molecular-b郑州大学分子生物学课件.ppt

,MolecularBiology,tianzengyua1.Centraldogmaanditsdevelopment,SomeRulesorSuggestions,TurnoffyourcellphonewheninclassEitheryoucomehereontimeoryouwontYoucansleepinclass,butdontsnore!DontaskmeformercywhenyougetpoorgradesinyourexamWhoeverauditingthisclasshastogetmypermission,Please.turnallcellphonesoff,turnpagersoff,Wheninclass,Tipsforsuccess,1Readbooksasmuchasyoucan.2Printouttheslides(1-2/page)andbringthemtolecture.3Takeadditionalnotesontheslidesinlecture.4Donttakenotestooseriously,Justfocusonlisteningandunderstanding.5Donthesitatetoaskquestions.ButbesurenottocontactmeoutofclasswhenyougettroublesstudyingMolecularBiologyIonlyknowonethingthatIknowNothing-Socrates(GreekPhilosopher)6Dontwaituntilthedaybeforetheexamtocram.,Contents,PartI:GenesPartII:GeneexpressionPartIII:GenomicsPartIV:Epigenetics,Whygenomics?,Genomicsrepresentsacompletechangeinthewayweareabletothinkaboutthelifesciences.Genomicsenablesrapidandefficientdiscoveryofimportantgenesrelatedtocommodityqualityandimprovement.Genomicsapproachesprovidetheabilitytolookatcomplextraitsandpathways.,Genomicscienceistheindustrializationofmolecularbiologytoaddresscomplexbiologicalquestions.Itistheintegrationofbiology,engineering,andstatisticstosolvethesequenceofacomplexgenomeandthenminethesequencedatatoobtainbiologicalinsights.AlthoughDNAsequenceiscentraltogenomics,itissimplythestartingpointforlarge-scalegenomeanalysis.,Whatisgenomics?,GenomesVmolecularbiology:reflectthemajorresearchissuesofthenewmillenniumratherthanthosetopicsthatwereinvogueduringthe1970sand1980s.Todaysmolecularbiologyisdrivenlessbyresearchintotheactivitiesofindividualgenesandmorebygenomesequencingandfunctionalanalysis.,Manyoftodaysmolecularbiologyundergraduateswillbeinvolvedingenomeresearchwhentheybegintheirgraduatecareersandallofthemwillfindtheirworkinfluencedinonewayoranotherbygenomeprojects.Itwouldofcoursebefoolishtosuggestthatgenesarenolongerimportant.,-SO,combinetheessentialelementsofthetraditionalmolecularbiologysyllabuswiththenewmaterialrelatingtogenomes.asubstantialpartofGenomesattemptstodescribetheexpressionpathwaysofindividualgenesinthecontextoftheactivityandfunctionofthegenomeasawhole.,Referencebooks,EssentialsofMolecularBiology(GeorgeM.Malacinski;DavidFreifelder)MolecularBiologyoftheGene6/E-Watsonetal.(2008)GenesIX(Lewin,B,OxfordUniversityPress)Genomes3(T.A.BROWN，2006.GarlandScience.),Phenotype,GenomesCentralDogma,第2基因组？,PartI:Genes,Chapter1GenesareDNAChapter2TheinterruptedgeneChapter3ThecontentofthegenomeChapter4Clustersandrepeats,PartI:Genes,Chapter1GenesareDNA1.1Introduction,Lifeasweknowitisspecifiedbythegenomesofthemyriadorganismswithwhichwesharetheplanet.Everyorganismpossessesagenomethatcontainsthebiologicalinformationneededtoconstructandmaintainalivingexampleofthatorganism.,Thenuclearandmitochondrialcomponentsofthehumangenome,UnitsoflengthforDNAmolecules,BecauseDNAisdouble-stranded,thelengthsofmoleculesaredescribedassomanybasepairs(bp).1kb=1000bp1Mb=1000kb=1000000bp1Gb=1000Mb=1000000kb=1000000000bpLengthsofRNAmoleculescannotbeexpressedinbpbecausemostRNAsaresingle-stranded:theirlengthsmustthereforebedescribedassomanynucleotides,中心法则的要点:,所谓遗传信息，是指核酸中的碱基序列以及蛋白质中的氨基酸序列。生物的全部遗传信息均包含于这种大分子的遗传信息中。,从DNA到RNA到蛋白质的遗传信息流是严格的单程路线。信息一旦进入蛋白质，就不可能再行输出。蛋白质是一切性状形成的工作分子。,序列假说是中心法则的核心，中心法则是序列转换的原则,中心法则体现的基本原则:,遗传信息的唯一性遗传物质的自决性信息表达的单程性序列转换的共线性,RNA研究高峰,1961DNA三联体1957美国生化学家Zemecnik发现tRNA1959rRNA分离1961MonodinE.coli,theymayfacilitaterapidresponsestoenvironmentalchangeandcouldservesimilarfunctionsinmammals.,Genomeandtranscription(tilingarraydata)(基因组和转录)Proteincodingsequence(编码蛋白序列)人(Human)2-3%ofgenome线虫(C.elegans)25%ofgenomeTranscriptionalactivity(基因组的转录水平)人(Human)60%(20-30X)ofgenome线虫(C.elegans)70%(2-3X)ofgenomeThemajorityoftranscriptsarenon-codingRNAsThemajordifferencesamongdifferentorganismsarencRNAs,Transcriptionaloutput/complexity基因组的转录情况,BiologicalDarkMatterNewfoundRNAsuggestsahiddencomplexityinsidecellsJohnTravis,Intheearly1990s,VictorAmbrosandhiscolleagueswereconductingagenehunt.Inparticular,theyweresearchingforthegenethatwasmutatedinaperplexingstrainofCaenorhabditiselegans,thesmallnematodewhosedevelopmentmanybiologistsstudy.Unlikemostgenes,theoneidentifiedbyAmbrosgroupdoesntencodeaprotein.ItspawnsasmallmoleculeofRNAachemicalrelativeofDNAthatsomehowturnsoffothergenesthatplayaroleinwormdevelopment.Severalgroups,includingoneledbyEddy,AmbrosteamandtwootherresearchgroupsreportedthatEscherichiacoli,worms,flies,andpeoplecontaindozensofpreviouslyundetectedgenesthatspawnRNAinsteadofprotein.TheRNAgenesfoundsofararejustthetipofahugeiceberg,saysRuvkun.,NoncodingRNA:fromdarkmattertowhitematter,sORFRNAImprintedRNAStressRNA双向转录微卫星RNA反转录转座子,TherapidlyincreasingnumberofmammalianncRNAsandncRNAcandidatesfrom1999to2004.,NcRNAsequence,人3号染色体上发现的非编码基因,ABSTRACTNONCODEisanintegratedknowledgedatabasededicatedtonon-codingRNAs(ncRNAs),thatistosay,RNAsthatfunctionwithoutbeingtranslatedintoproteins.AllncRNAsinNONCODEwerefilteredautomaticallyfromliteratureandGenBank,andwerelatermanuallycurated.ThedistinctivefeaturesofNONCODEareasfollows:(i)thencRNAsinNONCODEincludealmostallthetypesofncRNAs,excepttransferRNAsandribosomalRNAs.(ii)AllncRNAsequencesandtheirrelatedinformation(e.g.function,cellularrole,cellularlocation,chromosomalinformation,etc.)inNONCODEhavebeenconfirmedmanuallybyconsultingrelevantliterature:morethan80%oftheentriesarebasedonexperimentaldata.(iii)Basedonthecellularprocessandfunction,whichaivenncRNAisinvolvedin,weintroducedanovelclassificationsystem,labeledprocessfunctionclass,tointegrateexistingclassificationsystems.(iv)Inaddition,some1100ncRNAshavebeengroupedintonineotherclassesaccordingtowhethertheyarespecifictogenderortissueorassociatedwithtumorsanddiseases,etc.(v)NONCODEprovidesauser-friendlyinterface,avisualizationplatformandaconvenientsearchoption,allowingefficientrecoveryofsequence,regulatoryelementsintheflankingsequences,secondarystructure,relatedpublicationsandotherinformation.ThefirstreleaseofNONCODE(v1.0)contains5339non-redundantsequencesfrom861organisms,includingeukaryotes,eubacteria,archaebacteria,virusandviroids.Accessisfreeforallusersthroughawebinterfaceat.,ncRNA数据库,GenomicspaceforthediscoveryofnovelncRNAsinhighereukaryotes,Hard-wired,Soft-wiredorganism,ProcessesaffectedbyncRNAs,DifferentmechanismsofncRNAaction1,EukaryoticsnoRNAsdirectnucleotidemodifications(greenstar)byformingbasepairswithflankingsequences.,Directbase-pairingwithtargetRNA/DNAiscentraltothefunctionofsomencRNAs,TheE.coliOxySRNArepressestranslationbyformingbasepairswiththeShine-Dalgarnosequence(greenbox)andoccludingribosomebinding.,DifferentmechanismsofncRNAaction2,SomencRNAsmimicthestructureofothernucleicacids,BacterialRNApolymerasemayrecognizethe6SRNAasanopenpromoter,bacterialribosomesrecognizetmRNAasbothatRNAandanmRNA,DifferentmechanismsofncRNAaction3,ncRNAsalsocanfunctionasanintegralpartofalargerRNA-proteincomplex,suchasthesignalrecognitionparticle,whosestructurehasbeenpartiallydetermined,LE(消旋化酶),L-Phegeneral,D-Phespecial,HE(聚合酶)Pro：Val：Orn：Leu按1：1：1：1依次定位聚合的功能,短杆菌肽（GramicidS),其他多肽抗生素短杆酪肽（tyrocidin）伊短菌素(edeine)多粘菌素(polymyxin)大肠杆菌素（colistine）鹿铃菌素（surukacillin）环杆菌素（circulin）放线菌素（actinomycin）Amanitin.,糖基化、乙酰化、磷酸化、烃基化、intein切除、二硫键形成,修版,Post-translationprocessing,29aa信号肽N端半分子15aa连接肽C端半分子9aaC端,CanbeglucosylatedinAsnonly,Asn内切酶,N,N,C,C,MatureconA:氨基酸序列被大幅度地剪接重排完全破坏了与DNA序列的共线性关系,transcriptable,translatablegene(Z,Y,A),cisactionfactor,transcriptablebutnontranslatablegene(tDNA,rDNA),nontranscriptable,nontranslatablegene(promoter,operator),AffectstheactivityonlyofDNAsequencesonitsownmolecularofDNA,thispropertyusuallyimpliesthatthefactordoesnotcodeforprotein,通过核苷酸自身的特异二级结构控制与它紧密连锁的结构基因的表达，,一般不编码蛋白质。(无基因产物的DNA功能区),基因的类型,transactionfactor,cis-dominant,Affectstheactivityofanygenelocatedongenomebyitstranslatedproduct.,通过扩散自身表达产物（酶，调节蛋白）控制其他基因的表达可转录，可翻译调节蛋白的DNA功能区可通过互补测验体系确定其功能区域,Theabilityofasite(cis-factor)tocontroladjacentgeneirrespectiveofthepresenceinthecellofotherallelesofthesite.,cisactingfactor对与其紧密连锁基因的控制效应不受其等位基因的影响。,DNAisgeneticmaterial,Istheproteingeneticmaterial?,复制?,表面遗传信息?,RNAisgeneticmaterialalso,TobaccoMosaicVirus(TMV),Hersheylambdarphagecycle,Watsonbutthenumberofgenefamilymembershasexpandedinhumans,especiallyinproteinsinvolvedindevelopmentandimmunity.AlthoughhumansappeartohavestoppedaccumulatingrepeatedDNAover50millionyearsago,thereseemstobenosuchdeclineinrodents.Thismayaccountforsomeofthefundamentaldifferencesbetweenhominids(原始人类)androdents,althoughgeneestimatesaresimilarinthesespecies.Scientistshaveproposedmanytheoriestoexplainevolutionarycontrastsbetweenhumansandotherorganisms,includingthoseoflifespan,littersizes,inbreeding,andgeneticdrift.,Alternativesplicingresultsindifferentcombinationsofexonsbecominglinkedtogether,resultingindifferentproteinsbeingsynthesizedfromthesamepre-mRNA.,Whatdoesthedrafthumangenomesequencetellus?,VariationsandMutationsScientistshaveidentifiedabout3millionlocationswheresingle-baseDNAdifferences(SNPs)occurinhumans.Thisinformationpromisestorevolutionizetheprocessesoffindingchromosomallocationsfordisease-associatedsequencesandtracinghumanhistory.Theratioofgermline(spermoreggcell)mutationsis2:1inmalesvsfemales.Researcherspointtoseveralreasonsforthehighermutationrateinthemalegermline,includingthegreaternumberofcelldivisionsrequiredforspermformationthanforeggs.,23.2%Expressionreplicationmaintenance21.1%Signaltransduction17.5%Biochemicalfunctionsofthecell38.2%Other,Categorizationofgenes,Whatmakesahumanbeing?,Thegenecatalogcannottellus,andwillnotbeabletotellusthequestionevenwhenitiscomplete.Therearenoamazingrevelationsaboutwhatmakeshumansdifferentfromapes.Evenwhenthechimpanzeegenomehasbeencompletelysequenced,itmaystillnotbepossiblesimplyfromgenomecomparisonstodeterminewhatmakesushuman.-Nature409,814-816(15February2001)DavidBaltimore:Ourgenomeunveiled,Howsad,tooBad?&!,OnthebasisofgenenumberweareonlythreetimesmorecomplexthanafruitflyandonlytwiceascomplexasC.elegans.Moredetailedstudiesofhowthehumangenomefunctionsmayrevealkeyfeaturesthatunderliesomeofthespecialattributesofhumanbeings,butgenomicswillneverexplainwhyahumanwasabletocomposeMozarts40thsymphony,orindeedwhyitwascomposedbyMozartandnotbyanordinaryhuman.,WhyistheHumanGenomeProjectImportant?-1,First,thehumangenecatalog,containingadescriptionofthesequenceofeverygeneinthegenome,willbeimmenselyvaluable,evenifformanyyearsthefunctionsofsomeofthegenesremainunknown.-whentheyfunctionincorrectly,giverisetoageneticdisease.Thehumangenecatalogwillproviderapidaccesstothesegenes,enablingtheunderlyingbasistothesediseasestobestudied,hopefullyleadingtostrategiesfortreatmentandmanagement.,WhyistheHumanGenomeProjectImportant?-2,Whilethecatalogisbeingcompleted,attentionwillfocusmoreandmoreonthetranscriptomeandproteome,whicharethekeystounderstandinghowtheinformationcontainedinthegenomeisutilizedbythecell.,WhyistheHumanGenomeProjectImportant?-3,Thegenomeprojectswillhaveadditionalbenefitsthatatpresentcanonlybeguessedat.WethinkthatmostoftheintergenicDNAhasnofunction,butperhapsthisisbecausewedonotknowenoughaboutit.CouldtheintergenicDNAhavearole,butonethatatpresentistoosubtleforustograsp?ThefirststepinaddressingthispossibilityistoobtainacompletedescriptionoftheorganizationoftheintergenicDNAindifferentgenomes,sothatcommonfeatures,whichmightindicatearoleforsomeorallofthesesequences,canbeidentified.,WhyistheHumanGenomeProjectImportant?-4,Genomeanalysisthereforerepresentsthefrontierofmolecularbiology,territorythatwasinaccessiblejustafewyearsagoandwhichstilldemandsinnovativeapproachesandalotofsheerhardwork.Scientistshavealwaysstriventoachievethealmostimpossible,andthemotivationformanymolecularbiologistsinvolvedingenomeprojectsis,quitesimply,thechallengeoftheunknown.,IonlyknowonethingthatIknowNothing?!,-Socrates,BeyondtheHGP:WhatsNext?,HapMap,SystemsBiology,ExploringMicrobialGenomesforEnergyandtheEnvironment,Chartgeneticvariationwithinthehumangenome,Science1November2002:Vol.298.no.5595,p.947DNASEQUENCING:VENTERsNextGoal:1000HumanGenomes,J.CraigVenter,DNAsequencerJ.CraigVENTERisofferingsomethingmorepersonal.Peoplewhodonate$500,000tohisrecentlyformedJ.CraigVENTERScienceFoundationcanhavetheirgenomeanalyzedandgettheresultsonadisk.,HAPMAP,Tocreatethenextgenerationmapofthehumangenome.Goalistounderstandthe0.1%(100-99.9)differencethatmakeshumansdifferentfromeachother.Humangenomeisthoughttocontain10millionSNPs.DNAtakenfrombloodsamplesfromresearchersinNigeria,Japan,China,andUS(northern,westernEuropeancestory).Use200-400samplesinwidelydistributedgeographicregions.,HapMap,AnNIHprogramtochartgeneticvariationwithinthehumangenome,Begunin2002,theprojectisa3-yearefforttoconstructamapofthepatternsofSNPs(singlenucleotidepolymorphisms)thatoccuracrosspopulationsinAfrica,Asia,andtheUnitedStates.ConsortiumofresearchersfromsixcountriesResearchershopethatdramaticallydecreasingthenumberofindividualSNPstobescannedwillprovideashortcutforidentifyingtheDNAregionsassociatedwithcommoncomplexdiseasesMapmayalsobeusefulinunderstandinghowgeneticvariationcontributestoresponsesinenvironmentalfactors,IsallgeneticinformationcontainedinDNA?,Insteadofsayingonegene,onepolypeptide,wemaydescribetherelationshipasonepolypeptide,onegene.Thecentralparadigmofmolecularbiology:AsequenceofDNAfunctionseitherbydirectlycodingforaparticularproteinorbybeingnecessaryfortheuseofanadjacentsegmentthatactuallycodesfortheprotein,Developmentofmulticellularorganisms,restsontheuseofdifferentgenestogeneratethedifferentcellphenotypesofeachtissue.Theexpressionofgenesisdeterminedbyaregulatorynetworkthattakestheformofacascade.Whilesuchaseriesofinteractionsisalmostcertainlythemeansbywhichthedevelopmentalprogramisexecuted,wecanask:isitentirelysufficient?,Thenatureandroleofpositionalinformation,allpartsofafertilizedeggarenotequal:oneofthefeaturesresponsiblefordevelopmentofdifferenttissuepartsfromdifferentregionsoftheeggislocationofinformation(presumablyspecificmacromolecules)withinthecell.,leadsto,theexistenceofpositionalinformationintheegg:leadstothedifferentialexpressionofgenesinthecellssubsequentlyformedintheseregionswhichleadstothedevelopmentoftheadultorganismwhichleadstothedevelopmentofaneggwiththeappropriatepositionalinformation.,天問-屈原,曰：遂古之初，誰傳道之？上下未形，何由考之？冥昭瞢闇，誰能極之？馮翼惟像，何以識之？明明闇闇，惟時何為？陰陽三合，何本何化？圜則九重，孰營度之？惟茲何功，孰初作之？,天問屈原,請問：關於遠古的開頭，誰不能夠傳授？那時天地未分，能根據什麼來考究？那時是混混沌沌，誰能夠弄清？有什麼在迴旋浮動，如何可以分明？無底的黑暗生出光明，這樣為的何故？陰陽二氣，滲合而生，它們的來歷又在何處？穹窿的天蓋共有九層，是誰動手經營？這樣一個工程，何等偉大，誰是最初的工人？（引自屈原賦今譯，郭沫若譯）,?,someinformationneededfordevelopmentoftheorganismiscontainedinaformthatwecannotdirectlyattributetoasequenceofDNA(althoughtheexpressionofparticularsequencesmaybeneededtoperpetuatethepositionalinformation)?,whenwereadouttheentiresequenceofDNAcomprisingthegenomeofsomeorganismandinterpretitintermsofproteinsandregulatoryregions,couldweconstructanorganism(orevenasinglelivingcell)bycontrollingtheexpressionofpropergenes?,SYSTEMSBIOLOGY,Explorelifeattheultimatelevel:thatofthewholeorganisminsteadofindividualcomponentssuchassinglegenesorproteins.Thisnew“systemsbiology”approachcombinesDNAsequenceswithadvancedtechnologiestostudyhowPROTEINStheproductsofgenescarryoutalltheactivitiesofalivingcell.,GenomestoLife:ADOESystemsBiologyProgram,ExploringMicrobialGenomesforEnergyandtheEnvironment,Goalsidentifytheproteinmachinesthatcarryoutcriticallifefunctionscharacterizethegeneregulatorynetworksthatcontrolthesemachinescharacterizethefunctionalrepertoireofcomplexmicrobialcommunitiesintheirnaturalenvironmentsdevelopthecomputationalcapabilitiestointegrateandunderstandthesedataandbegintomodelcomplexbiologicalsystems,GTLApplicationsinEnergySecurityandGlobalClimateChange,生命体:一个复杂的动态变化的网络,网络中的节点是各种生物分子:DNA、RNA、蛋白质它们之间的关联表示为:边:无向边，如蛋白相互作用箭头:有向边，如基因调控关系-这种网络关系正是生命复杂性的源泉，各种复杂的生命现象都是由网络中各节点之间的不同相互作用、调控关系的组合和动态变化产生的。,NcRNAsequence,地图上的地道-？,生命复杂度的增加可以通过两种不同的途径,1)增加网络中的节点数目:即在网络中添加更多的蛋白质和核糖核酸从而扩大网络的规模2)通过加强网络中节点间相互作用动态调整的能力和引入新的节点间相互作用机制-对于生命的研究需要从整体出发，研究生命网络中的这些复杂的相互作用和调控关系，而不是仅仅孤立地研究一个个网络中的节点！,复杂生物网络与系统生物学,分析全基因组序列，预测编码蛋白质基因和非编码基因-迅速确定生物网络中大部分的节点(node)通过酵母双杂交技术大规模检测蛋白质之间的相互作用-找到生物网络中的无向边通过染色质免疫共沉淀微阵列技术检测转录因子在染色质上的特异结合位点-确定生物网络中的有向边通过全基因组芯片等基因芯片技术-动态地定量观测生物网络中各节点的表达水平-系统生物学:生命科学研究的工作模式:从多数据源整合出发，以网络分析为基础，通过统计学、信息学、人工智能等各种手段，对各种生命现象做出预测，并指导传统生物实验对预测做出验证。,edge,功能基因组学的主要研究思路,以前,研究基因的功能:针对单个基因来进行的“序列结构功能”一个基因的单独表达往往不能主宰一个生物学事件的发生生物的功能:通过一批基因的同时表达一批蛋白质的协同作用来实现在一个生物学事件中，存在着复杂的基因转录调控网络来控制相关基因的同时表达，还存在着各种蛋白质，甚至核糖核酸互相结合的相互作用网络Theremustbesomethingelselatentsomewhere！,-因此,改变原来的“一次一个基因”研究方式和“序列结构功能”思路；以系统生物学的观点，采用“相互作用网络功能”新思路：整合基因和蛋白质的不同方面、不同层次的信息，进行基因功能分析，已经成为当前功能基因组研究的新方向。网络聚类寻找功能模块根据网络邻居节点预测蛋白质功能研究网络模体的拓扑结构和信号传导特性,转录调控网络的调控模式倾向性分析,把转录调控网络简单地看作是一个有向图:transcriptionfactors(TF)transcriptiontargetgenes(TG）表示为图中的节点,转录调控网络中的模体示意图:Nodeedge,Networkschematicofpart(c.2600nodesand5890edges)ofthepredictedproteinproteininteractionsfromArabidopsisthaliana.InteractiondataasaCytoscape(Clineetal.,2007)filewasobtainedattheArabidopsisInformationResource(TAIR)(http:/www.A/)attheProteomicsResourcesPortal.,Hubsandbottlenecksinplantmolecularsignallingnetworks,Hubsintheredoxregulatorynetwork,(a)NetworkschematicofinteractionsinvolvingselectedproteinsandmetabolitesinredoxregulationinthechloroplastofArabidopsis2-Cysperoxiredoxin.,(b)ProteinproteininteractionsofArabidopsis2-Cysperoxiredoxin(c)Azoom-inintothesingleedgenetworkcentredc.2-CysPrxshownin(b).,Networkschematicofnearestneighbour(distanceofoneedge)proteinproteininteractionsofthecomponentsofthethreemultiproteincomplexesresponsibleforphotomorphogenesis,SchematicsofaproteininteractionnetworksgeneratedusingdataobtainedfromtheSTRINGdatabase(/)inAugust2010.,(a)Screencaptureofthenetwor