8b.Nucleus+and+Chromosomes(曲志才).ppt

Chap8NucleusandChromosomes,NucleusofaEukaryoticCellNuclearEnvelopeNuclearPoreComplexChromatinNucleolusandRibosomeBiogenesisNuclearMatrix,8.1TheNucleusofaEukaryoticCell,Theinnernuclearmembrane,Theouternuclearmembrane,Surroundedbytwoconcentricmembranes,8.1.1OrganizationoftheNucleus,chromosomes,nuclearmatrix,nucleoli,nucleoplasm,aninterphaseHeLacellnucleus,showingsomeofthemajorcomponentsofthenucleus,8.1.2InternalArchitecture,contrasttothecytoplasm,thenucleus:,notindividuallyenclosedbymembranes,notvisibleusingconventionallightorelectronmicroscopytechniques,revisedpictureofnuclearstructure,Nuclearmatrix:aproteinaceousscaffold-likenetworkNucleolus:thecenterforsynthesisandprocessingofrRNAmolecules,8.2TheNuclearEnvelope,Theseparationofacellsgeneticmaterialfromthesurroundingcytoplasmmaybesinglemostimportantfeaturethatdistinguisheseukaryotesfromprokaryotes.,Structureofthenuclearenvelope,outernuclearmembraneinnernuclearmembranenuclearlaminaperinuclearspacenuclearpore,nuclearlamina,Functionofthenuclearenvelope,abarrierbetweenthenucleusandcytoplasm,asadistinctbiochemicalcompartment,solechannelsthroughthenuclearenvelope,8.3NuclearPoreComplex,Vertebrate:50100proteinsDiameter:120nm,125MDabasketlikeapparatuseightfoldsymmetry,CompositionoftheNPC,CytoplasmicringNuclearringSpokeCentralplug,NuclearenvelopesofXenopusoocytesvisualizedbyfieldemissionin-lensSEM,Cut-awaymodeloftheNPC,Three-dimensionalmodelsoftheNPC,Astructurewitheightfoldsymmetry,TheNPCconsistsofanassemblyofeightspokesarrangedaroundacentralchannel,Thespokesareconnectedtoringsatthenuclearandcytoplasmicsurfaces,ElectronmicrographofNPC,NLSsdirectnuclearproteinstothenucleus,Thenuclearproteinsareselectivetrafficacrossthenuclearenvelopefromthecytoplasmtothenucleus,TheNLSsincludehistones,DNApolymerases,RNApolymerases,transcriptionfactors,splicingfactorstransportthroughNPC,核孔运输特点被动运输主动运输信号引导双向性,MoleculartrafficthroughNPC,核孔的被动运输,SmallmoleculartrafficthroughtheNPCbypassivediffusion,SmallmoleculesandsomeproteinswithMW50kDpassfreelyacrossthenuclearenvelopeineitherdirection.,MostproteinsandRNAspassthroughtheNPCbyanactiveprocessinonlyonedirection.,核孔的双向运输,mRNA的输出,2.1核蛋白运输机制,基本概念核蛋白(nuclearprotein)核定位信号(nuclearlocalizationsignals,NLS)核输出信号(nuclearexportsignals,NES)输入蛋白(importin)输出蛋白(exportin),核定位信号,Nuclearlocalizationsignals,核蛋白的输入核质蛋白(nucleoplasmin)实验核蛋白输入机理输入蛋白Ran蛋白:小GTPaseRanGAP:RanGTP激活蛋白:细胞质中RCC1：Rannucleotide-exchangefactor1.细胞核中,核质素的核定位信号及其作用,ReceptorsfortheNLStransportproteinstothenucleus,ProteinimportthroughtheNPCcanbedividedintotwosteps,distinguishedbywhethertheyrequireenergy.,Thefirststepdoesnotrequireenergy,proteinsthatcontainNLSsbindtotheNPCbutdonotpassthroughthepore.,Thesecondstepisanenergy-dependentprocessthatrequiresGTPhydrolysis.,ProteinimportthroughNPC,RoleoftheRanproteininnuclearimport,核蛋白输入机理,核内RNA与蛋白质输出,mRNA的输出异质核糖核蛋白(heterogeneousribonucleoprotein,hnRNP)信使RNP(messengerRNP,mRNP)mRNA的输出核内蛋白质输出snRNA的输出,TransportofRNAbetweennucleusandcytoplasm,active,energy-dependentprocessribonucleoproteincomplexesratherthannakedRNAs,mRNA的输出,snRNA的输出,核内蛋白质的输出,3分子伴侣(chaperones),3.1分子伴侣的发现及种类Theterm“chaperonewasfirstusedbyRonLaskeyandhiscolleaguestodescribeaprotein(nucleoplasmin)thatisrequiredfortheassemblyofnucleosomesfromhistonesandDNA.Nucleoplasminbindstohistonesandmediatestheirassemblyintonucleosomes,butnucleoplasminitselfisnotincorporatedintothefinalnucleosomestructure.Chaperonesthusactascatalyststhatfacilitateassemblywithoutbeingpartoftheassembledcomplex.,分子伴侣的概念及其特点1991年Ellis等人提出:由不相关类的蛋白质组成的一个家系它们介导其它蛋白质的正确装配但自己不成为最后功能结构中的组分。,该概念有以下特点:凡具有“介导”功能的蛋白,都称为分子伴侣,可以是完全不同的蛋白质;作用机理尚不清楚,故用“介导”二字,伸缩性较大；分子伴侣一定不是最终结构的组成部分,但不一定是一个分离的实体;装配的涵意比较广,包括:帮助新生肽的折叠,越膜定位,亚基组装等。,Itisimportanttonotethat:Chaperonesdonotconveyadditionalinformationrequiredforthefoldingofpolypeptidesintotheircorrectthree-dimensionalconformations.Thefoldedconformationofaproteinisdeterminedsolelybyitsaminoacidsequence.Rather,chaperonescatalyzeproteinfoldingbyassistingtheself-assemblyprocess.Theyappeartofunctionbybindingtoandstabilizingunfoldedorpartiallyfoldedpolypeptidesthatareintermediatesalongthepathwayleadingtothefinalcorrectlyfoldedstate.,分子伴侣的基本功能,分子内伴侣(intromolecularchaperones)分子伴侣的分布从细菌到人,从动物到植物细胞质、线粒体、叶绿体和微体分子伴侣结构上的共同特点家族成员具有高度保守性家族成员结构上具有相似性大部分在体内为组成型表达,在刺激条件下会被进一步诱导。,Hsp60的电镜三维镜象照片,3.2functionsofchaperones,帮助蛋白质折叠和装配蛋白质的转运和定位参与细胞器和细胞核结构的发生应激反应参与信号转导,Actionofchaperonesduringtranslation,ActionsofchaperonesGroEL,GroESofE.coliinproteinfolding,SequentialactionsofHsp70andHsp60chaperones,Actionofchaperonesinsignaling,Actionofchaperonesduringproteintransport,MolecularChaperones,应激反应Molecularchaperoneswereinitiallyidentifiedasheat-shockproteins,agroupofproteinsexpressedincellsthathavebeensubjectedtoelevatedtemperaturesorotherformsofenvironmentalstress.Theheat-shockproteins(abbreviatedHsp),whicharehighlyconservedinbothprokaryoticandeukaryoticcells,arethoughttostabilizeandfacilitatetherefoldingofproteinsthathavebeenpartiallydenaturedasaresultofexposuretoelevatedtemperature.However,manymembersoftheheat-shockproteinfamilyareexpressedandhaveessentialcellularfunctionsundernormalgrowthconditions.Theseproteinsserveasmolecularchaperones,whichareneededforpolypeptidefoldingandtransportundernormalconditionsaswellasincellssubjectedtoenvironmentalstress.,8.4Chromatin其中(H3-H4)2四聚体在一起,并且在两条子代双链上随机分布;原核小体中的H2A-H2B则是以两个二聚体存在,并相互分离;随机与子代双链上原或新合成的(H3-H4)2四聚体结合组成核小体。,核小体与DNA的转录即使正在转录的基因仍然有核小体结构,表明转录并不要求整个基因都处于无核小体状态。,NucleosomescontainsDNAwrappedaroundaproteincoreofeighthistonemolecules,NucleosomecoreparticleisreleasedfromchromatinbydigestionofthelinkerDNAwithanuclease.AfterdissociationoftheisolatednucleosomeintoitsproteincoreandDNA,thelengthoftheDNAthatwaswoundaroundthecorecanbedetermined.Itslengthof146nucleotidepairsissufficienttowrapalmosttwicearoundthehistonecore.,Thehigh-resolutionstructureofanucleosomecoreparticle,Thenucleosomecoreparticle,asdeterminedbyX-raydiffractionanalysis,revealshowDNAistightlywrappedaroundadisc-shapedhistonecore,making1.65turnsinaleft-handedcoil.,K.Lugeretal.Nature1997,389:251260,Histonedepletedmetaphasechromosomes,ChromosomeshaveseverallevelsofDNApacking,Packagingofnucleosomesintothe30-nmchromatinfiberdependsonhistoneH1,whichisthoughttopullthenucleosomestogetherintoaregularrepeatingarray.EachDNAmoleculeispackagedintoamitoticchromosomethatis10,000foldshorteritsextendedlength.,Chromatinfibersmaybepackedaccordingtoazigzagmodel,zigzagmodel,Thestructureofthe30-nmchromatinfibermaybeacombinationofthesezigzagvariations.Aninterconversionbetweenthesethreevariationsmayoccurthroughanaccordion-likeexpansionandcontractionofthefiber.,Problems,HowthelonglinearDNAmoleculesarepackagedintocompactchromosomes?,DNAPacking,EukaryoticDNAispackagedintoasetofchromosomesWhycompactionofDNAintochromosomeisessential?,SimpleCalculation,Human:3109bp,23chromosomes1.02m/haploid,2.04m/cellThenucleus:10mindiameterThemitoticchromosomeis1mIfnocompaction,nucleuswouldbetoosmalltoholdallDNA!,Compactionofchromatiniscell-stagedependent,A.Interphasechromatin,B.amitoticchromosome,whichisduplicatedalready,Question:Howthiscompactionisachieved?,ChangesinnucleosomestructureallowaccesstoDNA,Eukaryoticcellscontainchromatinremodelingcomplexes,proteinmachinesthatusetheenergyofATPhydrolysistochangethestructureofnucleosomestemporarilysothatDNAbecomeslesstightlyboundtothehistonecore.TheremodeledstatemayresultfrommovementoftheH2A-H2Bdimersinthenucleosomecore;theH3H4tetramerisparticularlystableandwouldbedifficulttorearrange.,Chromatinremodelingcomplexesalternucleosomestructure,Theremodelingofnucleosomestructurehastwoimportantconsequences,First,itpermitsreadyaccesstonucleosomalDNAbyotherproteinsinthecell,particularlythoseinvolvedingeneexpression,DNAreplication,andrepair.Second,theycancatalyzechangesinthepositionsofnucleosomesalongDNA;somecaneventransferahistonecorefromoneDNAmoleculetoanother.,MetaphaseChromosomes,Metaphasechromosomesaresohighlycondensedthattheirmorphologycanbestudiedusinglightmicroscope.Stainingtechniquesyieldcharacteristicpatternsofalternatinglightanddarkchromosomebands.Genescanbelocalizedtospecificchromosomebandsbyinsituhybridization.,Humanmetaphasechromosomes,Typicalappearanceofametaphasechromosome,Scanningelectronmicrographofseveralhumanmetaphasechromosomesshowingthepairedidenticalchromatidsassociatedalongtheirlengthandjoinedtightlyatthecentromere.Theconnectionsbetweenchromatidsconsistofaproteincalledcohesinthatcontainsanumberofhighlyconservedsubunits.,Thesisterchromotidsofamitoticpaireachconsistofafiber(30nmindiameter)compactlyfoldedintothechromosome.,染色体的主要结构着丝粒(centromere)主缢痕(Primaryconstriction)次缢痕(secondaryconstriction)动粒(kinetochore)核仁组织区(nucleolarorganizingregion,NOR)随体(satellite)端粒(telomere),四种不同位置着丝粒的染色体,Centromere,Theconstrictedregionofachromosomethatisthepositionatwhichthepairofchromatidsareheldtogether.Thecentromeresservebothasthesitesofassociationofsisterchromatidsandastheattachmentsitesformicrotubulesofthemitoticspindle.,着丝粒与动粒,Centromereandkinetochore,TheFunctionsofcentromeres,RequiredforchromosomestabilitySisterchromatidpairingMitoticandmeioticspindleattachmentChromosomemovementCellcyclecheckpointcontrol,主缢痕与主缢痕,Telomeres,allowcompletereplicationoftheendsofchromosomesprotectthemfromerosionandfusionwithotherDNAfragments,ThetelomereDNAsequencesofavarietyofeukaryotes,TelomeresignalsonchromosomesafterFISHwithCy3-labelled(CCCTAAA)3probe,Telomere-mediatedchromosomeintegrityinmammaliancellslackingtelomeraseorDNArepairfactorsAdicentric(Dic)chromosome(pointedarrow)inahumanmetaphasespreadshowingtelomeresignals(red)attheterminiandtwocentromeres(green)alongthechromosomearms.,端粒的形成,人工染色体(artificialchromosome)人工构建的含有稳定染色体的天然结构序列，即ARS、CEN、TEL序列的微小染色体，可以象天然染色体一样在寄主细胞中稳定复制和遗传，称为人工染色体。,DNA结构稳定遗传的功能序列ARS(autonomousreplicatingsequence)CEN(centromericsequence)ThecentromereisaspecializedregionofthechromosomethatplaysacriticalroleinensuringthecorrectdistributionofduplicatedchromosomestodaughtercellsduringmitosisTEL(telomericsequence)Thesequencesattheendsofeukaryoticchromosomes,calledtelomeres,playcriticalrolesinchromosomereplicationandmaintenance.,5.3Giantchromosone,多线染色体(polytenechromosome)概念时相：间期存在的组织双翅目昆虫的幼虫组织内,如唾液腺、气管等。体积也相应增大产生的原因：,Polytenechromosome,灯刷染色体(lampbrushchromosome)灯刷染色体是卵母细胞进行减数第一次分裂时,停留在双线期的染色体。它是一个二价体,含4条染色单体。它由轴和侧丝组成,形似灯刷。,灯刷染色体,8.5Nucleolusandribosomebiogenesis,Thenucleolusisthemostobviousstructureseeninthenucleusofaeukaryoticcellwhenviewedinthelightmicroscope.ItisthesiteofrRNAtranscriptionandprocessing,andofribosomeassembly.,Ultrastructureofnucleolus,fibrillarcenters,FCdensefibrillarcomponent,DFCgranularcomponent,GCnucleolarassociatedchromatinnucleolarmatrix,DNA稳定遗传的三种功能位点,Electronmicrographofathinsectionofanucleolusinahumanfibroblast,showingitsthreedistinctzones,Nucleolarfusion,Functionofthenucleolusinribosomeandotherribonucleoproteinsynthesis,Thenucleolusisaribosomeproductionfactory,designedtofulfilltheneedforlarge-scaleproductionofrRNAandassemblyoftheribosomalsubunits.Inadditiontoitsimportantroleinribosomebiogenesis,thenucleolusisalsothesitewhereotherRNAsareproducedandotherRNA-proteincomplexesareassembled.,Nucleolardynamics,Thenucleolusalsoplaysanimportantroleincell-cycleregulation,senescenceandstressresponses.Itisdemonstratedthatthenucleolarproteomechangessignificantlyovertimeinresponsetochangesincellulargrowthconditionsusingaquantitativeproteomicapproachforthetemporalcharacterizationofproteinfluxthroughcellularorganelles.,ThearrangementofrRNAgenes,Thenucleolusisorganizedaroundthechromosomalregionsthatcontainthegenesforthe5.8S,18S,and28SrRNA.,RibosomalRNAgenes,TherRNAtranscriptionunit,TranscriptionoftherRNAgenes,18S,5.8S,28SrRNARNApolI,asingleunit5SrRNARNApolIII,Processingofpre-rRNA,The45Spre-rRNAtranscriptcontainsexternaltranscribedspacers(ETS)atbothendsandinternaltranscribedspacers(ITS)betweenthesequencesof18S,5.8S,and28SrRNA.Thepre-rRNAisprocessedviaaseriesofcleavages(illustratedforhumanpre-rRNA)toyieldthematurerRNAspecies.,ProcessingofrRNA,RibosomeAssembly,Ribosomalproteinsareimportedtothenucleolusfromcytoplasmandbegintoassembleonpre-rRNApriortoitscleavage.Asthepre-rRNAisprocessed,additionalribosomalproteinsandthe5SrRNAassembletoformpreribosomalparticles.Thefinalstepsofmaturationfollowtheexportofpreribosomalparticlestothecytoplasm,yieldingthe40Sand60Sribosomalsubunits.,Nonhistoneproteins,Nonhistonechromosomalproteinsincludealargenumberofwidelydiversestructural,enzymatic,andregulatoryproteins.,非组蛋白的种类与性质序列特异性DNA结合蛋白(sequence-specificDNA-bindingprotein)。其他蛋白以DNA作为底物的酶作用于组蛋白的一些酶调节基因表达的蛋白因子等非组蛋白的特性:呈酸性、带负电荷。,非组蛋白的功能除了一些酶以外，非组蛋白还具有以下功能参与染色体的构建;参与DNA复制;调控基因的表达。,TranscriptionFactorMotifs,Trans-actingfactorandcis-actingelement,反式作用因子(trans-actingfactor)Theycanaffecttheexpressionofgeneslocatedonotherchromosomeswithinthecell.顺式作用元件(cis-actingelement)Theyaffecttheexpressionofonlylin