2013分子生物学工作基础CLASS.ppt

CloningandFunctionalAnalysisofGenesTranscriptionalRegulationofGeneExpression 梁静副教授liang jing HumanGenomeProject Goals identifyalltheapproximate30 000genesinhumanDNA determinethesequencesofthe3billionchemicalbasepairsthatmakeuphumanDNA storethisinformationindatabases improvetoolsfordataanalysis transferrelatedtechnologiestotheprivatesector and addresstheethical legal andsocialissues ELSI thatmayarisefromtheproject Milestones 1990 ProjectinitiatedasjointeffortofU S DepartmentofEnergyandtheNationalInstitutesofHealth June2000 Completionofaworkingdraftoftheentirehumangenome covers 90 ofthegenometoadepthof3 4xredundantsequence February2001 Analysesoftheworkingdraftarepublished April2003 HGPsequencingiscompletedandProjectisdeclaredfinishedtwoyearsaheadofschedule U S DepartmentofEnergyGenomePrograms GenomicsandItsImpactonScienceandSociety 2003 http doegenomes orghttp www sanger ac uk HGP overview shtml 3 Dr FrancisCollins FrancisSellersCollins bornApril14 1950 isanAmericanphysician geneticist notedforhisdiscoveriesofdiseasegenesandhisleadershipoftheHumanGenomeProject HGP HecurrentlyservesasDirectoroftheNationalInstitutesofHealthinBethesda Maryland Collinsacceptedaninvitationin1993tosucceedJamesD WatsonasDirectoroftheNationalCenterforHumanGenomeResearch whichbecameNationalHumanGenomeResearchInstitute NHGRI in1997 AsDirector heoversawtheInternationalHumanGenomeSequencingConsortium http www genome gov glossary index cfm id 528 4 Dr CraigVenter In1998 Dr VenterfoundedCeleraGenomicsandannouncedthatCelerawoulddecodethehumangenomefasterandmoreeconomicallythanthepubliclyfundedconsortiumofscientists Thatchallengeisnowcreditedwithcreatingtheclimateofurgencythatspurredcompetitionandsubstantiallyacceleratedtheproject ssuccessfulconclusion Dr VenterfoundedtheJ CraigVenterInstitute andheisnowworkingtheretocreatesyntheticbiologicalorganismsandtodocumentgeneticdiversityintheworld soceans Whatdoesthedrafthumangenomesequencetellus BytheNumbers Thehumangenomecontains3billionchemicalnucleotidebases A C T andG Theaveragegeneconsistsof3000bases butsizesvarygreatly withthelargestknownhumangenebeingdystrophinat2 4millionbases Thetotalnumberofgenesisestimatedataround30 000 muchlowerthanpreviousestimatesof80 000to140 000 Almostall 99 9 nucleotidebasesareexactlythesameinallpeople Thefunctionsareunknownforover50 ofdiscoveredgenes U S DepartmentofEnergyGenomePrograms GenomicsandItsImpactonScienceandSociety 2003 http doegenomes org 6 TheENCODEprojectprovidesinformationonthehumangenomefarbeyondthatcontainedwithintheDNAsequence ENCODEdescribesthefunctionalgenomicelementsthatorchestratethedevelopmentandfunctionofahuman TheprojectcontainsdataaboutthedegreeofDNAmethylationandchemicalmodificationstohistonesthatcaninfluencetherateoftranscriptionofDNAintoRNAmolecules ENCODEexamineslong rangechromatininteractions suchaslooping thataltertherelativeproximitiesofdifferentchromosomalregionsinthreedimensionsandalsoaffecttranscription ENCODEcataloguesthesequencesandquantitiesofRNAtranscripts frombothnon codingandprotein codingregions ENCODE theEncyclopediaofDNAElements 7 OneofthemoreremarkablefindingsofENCODEisthat80 ofthegenomecontainselementslinkedtobiochemicalfunctions dispatchingthewidelyheldviewthatthehumangenomeismostly junkDNA Theauthorsreportthatthespacebetweengenesisfilledwithenhancers regulatoryDNAelements promoters thesitesatwhichDNA stranscriptionintoRNAisinitiated andnumerouspreviouslyoverlookedregionsthatencodeRNAtranscriptsthatarenottranslatedintoproteinsbutmighthaveregulatoryroles ThePost GenomicEra GeneannotationGenefunctionGeneregulation TheHumanGenomeProject islikesendingamantothemoon Whenyouthinkaboutit sendingamantothemooniseasy it sgettinghimbackthat sdifficult SoIthinkwenowneedtogetthehumangenometoreturntowork SydneyBrenner CloningandFunctionalAnalysisofGenes GenestoProteins DifferentialgeneexpressionanddiseasesMicroarrayAnalysisDifferentiationDisplayRNA sequencingIdentifynovelprotein proteininteractionsYeastTwo hybridSystemPhageDisplay 11 Non codingRNA Anon codingRNA ncRNA isafunctionalRNAmoleculethatisnottranslatedintoaprotein Non codingRNAgenesencodehighlyabundantandfunctionallyimportantRNAssuchastRNAandrRNA aswellasRNAssuchassnoRNAs microRNAs piRNAsandthelongncRNAs MicroRNA AmicroRNA abbreviatedmiRNA isashortRNAmoleculefoundineukaryoticcells AmicroRNAmoleculehasveryfewnucleotides anaverageof22 comparedwithotherRNAs miRNAsarepost transcriptionalregulatorsthatbindtocomplementarysequencesontargetmessengerRNAtranscripts mRNAs usuallyresultingintranslationalrepressionortargetdegradationandgenesilencing Differentialgeneexpressionanddiseases MicroarrayAnalysisDifferentiationDisplayRNA sequencing DNAMicroarrays Microarrayexpressionprofiling 1 IsolateRNAsamples synthesizecDNAprobeformicroarrayhybridization 2 HybridizelabeledprobewithDNAmicroarrayonachip 3 Scanthechipandcollectrawdata 4 Analyzedata i e performhierarchicclusteringandcorrelatewithhistoclinicaldata Culturedcellsorclinicalsamples DNAmicroarraysinsystemsbiology ExperimentalDesign Repeat BiologicalRepeatedbiologicalsamplesRepeat TechnicalRepeatedchips DifferentialDisplay outdated AAAAA AAAAA AAAAA TotalRNA mRNAcDNA AAAAA TTTTT AAAAA TTTTT AAAAA TTTTT ControlTreatment SequenceAnalysis TestandcontrolmRNAareseparatelyreversetranscribedinthepresenceofanchoredoligo dT primerscontainingnucleotidesNandMinvariouscombinations whereNcanbeguanine G adenine A thymine T orcytosine C andMisG A orC ThesameprimerandanarbitrarydecamerarethenusedasprimersinaPCR Theproductsaresubjectedtoelectrophoresis PAGE Anadditionalband seearrow inthetestsamplerepresentsagenethathadnotbeenactivatedinthecontrolsample A11andT11denoteelevenAandTmolecules respectively PrincipleofaDifferentialDisplay Briefly longRNAsarefirstconvertedintoalibraryofcDNAfragmentsthrougheitherRNAfragmentationorDNAfragmentation seemaintext Sequencingadaptors blue aresubsequentlyaddedtoeachcDNAfragmentandashortsequenceisobtainedfromeachcDNAusinghigh throughputsequencingtechnology Theresultingsequencereadsarealignedwiththereferencegenomeortranscriptome andclassifiedasthreetypes exonicreads junctionreadsandpoly A end reads Thesethreetypesareusedtogenerateabase resolutionexpressionprofileforeachgene asillustratedatthebottom ayeastORFwithoneintronisshown ATypicalRNA SeqExperiment RNA Seq arevolutionarytoolfortranscriptomics NatRevGenet 2009Jan 10 1 57 63 RNA sequencing Quantification RNA Seq Transcriptomics AssessmentofsequencingScreeningofdifferentiallyexpressedgenes DEGs ExpressionpatternanalysisofDEGsGeneontologyanalysisofDEGsPathwayenrichmentanalysisofDEGsProteininteractionnetworkanalysis StandardBioinformaticsAnalysisofRNA seq Identificationofgenes denovotranscriptomeonly Identificationofuntranslatedregions UTRs intron exonboundaries alternativesplicing startcodonidentification andsimilarannotationdata Identificationofnon codingunits includingnon codingRNA microRNAprecursors andothertypesofRNAthatdonotcodeforproteins Determininggeneexpressionatthetranscriptionallevel Identificationofnoveltranscriptionunits RNA Seq Transcriptomics AdvantagesofRNA SeqComparedwithotherTranscriptomicsMethods RNA Seq arevolutionarytoolfortranscriptomics NatRevGenet 2009Jan 10 1 57 63 YeastTwo hybridSystemPhageDisplay Identifynovelprotein proteininteractions YeastTwo Hybrid GAL4DNABD Kan MCS Trp1 DNABindingDomainProteinX Bait YeastTwo hybridScreen GAL4AD Amp Leu2 2 ConstructcompatiblecDNAlibrary cDNAlibrary 1 ConstructaDNA BD BAITfusionexpressionvector 5 Isolatepositivecolonies amplifyplasmidDNAinE coliandsequence 3 Co transfect a cDNAlibrary b reportervector c GAL4ADcDNA expressingvectorintocompetentyeastcells 4 Plateonselectivemediumtoscreenforpositiveinteractions UAS Reportergene TATA BAIT ProteinX DNA BD GAL4AD Two hybridscreensarelow tech theycanbecarriedoutinanylabwithoutsophisticatedequipment Two hybridscreenscanprovideanimportantfirsthintfortheidentificationofinteractionpartners Theassayisscalable whichmakesitpossibletoscreenforinteractionsamongmanyproteins Yeasttwo hybriddatacanbeofsimilarqualitytodatageneratedbythealternativeapproachofcoaffinitypurificationfollowedbymassspectrometry AP MS Y2HStrengths Y2HWeaknesses Ahighnumberoffalsepositive andfalsenegative identifications Thehybridproteinsarefusionproteins andthefusedpartsmayinhibitcertaininteractions Aninteractionmaynothappeninyeast thetypicalhostorganismforY2H TheY2Htakesplaceinthenucleus Iftestproteinsarenotlocalizedtothenucleus twointeractingproteinsmaybefoundtobenon interacting Someproteinsmightspecificallyinteractwhentheyareco expressedintheyeast althoughinrealitytheyareneverpresentinthesamecellatthesametime PhageDisplay PhagevectorDNA LIGATION DNAencodingpeptideofinterest INFECTE coli fusionlibraryofmanyphage eachwithadifferentpeptidedisplayedonitssurface Geneencodingaphagecoatprotein Peptideinfusionproteindisplayedonsurfaceofphage AFFINITYCHROMATOGRAPHY ELUTECHARACTERIZATION ProteinstoGenes Protein proteininteractionProteomics Affinitypurification GSTPULL DOWN Invitro Y Z Cellextracts Sepharose4B GST BAIT Glutathione Sepharose4B GST BAIT x SDS PAGE Massspectrometry Proteinsequence Nucleotidesequence ChristopherRachezetal 1999Nature398 824 828 FLAGSystemAffinityPurification Invivo TransfectcellswithplasmidsencodingFLAG taggedprotei