真核甲基化酵母半乳糖类固醇激素植物

MolecularBiologyMolecularBiology‐InThelacOperonofThetrpOperonofRegulationofgeneexpressioninPhage‐InMutationandGeneticsofTransposableGeneManipulation&GenomicLevelsofgeneexpressionLevelsofgeneexpressioncontrolinPrecursor‐RNAmRNARNAProteinProteinTranscriptionTranscriptionGeneralaspectsoft/cChromosomechangesandt/cHistonesandgeneDNAmethylationandt/cT/ccontrolofgeneexpressioninGalactose‐utilizinggenesofSteroidhormoneregulationofgeneexpressioninHormonecontrolofgeneexpressionin•Proteincodinggenehas elementswhichbind ,and elementswhich•Proteincodinggenehas elementswhichbind ,and elementswhichbind •CombinatorialGene•••Transcriptionallyactivegenes‐‐‐looserchromosomestructure‐highDNaseI•HistoneTranscriptionTranscriptionGeneralaspectsoft/cChromosomechangesandt/cHistonesandgeneDNAmethylationandt/cT/ccontrolofgeneexpressioninGalactose‐utilizinggenesofSteroidhormoneregulationofgeneexpressioninHormonecontrolofgeneexpressionin DNA Adenine DNA AdenineandcytosinearemethylatedoftenthanguanineandsequencesorregionsofaDNAmolecule.AllknownDNAmethylasesuseadenosylmethionineasmethylgroup DNAMethylationinE. DNAMethylationinE. mechanismthathelpsthecelltodistinguishitsDNAfromforeignDNA DNAMethylationandT/C •5 DNAMethylationandT/C •5mC:Cytosinesaremethylatedshortlyafter•thepercentageof5mCvariesfromspeciestospecies.(3%formammalianDNA,littleornoinDrosophilaandyeast) DNAMethylationandT/C Thedistributionof DNAMethylationandT/C Thedistributionof5mCisnon‐random,Most5mCwasfoundinthesequenceCG(mCpGisland)•Inmammaliangenomes,CpGislandsaretypically3,000basepairsinlength.Theyareinandnearapproximately40%ofpromotersofmammaliangenes.•The"p"inCpGreferstothephosphodiesterbetweenthecytosineandtheguanine,whichindicatesthattheCandtheGarenexttoeachotherinsequenceregardlessofbeingsingle‐ordouble‐stranded.• DNAMethylationandT/C •Methylation DNAMethylationandT/C •MethylationcouldbeasignalforDNAinvolvedevents:replication,transcription,repairet.al.mCpGattractmethylationsensitiveDNAbindingproteins(MeCP1)recruitHistonede-acetylationenzyme(Sin3complex)histonede-acetylationgene•DNAMethylationisassociatedwithSilencedgenesmammalian Switchingageneoff Switchingageneoffthrough methylationandhistone Howismethylation •Therearespecificrestrictionenzymesthat5mCinvolved Howismethylation •Therearespecificrestrictionenzymesthat5mCinvolvedcleavage+‐++MspIisanisoschizomerofHpaII.WhentheexternalCinthesequenceCCGGismethylated,MspIandHpaIIcannotcleave.However,unlikeHpaII,MspIcancleavethesequencewhentheinternalCresidueismethylated. RFLPdetectionof RFLP:R RFLPdetectionof RFLP:RestrictionFragmentLengthRestrictionRestrictionLength MethylationandT/C •Hpa MethylationandT/C •HpaII/MspIRFLPstudyofgeneswithactivity(>30genesexamined)‐‐‐‐negativecorrelationbetweenDNAmethylationandt/cIsthisrelationshipageneralWillallmethylatedCdemethylatedinactivegene•••IsmethylationlevelchangeanecessityofactivityorabyproductofOverorUndermethylationmayOverorUndermethylationmayserious•Mutationofmethylaseinmiceis•FragileXsyndrome:FMR‐1generepeatoverexpansion(CGGrepeat#>200)andabnormalmethylationT/Csilenced•Abnormalmethylationandpromoteroftumorsuppressor BiologicalImportanceofDNA •DNAcondensation: BiologicalImportanceofDNA •DNAcondensation:chromatin•Geneexpressioncontrol:imprinting;X‐chromosome•Protectgenomenotbeinginvadedincompatibleforeign•Mis‐match epi:upon,inadditiontogenetics:generation,birth,origin“ChangesinfEpigeneticsTutorialluencephenotypewithoutalteringtheTheyconsistofchangesinthepropertiesofacellthatareinheritedbutthatdonotrepresentachangeingeneticinformation.” ExamplesofEpigenetic ExamplesofEpigenetic FactorsSustainEpigenetic FactorsSustainEpigenetic EpigeneticsEpigeneticsTranscriptionTranscriptionGeneralaspectsoft/cChromosomechangesandt/cHistonesandgeneDNAmethylationandt/cT/ccontrolofgeneexpressioninGalactose‐utilizinggenesofSteroidhormoneregulationofgeneexpressioninHormonecontrolofgeneexpressioninTranscriptionTranscriptionGeneralaspectsoft/cChromosomechangesandt/cHistonesandgeneDNAmethylationandt/cT/ccontrolofgeneexpressioninGalactose‐utilizinggenesofSteroidhormoneregulationofgeneexpressioninHormonecontrolofgeneexpressioninGalactose‐utilizinggenesof•ThreeGalactose‐utilizinggenesof•ThreespecificgenesrequiredforTheexpressionofthesethreegenesisresponsivetogalactosepresenceThreegenesareclosetogetherbutdonotformaAregulatoryproteinGAL4pis••Notethe•TheupstreamactivatorTheupstreamactivator galactose UASG(Upstreamactivatorsequence‐galactose)‐‐‐GAL4bindingsite,consistsoffour17‐bp,two‐foldsymmetryUASGwillplaceanystructuralgenedownstreamofitunderGAL4controlUASGfunctionsineither Gal4boundtoitssiteon Gal4boundtoitssiteon •Gal4hasaDNAbindingdomain(zincfinger)andaactivationdomain(bindsGAL80)Functionsasa••ActivatorsHaveSeparateActivatorsHaveSeparateDNABindingActivatingTheDNA-bindingdomainservesmerelytotethertheactivatingregiontotheOthereukaryotic(fromyeast,flies,ordomainsandactivatingregionsareseparable.GAL4anditsInthepresenceofGalactose,itsmetabolitebindstoGAL80GAL4anditsInthepresenceofGalactose,itsmetabolitebindstoGAL80andcauseGAL4undergophosphorylation.activeGAL4hastwoGAL4hastwoactivation•AnyoneactivationdomainofGAL4isforactivatingUASGdirectedLactoseLactose(Allolactose)VSgalactoseLacrepressor(DNAbindingdomainandinducerbindingdomain)GAL4(DNAbindingdomainandactivationTranscriptionTranscriptionGeneralaspectsoft/cChromosomechangesandt/cHistonesandgeneDNAmethylationandt/cT/ccontrolofgeneexpressioninGalactose‐utilizinggenesofSteroidhormoneregulationofgeneexpressioninHormonecontrolofgeneexpressioninWhatisWhatisA•AneffectormoleculethatisproducedlowconcentrationsbyonecellandcausesaphysiologicalresponseinanotherThefunctionofThefunctionof•Abacteriahastoadapttoitsenvironment(exposedtorapidHigherEukaryotecellsdifferentiatetocarryoutspecializedfunctions,theirenvironmentisrelativelyconstant(exposedtotheintercellularfluid).Thisconstancyispartiallymaintainedthroughtheactionofhormones.Hormoneproductioniselaborately•••HowdoesatargetHowdoesatargetcellbeing•DifferentHormonesspecificPeptideHormones:cannotfreelypassthroughareonthecellsurface.SteriodHormones:canmovefreelyacrosscellinsidethecell.•• SteroidHormonesand •Differenthormones SteroidHormonesand •DifferenthormonesdifferentsideEachinduces••Show氢化可的松（副肾荷尔蒙之一种醛固睾丸激孕酮,黄体 Tissue‐specificeffectsof Thespecificityof Tissue‐specificeffectsof ThespecificityoftheresponsetosteroidiscontrolledbythehormoneSteroidHormonesandSteroidHormonesand•SteroidhormoneshaveeffectsonstabilityofmRNAsandpossiblytheprocessingofmRNAprecursors.Steroidhormonesmayexistinacell,butitseffectiscontrolledbyreceptors’presence.Moststeroidhormonereceptors(SHRs)existinonlylimitedtargettissuesSteroidhormonesactaseffectormolecules,SHRsactasregulatorymolecules.••• Amolecularviewofreceptor •• Amolecularviewofreceptor ••SHRshavehighaffinityfortheirIntheabsenceofhormones:SHRsareprotectedbychaperones(largeproteincomplex),inactiveSteroidhormonereplacechaperonesandform•complexwith(SHR•Theactivecomplexbindstospecificregulatorysequence(Steroidhormoneresponseelements‐‐HREs)Allsteroidhormonesworkinthesamegeneral•Steroidhormoneresponseelements‐‐Steroidhormoneresponseelements‐‐•AllgenesregulatedbyaspecificsteroidhormonehavecommonDNAsequenceHREs.GREistheglucocorticoidresponseelement,EREistheestrogenresponseelementHREsarelocated,ofteninmultiplecopies,intheenhancerregionsofgenesGRE,forexample,islocatedabout250bpupstreamfromthetranscriptionstartpoint,theconsensussequenceisAGAACANNNTGTTCTEREconsensussequenceisAGGTCANNNTGACCTTwofoldsymmetry•••••Howishormone‐receptorHowishormone‐receptor (tissue‐specificity) ••MultiplecopiesofHREsforoneManyregulatoryproteinsbindstobothpromoterandSteroid‐receptorcomplexfunctiondependsonitsinteractionwithotherregulatoryfactorsAsteroid‐receptorcomplexcanactivat