitepolylinkertobeinsertedintoanexpressIBLegeneorgenefragmentwithintheplasmid(seeSection4.2.5).

TheplasmidvectorpUC19containsapolylinkerwithuniquecloningsitesformultiplerestrictionnucleasesandanampicillinresistancegenetopermitidentificationoftransformedcells(Figure4.10).Inaddition,selectionforrecombinantsisachievedbyinsertionalinactivationofacomponentoftheb-galactosidasegene,acomplementaryportionofthisgenebeingprovidedbyusingaspeciallymodifiedE.colihostcell.

ThemajordisadvantageofplasmidvectorsisthattheircapacityforacceptinglargeDNAfragmentsisseverelylimited:mostinsertsareafewkilobasesinlengthandinsertslargerthan510kbareveryrare.Additionally,standardmethodsoftransformationofbacterialcellswithplasmidvectorsarerelativelyinefficient.Toaddressthesedifficulties,attentionwasfocusedatanearlystageonthepossibilityofusingbacteriophagelambdaasacloningvector.Thewild-typelvirusparticle(virion)containsagenomeofcloseto50kboflineardouble-strandedDNApackagedwithinaproteincoatandhasevolvedahighlyefficientmechanismofinfectingE.colicells.Afterthelvirionattachestothebacterialcell,thecoatproteinisdiscardedandthelDNAisinjectedintothecell.AttheextremeterminiofthelDNAareoverhanging5endswhichare12nucleotideslongandcomplementaryinbasesequence.Becausetheselarge5overhangscanbase-pair,theyareeffectivelystickyends,similarto,butmorecohesivethan,thesmallstickyendsgeneratedbysomerestrictionnucleases(seeSection4.2.2).Suchcohesivepropertiesarerecognizedinthenamegiventothissequence-thecossequence.Onceinsidethebacterialcell,thecossequencesbase-pair,andsealingofnicksbycellularligasesresultsintheformationofadouble-strandedcircularDNA.ThereafterthelDNAcanenteroneoftwoalternativepathways(Figure4.11):

• Thelyticcycle.ThelDNAreplicates,initiallybidirectionally,andsubsequentlybyarollingcirclemodelwhichgenerateslinearmultimersoftheunitlength.Coatproteinsaresynthesizedandthelmultimersaresnippedatthecossitestogenerateunitlengthsoflgenomewhicharepackagedwithintheproteincoats.Someofthelgeneproductslysethehostcell,allowingthevirionstoescapeandinfectnewcells.
• Thelysogenicstate.ThelgenomepossessesageneattwhichhasahomologintheE.colichromosome.AppositionofthetwoattgenescanresultinrecombinationbetweenthelandE.coligenomesandsubsequentintegrationofthelDNAwithintheE.colichromosome.Inthisstate,thelDNAisdescribedasaprovirusandthehostcellasalysogenbecause,althoughthelDNAcanremainstablyintegratedforlongperiods,ithasthecapacityforexcisionfromthehostchromosomeandentryintothelyticcycle(Figure4.11).Genesrequiredforlysogenicfunctionarelocatedinacentralsegmentofthelgenome(Figure4.12).

Thedecisiontoenterthelyticcycleorthelysogenicstateiscontrolledbytworegulatorygenes,cIandcro.ThesetwogenesaremutuallyantagoNISTic:inthelyticstatethecroproteindominates,leADIngtorepressionofcI,whereasinthelysogenicstatethecIrepressordominatesandsuppressestranscriptionofotherlgenesincludingcro.Innormallygrowinghostcells,thelysogenicstateisfavoredandthelgenomereplicatesalongwiththehostchromosomalDNA.Damagetohostcellsfavorsatransitiontothelyticcycle,enablingthevirustoescapethedamagedcellandinfectnewcells.

Inordertodesignsuitablecloningvectorsbasedonl,itwasnecessarytodesignasystemwherebyforeignDNAcouldbeattachedtothelrepliconinvitroandfortheresultantrecombinantDNAtobeabletotransformE.colicellsathighefficiency.Thelatterrequirementwasachievedbydevelopinganinvitropackagingsystemwhichmimickedthewayinwhichwild-typelDNAispackagedinaproteincoat,resultinginhighinfectionefficiency(Figure4.13).

Severalmajortypesofcloningvectorthathavebeendevelopedbymodifyingphagel,orutilizingthesizeselectionimposedbycossequences,aredescribedinthefollowingsections.

OnlyDNAmoleculesfrom37to52kbinlengthcanbestablypackagedintothelparticle.Thecentralsegmentofthelgenomecontainsgenesthatarerequiredforthelysogeniccyclebutarenotessentialforlyticfunction.Asaresult,itcanberemovedandreplacedbyaforeignDNAfragment.Usingthisstrategy,itispossibletocloneforeignDNAupto23kbinlength,andsuchvectorsarenormallyusedformakinggenomicDNAlibraries.

LambdavectorsusedformakingCDNAlibrariesdonotrequirealargeinsertcapacity(mostcDNAsare<5kblong).DesignofinsertionvectorsofteninvolvesmodificationofthelgenometopermitinsertionalcloningintothecIgene.

Cosmidvectorscontaincossequencesinsertedintoasmallplasmidvector.Large(~3044kb)foreignDNAfragmentscanbeclonedusingsuchvectorsinaninvitropackagingreactionbecausethetotalsizeofthecosmidvectorisusuallyonlyabout8kb(seeFigure4.14).

Becausethehumanandothermammaliangenomesaresolarge,andbecausemanyindividualgenescanbeverylarge(seeFigure7.7),therewasaneedforthedevelopmentofnewcloningvectorsthatcouldacceptlargeDNAinserts.Anumberofsuchvectorshavebeendevelopedrecently(seeTable4.2)andhavefoundimmediateusesingeneralphysicalmappingofgenomesandinpermittingthecharacterizationandexpressionoflargegenesorgenecomplexes.Examplesofsuchvectorsarediscussedbelow.

ManyvectorswhicharepopularlyusedforDNAcloninginbacterialcellscontainhightomediumcopynumberreplicons.TheadvantageofvectorswhichcontainsuchrepliconsisthehighyieldofDNAtheyafford:eachcellinwhichavectormoleculeispropagatedwillhaveseveraltomultiplecopiesofthevectormolecule,dependingonthereplicationefficiencyofitsreplicon.However,animportantdisadvantageisthatsuchvectorsoftenshowstructuralinstABIlityofinserts,resultingindeletionorrearrangementofportionsoftheclonedDNA.SuchinstabilityisparticularlycommoninthecaseofDNAinsertsofeukaryoticoriginwhererepetitivesequencesoccurfrequentlyand,asaresult,itisdifficulttocloneandmaintainintactlargeDNAinbacterialcells.

Inordertoovercomethislimitation,attentionhasrecentlybeenfocusedonvectorsbasedonlowcopynumberreplicons,suchastheE.colifertilityplasmid,theF-factor.Thisplasmidcontainstwogenes,parAandparB,whichmaintainthecopynumberoftheFfactorat12perE.colicell.VectorsbasedontheFfactorsystemareabletoacceptlargeforeignDNAfragments(>300kb).Theresultingrecombinantscanbetransferredwithconsiderableefficiencyintobacterialcellsusingelectroporation(amethodofexposingcellstohighvoltagesinordertorelaxtheselectivepermeabilityoftheirplasmamembranes).However,becausetheresultingbacterialartificialchromosomes(BACs)containalowcopynumberreplicon,onlyverylowyieldsofrecombinantDNAcanberecoveredfromthehostcells(Shizuyaetal.,1992).

Certainbacteriophageshaverelativelylargegenomes,therebyaffordingthepotentialfordevelopingvectorsthatcanaccommodatelargeforeignDNAfragments.OnesuchisbacteriophageP1which,likephagel,packagesitsgenomeinaproteincoat,and110115kboflinearDNAispackagedintheP1proteincoat.P1cloningvectorshavethereforebeendesignedinwhichcomponentsofP1areincludedinacircularplasmid.TheP1plasmidvectorcanbecleavedtogeneratetwovectorarmstowhichupto100kbofforeignDNAcanbeligatedandpackagedintoaP1proteincoatinvitro.TherecombinantP1phagecanbeallowedtoadsorbtoasuitablehost,followingwhichtherecombinantP1DNAisinjectedintothecell,circularizesandcanbeamplified(Sternberg,1992)(seeFigure4.15).

AnimprovementonthesizerangeofinsertsacceptedbythebasicP1cloningsystemhasbeentheuseofbacteriophageT4invitropackagingsystemswithP1vectorswhichenablestherecoveryofinsertsupto122kbinsize.Morerecently,featuresoftheP1andF-factorsystemshavebeencombinedtoproduceP1-derivedartificialchromosome(PAC)cloningsystems(Iouannouetal.,1994).

ThemostpopularlyusedsystemforcloningverylargeDNAfragmentsinvolvestheconstructionofyeastartificialchromosomes(YACssee;Burkeetal.,1987;Schlessinger,1990).Cloninginyeastcellsoffers,inprinciple,someadvantagesovercloninginbacterialcells.Certaineukaryoticsequences,notablythosewithrepeatedsequenceorganizations,aredifficult,orimpossible,topropagateinbacterialcellswhichdonothavesuchtypesofDNAorganization,butwouldbeanticipatedtobetoleratedinyeastcellswhichareeukaryoticcells.However,themainadvantageofferedbyYACshasbeentheabilitytocloneverylargeDNAfragments.SuchadevelopmentproceededfromtherealizationthatthegreatbulkoftheDNAinachromosomeisnotrequiredfornormalchromosomefunction.AsdetailedinSections2.3.22.3.4,theessentialfunctionalcomponentsofchromosomesare:

Centromeres,requiredfordisjunctionofsisterchromatidsinmitosisandofhomologouschromosomesatthefirstmeioticdivision.Telomeres,requiredforcompletereplicationoflinearmoleculesandforprotectionoftheendsofthechromosomefromnucleaseattack.Autonomousreplicatingsequence(ARS)elements,requiredforautonomousreplicationofthechromosomalDNA.Theyarethoughttoactasspecificreplicationorigins.

Ineachcase,theDNAsegmentnecessaryforfunctionalactivityinvivoinyeastislimitedtoatmostafewhundredbasepairsofDNA(Figure2.8).Asaresult,itbecamepossibletoenvisageanovelcloningsystembasedontheuseofchromosomalreplicons(ARSelements)asanalternativetotheubiquitoususeofcloningvectorsbasedonextrachromosomalreplicons(thosefoundinplasmidsandbacteriophages).

TomakeaYAC,itissimplynecessarytocombinefourshortsequencesthatcanfunctioninyeastcells:twotelomeres,onecentromereandoneARSelement,togetherwithasuitablysizedforeignDNAfragmenttogivealinearDNAmoleculeinwhichthetelomeresequencesarecorrectlypositionedatthetermini(Figure4.16).Theresultingconstructcannotbetransfecteddirectlyintoyeastcells.Instead,yeastcellshavetobetreatedinsuchawayastoremovetheexternalcellwalls.Theresultingyeastspheroplastscanacceptexogenousfragmentsbutareosmoticallyunstableandneedtobeembeddedinagar.TheoveralltransformationefficiencyisverylowandtheyieldofclonedDNAislow(aboutonecopypercell).Nevertheless,thecapacitytoclonelargeexogenousDNAfragments(upto2Mb)hasmadeYACsavitaltoolinphysicalmapping(seeSection10.3).