Molecular analysis of the Oryzias latipes (Medaka) transcriptome [Elektronische Ressource] / vorgelegt von Anja Berger

Sujets

• Biologie

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Poids de l'ouvrage: 7 Mo

Extrait

hMolecularonanalysisFof2010theBiologie,OryziasBerlinlatipScesF(Medakereica)PharmazietranscriptomeUnivDissertationorgelegtzurBergerErlangungwdesimakacademiscbhenhGradesChemie,desderDoktorsreienderersit?tNaturwissenscvhaftenv(Dr.Anjarer.ausnat.)heingereicedt/OderhtProf.DissertationPDam1.MPI2.f?rDisputationMolekularehGenetikH.inhBerlin,R.Abteilung24.09.2010Prof.GutacDr.ter:LehracDr.h,HimmelbauerArbGutaceitsgruppter:eDr.PDMutzelDr.amHimmelbauer,23.04.2001-30.05.2005.Con.ten.ts.1.In.tro.duction.7.1.1.Fish.W.orldlibraries-.In.tro.ducing.teleosts.as.mo.del.organisms..Size...............2.1.....t...2.1.2......7.1.1.1.Dening.a.mo.del.organism..................40...........and...........2.1.1.........ts..8.1.1.2.DanioRadior.erio.(zebrash).-.theandp.opular.mo.del37...................39................10.1.1.32.1.9T.etrao.don.tiformes.(puershes).-.compact.genomic.mo.dels............2.ds....12.1.1.4.Oryzias.latip.es.(Medak.a).-.small.shoratorywith.big.ey.es..........and...............36.hemicals14.1.1.5.Redundan.t.sh.mo.del.systems?..2.1.4.................Media...................38..........19.1.2.Analysing.the.transcriptomeEnzymes...................2.1.8...................ucleotides.................PCR....24.1.2.1.Metho.ds.of.transcriptome40analysis..........................33.Materials.metho.35.Materials............25.1.2.2.OFP.normalisation.prior.to.sequencing................35.Lab.equipmen......................27.1.2.3.EST.sequence.analysis35.Chemicals.reagen...............................2.1.3.c......................29.1.2.3.1.EST.clustering......36.Buers.Solutions...............................2.1.5................29.1.2.3.2.Sequence.annotation..................2.1.6.standards................................31.1.2.42.1.7Iden.tifying.single.n.ucleotide.p.olymorphisms..........................39.cDNA........32.1.2.5.Comparing.transcriptomes....................40.Oligon..................................322.1.9.11.3primersOb.jectiv.e..........................1..2.1.9.2medakSequencing.primertology...........................................EST.eat...3.2.3.sequences.gene40.2.1.9.3.Oligon.ucleotides.usedmofor.OFP........of...pro...53.......of...........Annotated...hes41.2.2.Metho.ds....arian...3.5.....jectDB.........2.3.6.........annotation.......49.......2.3.9.........medak.......3.1.1.b...Com.cDNA...of....41.2.2.1.Oligon.ucleotide.ngerprin.ting.approac.hb.....b.............77.........tication..........41.2.2.1.1.Generation.of.PCR85pro.ducts................2.clustering.................2.3.7......41.2.2.1.2.Arra.ying.of.PCRIdenproeducts............up.database.............3.Creating.gene............41.2.2.1.3ofOligongastrulaucleotideOFPprob.e.selection56.EST.medak.......3.2.s.tigs.............3.2.1.ten......42.2.2.1.4.Oligon.ucleotide.h.ybridisation69.represen.gene.........urther.BLAST...........3.2.4.new............43Dieren2.2.1.5.Image.analysis............3.4.splice.................searc.....................Medak....43.2.2.1.6.Normalising.and.clustering.ngerprin.ting.data3.6.1...................49.EST44.2.2.1.7.Hybridisation.of.cDNA.con.trol.clones....................49.Sequence..........44.2.2.2.cDNA.sequencing................2.3.8.tication.alternativ.splices.......................50.Setting.a.ject..............45.2.2.3.Iden.tication.of.dieren.tially51expressedResultsOFP3.1clustera.a.catalogue......................45.2.3.Computational53resourcesNormalisationand26,880methoadsclones.y.............3.1.2.bined.analysis.119,040.a.clones...........60.Annotation.EST.and.con....46.2.3.1.Publicly.a.v.ailable.resources.applied........69.Rep.con.t................................46.2.3.23.2.2Pfunctionserltedscriptsy.on...............73.F.annotation.y.searc.....................75.New.-.information..................47.2.3.3.GCG.and3.3EMBOSStialsoftexpressionw.are.pac.k.ages......................78.Iden.of.v.ts............47.2.3.4.BLAST.algorithm..........83.SNP.h.........................................3.6.aPro......49.2.3.5.Sequence.analysis........................86.Database.del.................................86...3.6.2139Database.inmaterialterface..................onic.........4.3.8.3...all.A.1.......inserts.139.oundaries...ers...Medak...A...expressed..87.4102Discussion.914.3.104.1.AnalysisOligonof.a.transcriptomeucleotides...137...........D.splice.ts.....gene.................catalogue.....101.......transcripts....91to4.2.Oligon.ucleotide.ngerprinHoting......for.........132.........in.design.........139.........expressed.t.151.-.e.156.392.4.2.1.Lab.oratory4.3.7methoumds..............4.3.8.transcriptome.............100.a.............Dieren.............ely..92.4.2.2.Computational.metho.dsCon.a.............ok.to.a.........110.sequences.analysis.oligon.................Calculated....94.4.2.3.OFP.analysis.as.a.methocDNAdvforVtranscriptome.analysis..................Cloning.to......95.4.3.Analysis.of.theDierenMedakclusteraimptranscriptomeem.elopmen.Alternativ.en.tron.F.ev.PIP.Supplemen.CD...............99.Estimating.n.b..........96.4.3.1.EST.sequencing............99.The.a.............................4.3.8.1.Medak.gene..................96.4.3.2.Sequence.qualit4.3.8.2ytially.genes.....................101.Alternativ.spliced.....................4.3.9.tribution.Medak.resources..........97.4.3.3.EST.clustering......109.Outlo.-.w.obtain.Medak.genes?...................A.ucleotide.used.OFP.132.Standard.ucleotides..................97.4.3.4.EST.annotation........A.2.oligon.................................B.libraries.pCS2.ector.B.1.ector............98.4.3.5.GO.annotation......................B.2.cDNA.in.pCS2.............................C.tially.OFP.140.Genes.ortan98for4.3.6bryNon-annotateddevESTtsE.e.ev.ts.exon-in.b.154.Alternativ.splice.en.-.plots.G.tal.on.158..usAbstractwBasedtoonnoligonereucleotidesequencengerprinprotingere,(OFP)yanalysisfunctions,andandsubsequenvtanotherESTpublishedproTheductiontoareducednon-Tredundansimilarittwhicsetboftherefore10,0165medakneurulaaromcDNA5'ESTclonessequencewdatabaseasfromestablishedse-fromclustering,three7274dierensequences.t10,016emsingletonsbrytheseonicystageswith(gastrula,elopmenneurulaedandknoorganogenesis)amounandinformationoneasadultdingtissuethe(otissuevorganogenesisary)included.asapproacahigh-qualitresourcewofAllhighwvGenBankaluetheforbfurthertoresearchigh-qualithobtainedonytheedmedakclustersaprotranscriptome.10542Inurtherasetrstsequences.roundclusters26,880eremedak8155afunctionsgastrulawithclonesshowtoereortansubinjectedESTtoshoOFPsimilaritclusteryanalysisproteinsandarepre-vsenytativmeseofMedakeaceitherhnon-coclusteroforoclonesaryleftandasandsingletonsstageswereereFcthishosenhfor11,468proyducingsESTeres.duced.InESTtotaldata7680ascDNAinclonesESTwwithereaccessionsequencedumanders6909AM137442high-qualitAM156757.y18,3775'readsywquenceserewobtained.bTheESTadvgroupanintage3268ofandOFPsingletonsliesvidingnotwithonlyuniqueinFtheclusteringnormalisationthisbuttoituniqueisHigh-qualitalsoESTpandossiblewtoannotated.getoinsighoftsequencesinwtoassigned,dierenmantialsequencesexpressionwingbyyproteinssubimpjectingtcDNAe.g.librariesdevoft.dierendatathdevwelopmennotalystagesanorothertissueswntoincludesngerprinytinglargeanalysis.tThereforealuableinhigh-qualitasequencesecondandroundustinbadditionseentonewtheagastruladata,clones,protein-cocDNAorinsertsding.fromlibrariesAM137442ZusammenfassungderMitSequenzenHilfebdereitereOligonzugewiesenukleotidvFingerprinEST-Sequenzentinganalyse3.268(OFP)8.155undtionanscoller,hlie?enderzus?tzlicESTNeurulaSequenzierungeiterewurdeuneinhohernicvhreduziert.tunktionredundanProteinenterklung.Satzzuv?nnenonSequenzinformation10.016auccDNABibliothekKloneninvononsdreiGenBankvccessionerscDiehiedenenhemSingletonsbrydarstellen.onalenDatensatzStadienwurden(Gastrula,onnNeurulawundzuOrganogenese)ProteinenundbryeinemkadultenonnGeweilebereproteink(Ovoar)den.erstellt.zuDiesercDNADatensatzdreistelltOveineOrgano-wAnalyseertvDaolledannResource5'f?rduziert.dieinwDateneiteredenArbbeitAM156757amESTMedakwurdenayseTundranskriptomdiedar.hiedeneW?hrendhersterwurdeExp10.016erimenSe-teundwurdenSequenzen26.880eineMedakwaeiGastrulahkKloneheinerz.B.OFPFClusteranalysederun-tterzogenDaten,undFRepr?senerdentanbtengro?enresultierenderwOFPcClusterundundalsSingletonsdierendewurdenhf?rMedakdiewProtenduktionhvGastrulaklonenonhESTKlonesonausgewanderen?hlt.en,Insgesamar,tundwurdengenese,7.680diecDNAeinKloneezogen.sequenziertvundwurdendarauswen11.468tstandenEST6.909proqualitativDiehowurdencderhESTwbankertigeter5'ASequenzen.NumDerersVbisorteilpubliziert.der18.377OFPSequenzenAnalyseQualit?tliegtdurcabClusteranal-erinnicClusterh7.274tgruppiert,n10.542urerscinSequenzenderDurcNormalisierungwvClusteranalyseondiesercDNAaufBibliothekSequenzenen,DiesesondernquenzendieseannotiertMethof?rdedieserbietetkauctehFdiezugeordnetM?glicerden,hkobeitvieleeinen?hnlicEineitblicwicktigeninaufwiesen,dierenzutiellemitExpressionunk-zuinerhalten,EmwonalenennwiccDNAESTBibliothekdeneneneinevunktionerscwhiedenerkEnte,testehenwiceinemk-TlungsstadienausoertvderhoGewh-qualitativebSequenzinformationekvsomiterwneue,endetowoerden.nicDeshalbt-kwurdendierende,inawgeseheneiterener-Exp6erimenecologicalChapteron1dataInatroofductionerformed1.1al.,FishonWleadingorldshes-videInevtro1995]),ducing2002,teleostsareasymonotdelMeyorganismsresearcAmongscienvbertebratesregulation.shesariouscomprisetionedtheordtailsmostarevXiphophorusersatileandgroupandassections.around2002],28,900ccupationshsospevecieseciesareonlydescribmakedgenetic[FforrofurthereseshandthatPgenes,aulyts,y2005],expwhicusinghhlids,haplatvXiphophoruseandevklebacolvved[Kinarejuststudies450(see[KumartsandedHedges,eral1998]fugumil-thereforeliontheydierenears.hesFishesspexhibittothelutionaryhighestshsp[Salzburgereciation2004].ratesextremeknoevwnclearsoonfarersicationinessenveconomicalertebrates,reasons.andonshodierenweciesthereforeessenamazingydierencesinformationinsequences,theirexpmorphologyereandtheecologicalonandspbtsehaaspviouralalreadyadaptationsAfrican[Vsearcenkoftenatesh,and2003].tiformes,T[Scoofget