专业基础课-《分子生物学》课程教学大纲（英文班）

《MolecularBiology》课程教学大纲适用对象：本科药学专业全英班（学分：3学时：54）一、课程的性质和任务：Molecularbiologyisdefinedverybroadlyastheattempttounderstandbiologicalphenomenainmolecularterms.Butthisdefinitionmakesmolecularbiologydifficulttodistinguishfromanotherwell-knowndiscipline,biochemistry.Anotherdefinitionismorerestrictiveandthereforemoreuseful:thestudyofgenestructureandfunctionatthemolecularlevel.Thisattempttoexplaingenesandtheiractivitiesinmoleculartermsisthesubjectmatterofthiscourse.Molecularbiologygrewoutofthedisciplinesofgeneticsandbiochemistry.Ofthediversewaystostudythelivingworld,molecularbiologyhasbeenmostremarkableinthespeedandbreadthofitsexpansion.Newdataareacquireddaily,andnewinsightsintowell-studiedprocessescomeonascalemeasuredinweeksormonthsratherthanyears.It'sdifficulttobelievethatthefirstcompleteorganismallgenomesequencewasobtainedlessthan20yearsago.Thestructureandfunctionofgenesandgenomesandtheirassociatedcellularprocessesaresometimeselegantlyanddeceptivelysimplebutfrequentlyamazinglycomplex.Thiscourseisaimedatundergraduatestudentsinmoleculargeneticsandmolecularbiologytogetclosertotherealitiesanddiversitiesofnaturalgeneticsystems.Thiscourseisorganizedintofourparts,includingGeneandGenomeStructure,DNAReplication,TranscriptionandPosttranscriptionalMechanisms,andRegulationofGeneExpression.二、教学内容和要求（含每章教学目的、基本教学内容和教学要求）：IntroductionofMolecularBiology【教学基本要求】掌握分子生物学的概念和研究范畴，熟悉教材的布局和内容。【教学具体内容】1.IntroductionofMolecularBiologyWhatismolecularbiology?Genetics？Biochemistry？Somedefineitverybroadlyastheattempttounderstandbiologicalphenomenainmolecularterms.Anotherdefinitionismorerestrictiveandthereforemoreuseful:thestudyofgenestructureandfunctionatthemolecularlevel.Thisattempttoexplaingenesandtheiractivitiesinmoleculartermsisthesubjectmatteroftheserecommendedbooks.2.ThecoursemaincontentsDNAReplicationTranscriptionTranslationRegulationThemethodsHumanhealth3.Introductionoftextbook-GeneVIII,X,XI4.GenesXContentsPart1GenesandChromosomes

Part2DNAReplicationandRecombination

Part3TranscriptionandPosttranscriptionalMechanisms

Part4GeneRegulationChapter1GenesAreDNA【教学基本要求】掌握基因的本质是核酸，并了解这个现象的发现过程相关的科研故事及其逻辑，掌握核酸的组成和特点。【教学具体内容】1.1IntroductionChromosome–Adiscreteunitofthegenomecarryingmanygenes.structuralgene–AgenethatcodesforanyRNAorpolypeptideproductotherthanaregulator.1.2DNAIstheGeneticMaterialofBacteriaandVirusesBacterialtransformation转化transformingprinciplePhageinfection(噬菌体侵染).DNAIstheGeneticMaterialofEukaryoticCellsInsomeviruses,thegeneticmaterialisRNA.1.4PolynucleotideChainsHaveNitrogenousBasesLinkedtoaSugar–PhosphateBackboneAnucleoside(核苷)consistsofapurineorpyrimidinebaselinkedtothe1carbonofapentosesugar(戊糖).1.5SupercoilingAffectstheStructureofDNASupercoiling(超螺旋)occursonlyin“closed”DNAwithnofreeends.1.6DNAIsaDoubleHelixTheB-formofDNAisadoublehelixconsistingoftwopolynucleotidechainsthatrunplementaryThediameterofthedoublehelixis20Å,andthereisacompleteturnevery34ÅThedoublehelixhasamajor(wide)grooveandaminor(narrow)groove.1.7DNAReplicationIsSemiconservativesemiconservativereplication1.8PolymerasesActonSeparatedDNAStrandsattheReplicationFork(复制叉)ReplicationofDNAisundertakenbyacomplexofenzymesthatseparatetheparentalstrandsandsynthesizethedaughterstrands.denaturationrenaturation1.9GeneticInformationCanBeProvidedbyDNAorRNACellulargenesareDNA,butvirusesmayhavegenomesofRNA.DNAisconvertedintoRNAbytranscription,andRNAmaybeconvertedintoDNAbyreversetranscription.RNApolymerase.centraldogma(中心法则)1.10NucleicAcidsHybridizebyBasePairingThemeltingtemperature(Tm)isthemidpointofthetemperaturerangefordenaturation.Denaturationandrenaturation/hybridization杂交1.11MutationsChangetheSequenceofDNA1.12MutationsMayAffectSingleBasePairsorLongerSequencesAtransition(转换),Atransversion(颠换)1.13TheEffectsofMutationsCanBeReversedForwardmutationsalterthefunctionofagene,andbackmutations(orrevertants)reversetheireffects.回复突变1.14MutationsAreConcentratedatHotspots突变热点1.15ManyHotspotsResultfromModifiedBases1.16SomeHereditaryAgentsAreExtremelySmallprion(朊病毒)Chapter2Chromosomeandgenome【教学基本要求】掌握染色体的结构及其组成，了解C-值和C-值矛盾，熟悉原核生物和真核生物基因组的特点。【教学具体内容】Part1.Thechromosomeofvirus，prokaryoteandeukaryoteIntroductionPart1Thechromosomeofvirus，prokaryoteandeukaryotenucleoid类核,chromatin,packingratio1.1ViralGenomesArePackagedintoTheirCoatscapsidNucleicacidwithintheheadshellisextremelycondensed.FilamentousRNAvirusescondensetheRNAgenomenucleationcenter成核中心1.2TheBacterialGenomeIsaNucleoidThebacterialnucleoid类核.TheBacterialGenomeIsSupercoiledThenucleoidhas~400independentnegativelysupercoileddomains.1.4EukaryoticDNAHasLoopsandDomainsAttachedtoaScaffold1.5ChromatinIsDividedintoEuchromatinandHeterochromatin常/异染色质TheEukaryoticChromosomeIsaSegregationDevice1.7Telomeres端粒Thetelomereisrequiredforthestabilityofthechromosomeend.TelomeresAreEssentialforSurvival:Part2.Chromatinstructure：染色质结构DNAIsOrganizedinArraysofNucleosomesThelengthofDNApernucleosome:154to260bp.TheNucleosomeIstheSubunitofAllChromatinAnucleosomecontains~200bpofDNAandtwocopiesofeachcorehistone(H2A,H2B,H3,andH4).NucleosomesAreCovalentlyModifiedHistonesaremodifiedbymethylation,acetylation,phosphorylation,andothermodifications.HistoneVariantsProduceAlternativeNucleosomesThePathofNucleosomesintheChromatinFiber10nmchromatinfibers,30nmfibershavesixnucleosomes/turn,whichareorganizedintoatwo-starthelix.Part3.Genome基因组Genomesize：C-value，C-valueparadox.genomefeaturesofprokaryoteandeukaryote1.GenomesizesandGeneNumbersThenumberofgenesinbacterialandarchaealgenomesisproportionaltogenomesize2.TotalGeneNumberIsKnownforSeveralEukaryotes3.TheGenomesizeandthegeneticcomplexityC-value–ThetotalamountofDNAinthegenome(perhaploidsetofchromosomes)C-valueparadox–ThelackofrelationshipbetweentheDNAcontent(C-value)ofanorganismanditscodingpotential.4.ThegenomefeatureofProkaryotes5.ThegenomefeatureofEukaryotes6.AnInterruptedGeneConsistsofExonsandIntronsinterruptedgene；RNAsplicing；intron7.PseudogenesAreNonfunctionalGeneCopies假基因：失去功能的基因拷贝8.EukaryoticGenomesContainBothNonrepetitiveandRepetitiveDNASequences9.SomeOrganellesHaveDNA10.原核生物与真核生物基因组结构特点的比较Chapter3DNAReplication【教学基本要求】掌握DNA复制相关概念，了解复制的几种类型，掌握复制相关蛋白，熟悉DNA复制的特点，及其复制起始延伸终止的步骤。【教学具体内容】Part0Introduction:Thesemi-conservativereplication半保留复制：Replicon复制子，Origin复制起点，Terminus复制终止位点Part1.Thereplicon复制子1.1ThetypesofrepliconRepliconsCanBeLinearorCircularThestructureofreplicatedDNA：replicationbubble，replicationforkRepliconsCanBeunidirectionalorbidirectionalOriginsCanBeMappedbyAutoradiographyandElectrophoresis1.2ThereplicationtypesofcircularrepliconTheBacterialGenomeIs(Usually)aSingleCircularRepliconRollingCirclesProduceMultimersofaRepliconDLoopsMaintainMitochondrialOriginsThenumbers/copiesofrepliconThecopyofrepliconsinbacteriumEachEukaryoticChromosomeContainsManyRepliconsPart2.TheDNAreplicationprocess：initiation,prolongation,andterminationDNA复制的阶段：起始，延伸和终止TheproteinsforDNAreplicationDNA复制所需蛋白Topoisomerase拓扑异构酶，Primase引发酶，helicase解链酶Asingle-strandedbindingprotein(SSB单链结合蛋白)DNAPolymerases(DNA聚合酶)aretheenzymesthatsynthesizeDNA.semiconservativereplication2.2Theinitiation,elongationandterminationDNA复制的几个阶段2.2.1Initiation:CreatingtheReplicationForksattheOriginoriCPrimingIsRequiredtoStartDNASynthesisTheelongationTheTwoNewDNAStrandsHaveDifferentModesofSynthesisTheleadingstrand前导链，thelaggingstrand滞后链bymakingshortfragments(Okasakifragments冈崎片段)thataresubsequentlyjoinedtogether.CoordinatingSynthesisoftheLaggingandLeadingStrandsDNAPolymeraseHoloenzymeConsistsofSubcomplexesOkazakiFragmentsAreLinkedbyLigase2.2.3TheterminationTheTerminationoftheReplicationoftheCircularRepliconinBacteriaTheEndsofLinearDNAAreaProblemforReplicationTerminalProteinsEnableInitiationattheEndsofViralDNAsTelomeresAreSynthesizedbyaRibonucleoproteinEnzymeLesionBypassRequiresPolymeraseReplacementPart3.TheDNAreplicationandcellcycleDNA复制与细胞周期之间的关系3.1BacterialReplicationandCellCycle3.2TheReplicationofEukaryoticChromosomeisconnectedtotheCellCycleChapter4DNARepairsystem（ChapterATransposableElementsandRetroviruses）【教学基本要求】掌握DNA复制修复的几种类型，了解复制修复的分子机制，熟悉复制相关的关键蛋白例子。掌握转座和转座子的概念，并了解其衍生类型和功能。【教学具体内容】1.Introductionmismatchrepair(错配修复)Photoreactivation光复活作用excisionrepair切除修复baseexcisionrepair(BER)nucleotideexcisionrepair(NER)2.RepairSystemsCorrectDamagetoDNARepairsystems修复系统,Excisionsystems切除系统Recombination-repairsystems3.ExcisionRepairSystemsinE.coli4.ControllingtheDirectionofMismatchRepair5.Recombination-RepairSystemsinE.coli6.RecombinationIsanImportantMechanismtoRecoverfromReplicationErrors7.Recombination-RepairofDouble-StrandBreaksinEukaryotes8.NonhomologousEnd-JoiningAlsoRepairsDouble-StrandBreaksThenonhomologousend-joining(NHEJ)pathwaycanligatebluntends(平末端/钝端)ofduplexDNA.9.DNARepairinEukaryotesOccursintheContextofChromatinBothhistonemodification(组蛋白修饰)andchromatinremodeling(染色质重塑)Remodelers(重塑者)andchaperones(分子伴侣)10.ThecellresponsetoDNAdamage11.RecATriggerstheSOSSystemChapterATransposableElementsandRetroviruses1.Introductiontransposon转座子(transposableelement)Retrovirus逆转录病毒retrotransposon(retroposon)2.InsertionSequencesAreSimpleTranspositionModulesAninsertionsequence(IS插入序列)isatransposonthatcodesfortheenzyme(s)neededfortranspositionflankedbyshortinvertedterminalrepeats.ThetargetsiteTransposase转座酶3.TranspositionOccursbyBothReplicativeandNonreplicativeMechanismsResolvase解离酶compositetransposons复合转座子4.TransposonsCauseRearrangementofDNA5.ReplicativeTranspositionProceedsthroughaCointegrate6.NonreplicativeTranspositionProceedsbyBreakageandReunionChapter5AProkaryoticTranscriptionChapter5BEukaryoticTranscription【教学基本要求】掌握转录过程中涉及的各个基本元件。了解转录的过程，掌握起始和终止的协助因子。掌握关键的终止类型。【教学具体内容】Chapter5AProkaryoticTranscriptionPart0Introductioncoding(nontemplate)strand编码链,RNApolymerase,promoter启动子,Startpoint起始位点,Terminator终止子,transcriptionunit转录单元Part1.Thethreestagesoftranscriptionreaction转录过程的三个阶段概述1.1TranscriptionOccursbyBasePairingina“Bubble”ofUnpairedDNA1.2TheTranscriptionReactionHasThreeStagesRNApolymeraseinitiatestranscription(initiation起始)Duringelongation延伸.Transcriptionstops(termination终止)Part2.TheRNApolymeraseRNA聚合酶及与转录起始阶段2.1BacterialRNAPolymeraseConsistsofMultipleSubunitsholoenzyme:coreenzymeandσfactor.2.2RNAPolymeraseHoloenzymeConsistsoftheCoreEnzymeandSigmaFactor2.3TheHoloenzymeGoesthroughTransitionsintheProcessofRecognizingandEscapingfromPromotersternarycomplex三元复合物Theremaybeacycleofabortiveinitiations流产式起始CompetitionforSigmaFactorsCanRegulateInitiationPart3.Thepromoterelements启动子及与转录起始识别/启动子解脱3.1SigmaFactorControlsBindingtoDNAbyRecognizingSpecificSequencesinPromoterspromoter启动子conservedsequence保守序列consensussequences一致性序列(–10elementorTATAbox)(–35element).3.2PromoterEfficienciesCanBeIncreasedorDecreasedbyMutationDownmutations,upmutations3.3InteractionsbetweenSigmaFactorandCoreRNAPolymeraseChangeDuringPromoterEscapePart4.Theterminationandantitermination 转录终止与抗终止及其转录本命运4.1BacterialRNAPolymeraseTerminatesatDiscreteSites“intrinsicterminators内部终止子.”:“rho-dependentterminators.”4.2HowDoesRhoFactorWork?4.3AntiterminationCanBeaRegulatoryEventNantiterminationfactor抗终止N蛋白.4.4TheCycleofBacterialMessengerRNATranscriptionandtranslationoccursimultaneouslyinbacteria(coupledtranscription/translation转录翻译偶联).Chapter5BEukaryoticTranscription1.Introductionbasaltranscriptionfactors基础转录因子TATA-bindingprotein(TBP)corepromoterenhancersilencer2.EukaryoticRNAPolymerasesConsistofManySubunitsRNApolymeraseIsynthesizesrRNAinthenucleolus.RNApolymeraseIIsynthesizesmRNAinthenucleoplasm.heterogeneousnuclearRNA(hnRNA)RNApolymeraseIIIsynthesizestRNAandsmallRNAsinthenucleoplasm.3.TheStartpointforRNAPolymeraseIITheTATAbox,located~25bpupstreamofthestartpoint.4.TBPIsaUniversalFactorTBP(TATAboxbindingprotein)5.TheBasalApparatusAssemblesatthePromoter6.InitiationIsFollowedbyPromoterClearanceandElongation7.EnhancersContainBidirectionalElementsThatAssistInitiation8.GeneExpressionIsAssociatedwithDemethylation9.CpGIslandsAreRegulatoryTargetsChapter6RNASplicingandProcessing【教学基本要求】掌握RNA的基本加工过程，了解RNA剪接类型及其经典类型的剪接机制，掌握RNA剪接的结果，了解RNA编辑的定义及其意义。【教学具体内容】Part1.The5′EndofEukaryoticmRNAIsCapped -真核生物mRNA的5‘加帽修饰The5′capofmostmRNAismonomethylated单甲基化influencemRNAstability,splicing,export,andtranslationPart2.ThePolyadenylationofthe3′EndsofmRNAsmRNA的3‘端加poly-A尾anendonuclease,andpoly(A)polymerase.Thepoly(A)tailcontrolsmRNAstabilityandinfluencestranslation.Part3.TheSplicingofEukaryoticmRNA真核生物mRNA的剪接（内含子去除）GroupGU-AG/AU-ACIntrons,GroupIIntronsGroupIIIntronsalternativesplicing3.1RNAsplicing/RNA的剪接3.2snRNAs/snRNPsAreRequiredforSplicing3.3ThesitesofRNAsplicing3.4NuclearSpliceJunctionsAreShortSequences3.5Pre-mRNASplicing——theGU-AGintronPre-mRNASplicingProceedsthroughaLariat3.6GroupIIntronsUndertakeSelf-SplicingbyTransesterification3.7GroupIIntronsFormaCharacteristicSecondaryStructure3.8GroupIIAutocatalyticIntronsLikelySharestheMechanismwithPre-mRNASplicing3.9SplicingUsesTransesterification转酯反应3.10AlternativeSplicingIsaRule,RatherThananException,inMulticellularEukaryotesPart4.TheRNAeditingRNA的编辑4.1RNAEditingOccursatIndividualBasesRNAediting–AchangeofsequenceatthelevelofRNAfollowingtranscription.4.2RNAEditingCanBeDirectedbyGuideRNAsPart5.TheSplicingofrRNAandtRNA rRNA和tRNA的剪接成熟5.1tRNASplicingInvolvesCuttingandRejoininginSeparateReactions5.2ProductionofrRNARequiresCleavageandModificationEventsChapter7Translation【教学基本要求】掌握蛋白翻译过程的步骤，尤其起始步骤，了解翻译过程中相关协助因子，熟悉核糖体的结构组成和活性中心。掌握蛋白转运机制和类型。【教学具体内容】Part1.TheGeneticCode——theTriplet 遗传密码——三联子1.1TheDecodingoftheGeneticCode1.2Thegeneticcodeistriplet1.3Theutilizationofgeneticcode1.4Codon–AnticodonRecognitionInvolvesWobblingPart2.TheStructureandFunctionoftRNA tRNA的结构与功能2.1TransferRNAformsaclover-leaf 三叶草结构2.2ThesecondarystructureoftRNA:thecloverleaf2.3AlltRNAsshareaL-shapedtertiarystructure2.4ThefunctionoftRNA2.5Thesequenceoftheanticodonissolelyresponsibleforthespecificityoftheaminoacyl-tRNA.2.6tRNAsAreSelectivelyPairedwithAminoAcidsbyAminoacyl-tRNASynthetases2.7MutatedAnticodonsThatReadNewCodons2.8SuppressortRNAsHaveMutatedAnticodonsThatReadNewCodons2.9ThereAreNonsenseSuppressorsforEachTerminationCodon2.10FrameshiftingOccursatSlipperySequences——programmedframeshifting程序性移码.Part3.TheStructureandFunctionofRibosome 核糖体的结构与功能3.1Ribosome3.2RibosomalRNAPervadesBothRibosomalSubunits3.3RibosomesHaveSeveralActiveCenters3.4TheribosomehasthreetRNA-bindingsites.Aminoacylsiteàpeptidylsiteàexitsite3.5大小亚基的生物学功能：Part4.TheProcessofProteinSynthesis 蛋白质合成的过程4.1TheThreetRNA-BindingSitesontheRibosome4.2TranslationOccursbyInitiation,Elongation,andTermination4.3TheProcessofProteinSynthesis4.3.1TheInitiationStageInitiationinBacteriaNeeds30SSubunitsandAccessoryFactorsInitiationInvolvesBasePairingbetweenmRNAandrRNAASpecialInitiatortRNAStartsthePolypeptideChainSmallSubunitsScanforInitiationSitesonEukaryoticmRNA4.3.2TheElongationStageElongationFactorTuLoadsAminoacyl-tRNAintotheASiteThePolypeptideChainIsTransferredtoAminoacyl-tRNATranslocationMovestheRibosome4.3.3TheTerminationStage——ThreeCodonsTerminateTranslation4.3.4ThemodificationofnascentProteinPart5.TheTransportionofProtein 蛋白质的运转机制1)Proteinsthatarelocalizedpost-translationallyPost-translationalmembraneinsertiondependsonleadersequences2.)Proteinsthatarelocalizedco-translationallyChapter8MethodsinMolecularBiologyandGeneticEngineering【教学基本要求】掌握分子生物学中的基本技术方法的原理和应用，了解各种方法的应用例子分析，熟悉基因研究方法的选择。【教学具体内容】Part1TheManipulationofDNATwoessentialitemsinthemolecularbiologist’stoolkitare：restrictionendonucleases限制性核酸内切酶cloningvectors克隆载体1.1Nucleases核酸酶Endonuclease核酸内切酶Exonuclease核酸外切酶.restrictionendonuclease限制性内切酶1.2CloningcloningvectorrecombinantDNA重组DNA–ligating(orligationDNA连接).1.3vector载体Plasmid质粒Shuttlevectors穿梭载体Expressionvectors表达载体.Subclone亚克隆重组DNA操作过程：1.4Cloning：transformationandselection转化与筛选pUC载体：（α互补）Transformation–Blue/whiteselection细菌转化及蓝白斑筛选Part2Thelargescalecloning：GenomicandcDNALibraries基因组文库和cDNA文库Part3Polymerasechainreaction(PCR)聚合酶链式反应TheprincipleofPCR：denaturation变性、renaturation复性、semiconservativereplication半保留复制Part4RT-PCRRT-PCRusesreversetranscriptasetoconvertRNAtoDNAforuseinaPCRreaction.MakingacDNAlibraryPart5Real-timePCR实时定量PCR应用TaqMan探针实时定量PCR技术荧光染料SYBRGreenⅠ探针的实时定量PCRPart6DNASeparationTechniquesGelelectrophoresis电泳。6.2Thefluorescentdye:EB（Ethidiumbromide，溴化乙锭）6.3PFGE：pulse-fieldgelelectrophoresisDNA的脉冲电泳技术分离超大片段的DNA6.4DNAcanalsobeisolatedusingdensitygradientcentrifugation.密度梯度离心法Part7Theproteomicanalysis蛋白组的分析7.1双向电泳（two-dimensionalelectrophoresis）isoelectricfocusing(等电聚焦)inthefirstdimensionandSDSinthesecond.7.2双向电泳（two-dimensionalelectrophoresis）双荧光标记-差异电泳7.3massspectrometry：蛋白质的质谱分析技术Sequencingapeptidebymassspectronomy基于肽段的核质比进行分析。Part8Thegenomicanalysis基因组的分析8.1SNP:SingleNucleotidePolymorphisms（单核苷酸多态性）Transitions置换Transversions颠换8.2DNASequencingdideoxynucleotides(ddNTPs)双脱氧核苷酸DNA测序技术：Sanger双脱氧技术8.3DNAfingerprint(DNA指纹图谱)CuttheDNAwitharestrictionenzyme.Part9Genecloning基因克隆技术9.1RACE技术（rapidamplificationofcDNAends(RACE)cDNA末端快速扩增技术）Part10NucleicAcidDetection核酸的检测insituhybridization原位杂交荧光原位杂交(fluorescenceinsituhybridization，FISH)10.2BlottingMethods印迹法SouthernblottingNorthernblottingWesternblottingepitopetag表位标签10.3DNAMicroarrays（DNA微阵列）Part11GeneKnockouts基因敲除技术11.1GeneKnockoutsandTransgenicsTransgenics转基因ES(embryonicstem)cells胚胎干细胞transfectedgeneusinghomologousrecombination同源重组.usingtwoselectablemarkersKnockout敲除knock-in敲入11.2RNAi（RNAinterference,RNA干涉）RNAinterference(RNAi)11.3基因定点突变(site-directedmutagenesis)PCR-basedsite-directedmutagenesis：，重叠延伸技术和大引物诱变法Part12Protein–Protein/DNAInteractionsanalysisProtein–ProteinInteractions12.1TheTwo-HybridAssay酵母双杂12.2Co-IP：Co-Immunoprecipitation免疫共沉淀Protein–DNAInteractions12.3ElectrophoreticmobilityshiftassayDNasefootprintingassay12.5Chromatinimmunoprecipitation.12.6YeastOne-hybridSystem(Y1H)12.7Bacterialone-hybridsystem(B1H)，StructuredeterminationusingX-raycrystallographyhasbeenusedtogiveahighlydetailedatomicviewofprotein-DNAinteractions.12.8噬菌体展示技术(PhageDisplay)Chapter9RegulationofGeneExpressioninProkaryota——TheOperon原核基因表达调节——操纵子模型【教学基本要求】掌握原核生物基因表达调控的基本规律；理解基因表达调节相关名词和类型；熟悉典型的操纵子——乳糖操纵子和色氨酸操纵子。【教学具体内容】Part1RegulationofGeneExpression基因表达调控总论Geneexpressioncanbecontrolledatanyofseveralstages：transcription,processing,andtranslation:1)、转录水平上的调控（transcriptionalregulation）2)、转录后水平上的调控（post-transcriptionalregulation）Part2IntroductionofRegulationofGeneExpressioninProkaryotacoupledtranscription/translation转录与翻译偶联Operon操纵子.2.1Theparticipatorsingeneregulationtrans-acting反式作用.cis-acting顺式作用2.2Thesimplestformoftheregulatorymodelregulatorgene调节基因structuralgene结构基因2.3RegulationcanbenegativeorpositiveInnegativeregulation负调控,Inpositiveregulation正调控,2.4ThePatternsofGeneExpression1）、constitutiveexpression组成型表达：管家基因(housekeepinggene)。2）、调节型（适应型）表达(regulated/adaptiveexpression）2.5TheresponseofregulatorstothesubstratesIninducibleregulation可诱导调节：(theinducer诱导物).Inrepressibleregulation可阻遏调节：(thecorepressor辅阻遏物).2.6DistinguishingpositiveandnegativecontrolPart3Operon操纵子3.1Structuralgenes+controlregion(Promoter，P+Operator，O)3.2StructuralGeneClustersAreCoordinatelyControlledPart4乳糖操纵子(lacoperon)4.1ThelacOperonIsNegativeInducible4.2Theinducer诱导物：半乳糖苷（实际为异乳糖lacRepressorIsControlledbyaSmall-MoleculeInducer4.3Theoperator操纵基因：LacOcis-ActingConstitutiveMutationsIdentifytheOperatortrans-ActingMutationsIdentifytheRegulatorGene4.4Therepressor抑制蛋白：LacILacrepressormonomerhasseveraldomains4.5lacRepressorBindingtotheOperatorIsRegulatedbyanAllostericChangeinConformationTheOperatorCompeteswithLow-AffinitySitestoBindRepressor4.6乳糖操纵子调控模型、大肠杆菌对乳糖的反应4.7ThelacOperonHasaSecondLayerofControl:CataboliteRepressionLac操纵子的第二层面调节：分解代谢物阻遏cataboliterepressionCataboliterepressorprotein(CRP),cyclicAMP(cAMP).cAMP-CRP为激活物两个层面之间的协调调节Part5OtherOperons——TheTrpOperon（Trp：tryptophan色氨酸5.1ThetrpOperonIsaRepressibleOperonwithThreeTranscriptionUnits5.2ThetrpOperonIsAlsoControlledbyAttenuationAttenuation弱化作用.Anattenuator弱化子(intrinsicterminator)5.3AttenuationCanBeControlledbyTranslationleaderpeptide（前导肽）,ribosomestalls（核糖体停滞）\Part6Post-transcriptionalregulation转录后调控6.1AdversegrowthconditionsprovokethestringentresponseIdlingreaction空运转:unchargedtRNA空载tRNAispresentintheAsite;triggerstheStringentresponse严紧反应.6.2r-ProteinSynthesisIsControlledbyAutoregulation6.3TranslationCanBeRegulated6.4SmallRNAmoleculescanregulatetranslationAntisenseRNAcanaffectfunctionorstabilityofanRNAtarget.6.5mRNA稳定性对转录水平的影响Chapter10EukaryoticTranscriptionRegulation真核生物基因表达调节【教学基本要求】熟悉真核基因组结构特点，了解真核基因表达调节基本规律，掌握真核生物中基因表达调节的发生的不同阶段。【教学具体内容】Part1IntroductionofEukaryoticTranscriptionRegulation真核生物基因表达调节1）、DNA水平上的调控（DNAreplicationandrearrangement）2）、转录水平上的调控（transcriptionalregulation）3）、转录后水平上的调控（post-transcriptionalregulation）Part2TheregulatorsintheEukaryoticTranscriptionRegulation2.1MechanismofActionofActivatorsandRepressorspositivecontroltrueactivatorAntirepressor抗阻遏物negativecontrol2.2IndependentDomainsBindDNAandActivateTranscriptionDNA-bindingdomain,transcription-activationdomainThetwo-hybridassay2.3ActivatorsInteractwiththeBasalApparatusPart3TheBasalApparatus3.1TheGeneStructureofEukaryota3.2PromoterelementsaredefinedbymutationsandfootprintingTATAboxes,CAATboxes,GCboxes,andotherelements.3.3Enhancerelement3.4Transcriptioninitiationcomplex真核细胞中3种RNA聚合酶:PolI,PolII,PolIIIEukaryoticRNApolymerasesconsistofmanysubunits3.4.1RNApolymeraseIhasabipartitepromoter3.4.2RNApolymeraseIIIusesbothdownstreamandupstreampromoters3.4.3EukaryoticRNApolymeraseIIhas>10subunits.ThetranscriptionalfactorforRNApolymeraseII：ThestartpointforRNApolymeraseII:3.4.4TBPisauniversalfactor(TATAboxbindingprotein)3.4.5Thebasalapparatusassemblesatthepromoter3.5Trans-actingelements反式作用因子makingcontact,directorindirect,withthecis-actingelementstocontrolthetranscriptionlevel.IndependentDomainsoftranscriptionfactors:BindDNAandActivateTranscription3.5.1ThereAreManyTypesofDNA-BindingDomainszincfinger锌指结构.helix-turn-helixHTH螺旋-转角-螺旋helix-loop-helix(bHLH碱性螺旋-环-螺旋)leucinezipper亮氨酸拉链3.5.2Theactivatingdomain转录活化结构域特征性结构：Negativechargedhelix带负电荷的螺旋结构DomainrichinGlutamine富含谷氨酰胺的结构DomainrichinProline富含脯氨酸的结构Part4Transcriptioninitiationrequireschangesinchromatinstructure转录前调控：染色质结构与基因表达调控4.1ChromatinRemodelingIsanActiveProcesschromatinremodeling染色质重塑ATP-dependentchromatinremodelingcomplexest4.2NucleosomeOrganizationorContentMayBeChangedatthePromoter4.3HistoneAcetylationIsAssociatedwithTranscriptionActivationlysine(K)acetyltransferase(KAT),histoneacetyltransferase(HAT).赖氨酸残基的乙酰化修饰histonedeacetylase(HDAC)–repressoractivity.4.4MethylationofHistonesandDNAIsConnected4.5PromoterActivationInvolvesMultipleChangestoChromatin4.6HistonePhosphorylationAffectsChromatinStructureHowIsaGeneTurnedOn?三、课程的重点和难点：1、理解基因和基因组的结构、组成和功能；从分子机制角度认识生命遗传信息的存储者的本质。2、掌握复制、转录、翻译及各种RNA产物及蛋白产物实现功能前的加工步骤，了解参与的各种蛋白和其他因子的功能，认识从微小单细胞生物到复杂高等生物的生命活动的本质。3、理解遗传信息的表达过程中的多层面表达调节，从而构成一个复杂而精细的表达调控网络。4、掌握分子生物学的基本操作技术，包括核酸操作、蛋白操作及其功能研究的有关方法。四、参考性教学时间安排：教学内容授课学时实验（上机）学时实践学时IntroductionofMolecularBiology0.5Chapter1GenesAreDNA1.5Chapter2Chromosomeandgenome2Chapter3DNAReplication2Chapter4DNARepairsystem，ChapterATransposableElementsandRetroviruses2Chapter5AProkaryoticTranscriptionChapter5BEukaryoticTranscription2Chapter6RNASplicingandProcessing2Chapter7Translation5Chapter8MethodsinMolecularBiologyandGeneticEngineering9Chapter9RegulationofGeneExpressioninProkaryota——TheOperon4Chapter10EukaryoticTranscriptionRegulation4实验一大肠杆菌感受态细胞的制备及转化5实验二聚合酶链反应技术实验三DNA的琼脂糖凝胶电泳及其切胶回收5实验四：质粒DNA的分离纯化及酶切5实验五：质粒DNA的酶切及电泳分析鉴定3合计3418五、实践（实验）教学环节（含实验项目、实践内容）：实验课实验一：大肠杆菌感受态细胞的制备及转化一、实验目的1、掌握CaCl2法制备大肠杆菌感受态细胞的原理和方法2、掌握热激法转化大肠杆菌的原理和方法。二、实验原理细菌处于容易吸收外源DNA的状态叫感受态。转化是指质粒DNA或以它为载体构建的重组子导人细菌的过程。受体细胞经过一些特殊方法（如：电击法，CaCl2等化学试剂法）处理后，使细胞膜的通透性发生变化，成为能容许外源DNA分子通过的感受态细胞。进入细胞的DNA分子通过复制、表达实现遗传信息的转移，使受体细胞出现新的遗传性状。大肠杆