基因的分子生物学Transposon

TransposonAtransposon(transposableelement)isaDNAsequenceabletoinsertitself(oracopyofitself)atanewlocationinthegenome,withouthavinganysequencerelationshipwiththetargetlocus.

DNAsegmentsthatcarrythegenesrequiredforthisprocessandconsequentlymoveaboutchromosomesaretransposableelementsortransposons.ThesimplesttransposableelementsareinsertionsequencesorISelements.Transposableelementsalsocancontaingenesotherthanthoserequiredfortransposition(forexample,antibioticresistanceortoxingenes).Theseelementsoftenarecalledcompositetransposonsorelements.InsertionSequencesandTransposons.Thestructureofinsertionsequences(a),compositetransposns(b),andtargetsites(c),IRstandsforinvertedrepeat.In(c),thehighlightedfive-basetargetsiteisduplicatedduringTn3transpositiontoformflankingdirectrepeats.TheremainderofTn3liesbetweentheinvertedrepeats.Thesuddenintroductionofnewsequencesresultsfromtheabilityofvectorstocarryinformationbetweengenomes.Extrachromosomalelementsmoveinformationhorizontallybymediatingthetransferof(usuallyrathershort)lengthsofgeneticmaterial.Inbacteria,plasmidsmovebyconjugation,whilephagesspreadbyinfection.Bothplasmidsandphagesoccasionallytransferhostgenesalongwiththeirownreplicon.DirecttransferofDNAoccursbetweensomebacteriabymeansoftransformation.Ineukaryotes,someviruses,notablytheretroviruses,cantransfergeneticinformationduringaninfectivecycle.Unequalrecombinationresultsfrommispairingbythecellularsystemsforhomologousrecombination.Anothermajorcauseofvariationisprovidedbytransposableelementsortransposons.

GenerationofDirectRepeatsinHostDNAFlankingaTransposon.

(a)ThearrowsindicatewherethetwostrandofhostDNAwillbecutinastaggeredfashion,9basepairsapart.

(b)Aftercutting.

(c)Thetransposon(pink)hasbeenligatedtoonestrandofhostDNAateachend,leavingtwo9basegaps.

(d)Afterthegapsarefilledin,thereare9basepairrepeatsofhostDNA(purpleboxes)ateachendofthetransposon.

KeyTermsAninsertionsequence(IS)isasmallbacterialtransposonthatcarriesonlythegenesneededforitsowntransposition.Invertedterminalrepeatsaretheshortrelatedoridenticalsequencespresentinreverseorientationattheendsofsometransposons.Directrepeatsareidentical(orcloselyrelated)sequencespresentintwoormorecopiesinthesameorientationinthesamemoleculeofDNA;theyarenotnecessarilyadjacent.Atransposaseistheenzymeactivityinvolvedininsertionoftransposonatanewsite.KeyConceptsAninsertionsequenceisatransposonthatcodesfortheenzyme(s)neededfortranspositionflankedbyshortinvertedterminalrepeats.Thetargetsiteatwhichatransposonisinsertedisduplicatedduringtheinsertionprocesstoformtworepeatsindirectorientationattheendsofthetransposon.Thelengthofthedirectrepeatis5-9bpandischaracteristicforanyparticulartransposon.KeyTermsBacterialtransposonsthatcontainmarkersthatarenotrelatedtotheirfunction,e.g.drugresistance,arenamedasTnfollowedbyanumber.Compositetransposons(Compositeelement)haveacentralregionflankedoneachsidebyinsertionsequence,eitherorbothofwhichmayenabletheentireelementtotranspose.KeyConceptsTransposonscancarryothergenesinadditiontothosecodingfortransposition.CompositetransposonshaveacentralregionflankedbyISelementateachend.抗菌素与细菌耐药性移动因子(mobileDNAelements-trojan)传播抗性基因插入序列（InsertedSequences）转座因子（Transposons）整合子（Integrons）致病岛（PathogenicIslands）Interstraintransferofthelargepathogenicityisland(PAPI-1)ofPseudomonasaeruginosa

PNAS2006(103)19830

Modularschematicstructureofthebackboneofclass1integronscontainingblaCTX-Mgenes,onthebasisofavailableinformationfromsequencesofIn117,InV117(blaCTX-M-2)andIn60variants(blaCTX-M-9).Eachintegronincludestheconservedregions5’CSand3’CSflankingavariablenumberofgenecassettes,followedbyISCR1,blaCTX-Mgenes.

KeyTermsAdeletionistheremovalofasequenceofDNA,theregionsoneithersidebeingjoinedtogetherexceptinthecaseofaterminaldeletionattheendofachromosome.Preciseexcisiondescribestheremovalofatransposonplusoneoftheduplicatedtargetsequencesfromthechromosome.Suchaneventcanrestorefunctionatthesitewherethetransposoninserted.Impreciseexcisionoccurswhenthetransposonremovesitselffromtheoriginalinsertionsite,butleavesbehindsomeofitssequence.KeyConceptsHomologousrecombinationbetweenmultiplecopiesofatransposoncausesrearrangementofhostDNA.Homologousrecombinationbetweentherepeatsofatransposonmayleadtopreciseorimpreciseexcision.KeyTermsReplicativetranspositiondescribesthemovementofatransposonbyamechanisminwhichfirstitisreplicated,andthenonecopyistransferredtoanewsite.Atransposaseistheenzymeactivityinvolvedininsertionoftransposonatanewsite.Resolvaseistheenzymeactivityinvolvedinsite-specificrecombinationbetweentwotransposonspresentasdirectrepeatsinacointegratestructure.Nonreplicativetranspositiondescribesthemovementofatransposonthatleavesadonorsite(usuallygeneratingadouble-strandbreak)andmovestoanewsite.Conservativetranspositionreferstothemovementoflargeelements,originallyclassifiedastransposons,butnowconsideredtobeepisomes.Themechanismofmovementresemblesthatofphageexcisionandintegration.KeyConceptsAlltransposonsuseacommonmechanisminwhichstaggerednicksaremadeintargetDNA,thetransposonisjoinedtotheprotrudingends,andthegapsarefilled.TheorderofeventsandexactnatureoftheconnectionsbetweentransposonandtargetDNAdeterminewhethertranspositionisreplicativeornonreplicative.KeyConceptsTranspositionstartsbyformingastrandtransfercomplexinwhichthetransposonisconnectedtothetargetsitethroughonestrandateachend.TheMutransposaseformsthecomplexbysynapsingtheendsofMuDNA,followedbynicking,andthenastrandtransferreaction.Replicativetranspositionfollowsifthecomplexisreplicatedandnonreplicativetranspositionfollowsifitisrepaired.

KeyTermsAcointegratestructureisproducedbyfusionoftworeplicons,oneoriginallypossessingatransposon,theotherlackingit;thecointegratehascopiesofthetransposonpresentatbothjunctionsofthereplicons,orientedasdirectrepeats.Resolutionoccursbyahomologousrecombinationreactionbetweenthetwocopiesofthetransposoninacointegrate.Thereactiongeneratesthedonorandtargetreplicons,eachwithacopyofthetransposon.Resolvaseistheenzymeactivityinvolvedinsite-specificrecombinationbetweentwotransposonspresentasdirectrepeatsinacointegratestructure.KeyConceptsReplicationofastrandtransfercomplexgeneratesacointegrate,whichisafusionofthedonorandtargetreplicons.Thecointegratehastwocopiesofthetransposon,whichliebetweentheoriginalreplicons.Recombinationbetweenthetransposoncopiesregeneratestheoriginalreplicons,buttherecipienthasgainedacopyofthetransposon.Therecombinationreactioniscatalyzedbyaresolvasecodedbythetransposon.KeyConcepts

Nonreplicativetranspositionresultsifacrossoverstructureisnickedontheunbrokenpairofdonorstrands,andthetargetstrandsoneithersideofthe

transposonareligated.

Twopathwaysfornonreplicativetranspositiondifferaccordingtowhetherthefirstpairoftransposonstrandsarejoinedtothetargetbeforethesecondpairarecut(Tn5),orwhetherallfourstrandsarecutbeforejoiningtothetarget(Tn10).KeyConceptsReplicativetranspositionofTnArequiresatransposasetoformthecointegratestructureandaresolvasetoreleasethetworeplicons.TheactionoftheresolvaseresembleslambdaIntproteinandbelongstothegeneralfamilyoftopoisomerase-likesite-specificrecombinationreactions,whichpassthroughanintermediateinwhichtheproteiniscovalentlyboundtotheDNA.KeyConceptsMulticopyinhibitionreducestherateoftranspositionofanyonecopyofatransposonwhenothercopiesofthesametransposonareintroducedintothegenome.Multiplemechanismsaffecttherateoftransposition.KeyTermsAretrovirusisanRNAviruswiththeabilitytoconvertitssequenceintoDNAbyreversetranscription.Aretroposon(retrotransposon)isatransposonthatmobilizesviaanRNAform;theDNAelementistranscribedintoRNA,andthenreverse-transcribedintoDNA,whichisinsertedatanewsiteinthegenome.Thedifferencefromretrovirusesisthattheretroposondoesnothaveaninfective(viral)form.RETROVIRUSESANDRETROPOSONSTheretroviruslifecycleinvolvestransposition-likeevents

KeyTermsProvirusisaduplexsequenceofDNAintegratedintoaeukaryoticgenomethatrepresentsthesequenceoftheRNAgenomeofaretrovirus.Reversetranscriptaseisanenzymethatusesatemplateofsingle-strandedRNAtogenerateadouble-strandedDNAcopy.Anintegraseisanenzymethatisresponsibleforasite-specificrecombinationthatinsertsonemoleculeofDNAintoanother.KeyConcepts·Aretrovirushastwocopiesofitsgenomeofsingle-strandedRNA.·Anintegratedprovirusisadouble-strandedDNAsequence.·Aretrovirusgeneratesaprovirusbyreversetranscriptionoftheretroviralgenome.Twonoveldrugsshowpromiseinhard-to-treatpatients,butbreastfeedingstudiesunderscorethedifficultyofapplyingadvancesintherealworld.Nearlytwo-thirdsofinfectedmothersinBotswananowuseformula,whichisprovidedfreebythegovernment.Butthatpolicyappearstohavehadatragicdownside.Researchersstudyinganespeciallydeadlyoutbreakofdiarrhealdiseaseslastyearreportedthatinfantformula,ascomparedtobreast-feeding,increasedachild’sriskofdeathfromdiarrhealdisease50times,likelybecauseaseverefloodcontaminatedthewaterusedtomaketheformula.RETROVIRUSMEETING

Science(2007)315:1357

HopeonNewAIDSDrugs,butBreast-FeedingStrategyBackfires

HumanT-cellleukaemiavirustype1(HTLV-1)infectivityandcellulartransformation

NatureReviewsCancer2007(7)270HumanT-cellleukaemiavirustype1(HTLV-1),aretrovirusthatinfects20millionpeopleworldwide,wasthefirstretrovirustobeshowntobecausalforahumancancer,adultT-cellleukaemia(ATL).TheinfectivityofHTLV-1istightlycell-associated,andismediatedthroughavirologicalsynapse.Cell-freevirusislargelynon-infectious.HTLV-1doesnotuseviralcaptureofacellularproto-oncogeneforoncogenesis.Itsviraloncoprotein,Tax,isneededtoinitiatebutnotmaintaincellulartransformation.Taxtransformscellsthroughvariousmechanisms,includingthecreationofchromosomalinstability,theamplificationofcentrosomes,theabrogationofDNArepair,theactivationofcyclin-dependantkinasesandnuclearfactorκB(NFκΒ)andAktsignalling,andthesilencingofp53andspindle-assemblycheckpoints.ThemaintenanceofATLtransformationseemstorequirethefunctionofanovelantisenseproteinandRNA,termedHTLV-1basicleucinezipperfactor(HBZ)Aretroviralstrategythatefficientlycreateschromosomaldeletionsinmammaliancells

NatureMethods2007(3)263

Chromosomaldeletions,asagenetictoolforfunctionalgenomics,remainunderexploitedforvertebratestemcellsmostlybecausepresentlyavailablemethodsaretoolaborintensive.Toaddressthis,wedevelopedandvalidatedasetofcomplementaryretrovirusesthatcreatesawiderangeofnestedchromosomaldeletions.ViralDNAisgeneratedbyreversetranscriptionKeyTermsAplusstrandvirushasasingle-strandednucleicacidgenomewhosesequencedirectlycodesfortheproteinproducts.MinusstrandDNAisthesingle-strandedDNAsequencethatiscomplementarytotheviralRNAgenomeofaplusstrandvirus.PlusstrandDNAisthestrandoftheduplexsequencerepresentingaretrovirusthathasthesamesequenceasthatoftheRNA.TheRsegmentsarethesequencesthatarerepeatedattheendsofaretroviralRNA.TheyarecalledR-U5andU3-R.U5istherepeatedsequenceatthe5’endofaretroviralRNA.U3istherepeatedsequenceatthe3’endofaretroviralRNA.Thelongterminalrepeat(LTR)isthesequencethatisrepeatedateachendoftheintegratedretroviralgenome.CopychoiceisatypeofrecombinationusedbyRNAviruses,inwhichtheRNApolymeraseswitchesfromonetemplatetoanotherduringsynthesis.KeyConcepts·Ashortsequence(R)isrepeatedateachendoftheviralRNA,sothe5’and3’endsrespectivelyareR-U5andU3-R.·ReversetranscriptasestartssynthesiswhenatRNAprimerbindstoasite100-200basesfromthe5’end.·Whentheenzymereachestheend,the5’–terminalbasesofRNAaredegraded,exposingthe3’endoftheDNAproduct.·Theexposed3’endbasepairswiththe3’terminusofanotherRNAgenome.·Synthesiscontinues,generatingaproductinwhichthe5’and3’regionsarerepeated,givingeachendthestructureU3-R-U5.·SimilarstrandswitchingeventsoccurwhenreversetranscriptaseusestheDNAproducttogenerateacomplementarystrand.·Strandswitchingisanexampleofthecopychoicemechanismofrecombination.RetroviralgenescodesforpolyproteinsKeyConcepts·Atypicalretrovirushasthreegenes,gag,pol,env.·GagandPolproteinsaretranslatedfromafull-lengthtranscriptofthegenome.·TranslationofPolrequiresaframeshiftbytheribosome.·EnvistranslatedfromaseparatemRNAthatisgeneratedbysplicing.·Eachofthethreeproteinproductsisprocessedbyproteasestogivemultipleproteins.StructuralandBiochemicalStudiesofALIX/AIP1andItsRoleinRetrovirusBudding

Cell2007(128)841ManyenvelopedRNAvirusesuseshortpeptidemotifs,termed‘‘latedomains,’’torecruitcellularfactorsthatfacilitatebudding.ALIXperformsaseriesofimportantfunctionsintheendosomalpathway,incytoskeletaldynamics,andinenvelopedvirusbudding.However,themechanisticbasesfortheseseeminglydiverseALIXfunctionsarenotknown.OurstudieswerethereforeundertakenwiththegoalsofelucidatingthestructureofALIX,determininghowitinteractswithretroviralGagproteins,andtestingitsrequirementsforfunctioninginvirusbudding.ViralDNAintegratesintothechromosomeKeyConcepts·TheorganizationofproviralDNAinachromosomeisthesameasatransposon,withtheprovirusflankedbyshortdirectrepeatsofasequenceatthetargetsite.·LinearDNAisinserteddirectlyintothehostchromosomebytheretroviralintegraseenzyme.·TwobasepairsofDNAarelostfromeachendoftheretroviralsequenceduringtheintegrationreaction.RetrovirusesmaytransducecellularsequencesKeyTermsAtransducingviruscarriespartofthehostgenomeinplaceofpartofitsownsequence.ThebestknownexamplesareretrovirusesineukaryotesandDNAphagesinE.coli.Areplication-defectiveviruscannotperpetuateaninfectivecyclebecausesomeofthenecessarygenesareabsent(replacedbyhostDNAinatransducingvirus)ormutated.Ahelpervirusprovidesfunctionsabsentfromadefectivevirus,enablingthelattertocompletetheinfectivecycleduringamixedinfection.Transformation(Oncogenesis)ofeukaryoticcellsreferstotheirconversiontoastateofunrestrainedgrowthinculture,resemblingoridenticalwiththetumorigeniccondition.KeyConcepts·TransformingretrovirusesaregeneratedbyarecombinationeventinwhichacellularRNAsequencereplacespartoftheretroviralRNA.YeastTyelementsresembleretrovirusesKeyTermsTystandsfortransposonyeast,thefirsttransposableelementtobeidentifiedinyeast.Aretroposon(retrotransposon)isatransposonthatmobilizesviaanRNAform;theDNAelementistranscribedintoRNA,andthenreverse-transcribedintoDNA,whichisinsertedatanewsiteinthegenome.Thedifferencefromretrovirusesisthattheretroposondoesnothaveaninfective(viral)form.KeyConcepts·Tytransposonshaveasimilarorganizationtoendogenousretroviruses.·Theyareretroposons,withareversetranscriptaseactivity,thattransposeviaanRNAintermediate.ManytransposableelementsresideinD.melanogasterKeyConcepts·

copiaisaretroposonthatisabundantinD.melanogaster.RetroposonsfallintothreeclassesKeyTermsTheviralsuperfamilycomprisestransposonsthatarerelatedtoretroviruses.Theyaredefinedbysequencesthatcodeforreversetranscriptaseorintegrase.Thenonviralsuperfamilyoftransposonsoriginatedindependently