《研人类基因组学》PPT课件.ppt

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1、基因组与基因作图基因组与基因作图Lecture2向光盛生物化学教研室2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院1.Objective2.Review3.Concepts4.PositionofgenesOrganizationofthehumangenome2011-10-25向光盛长江大学医学院HUMANNUCLEARGENOME24chromosomes(haploid)3200Mbp30,000genesMitochondrialgenome16569bp37genesHumangenome=nucleargenome+mitochondrialge

2、nome2011-10-25向光盛长江大学医学院基因组(genome)泛指一个有生命体、病毒或细胞器的全部遗传物质；在真核生物，基因组是指一套染色体（单倍体）DNA。基因组学(genomics)就是发展和应用DNA制图、测序新技术以及计算机程序，分析生命体（包括人类）全部基因组结构及功能。2011-10-25向光盛长江大学医学院基因组学包括3个不同的亚领域结构基因组学(structural genomics)功能基因组学(functional genomics)比较基因组学(comparative genomics)基因组学概念2011-10-25向光盛长江大学医学院结构基因组学(struct

3、ural genomics)是通过HGP的实施来完成的。HGP的内容就是制作高分辨率的人类遗传图和物理图，最终完成人类和其它重要模式生物全部基因组DNA序列测定，因此HGP属于结构基因组学范畴2011-10-25向光盛长江大学医学院HGP包括以下研究内容（一）物理制图（二）遗传制图（三）基因组DNA序列测定（四）创建计算机分析管理系统 2011-10-25向光盛长江大学医学院HGP主要任务及内容2011-10-25向光盛长江大学医学院通过HGP获得的广泛基因组信息组成了结构基因组学的基本内容，是开展功能基因组学的研究的基础；同时为详尽研究每一个单基因遗传病提供“平台”，并将成为复杂的多基因遗传

4、病研究的发端。2011-10-25向光盛长江大学医学院功能基因组学功能基因组学完成一个生物体全部基因组测序后即进入后基因组测序阶段详尽分析序列，描述基因组所有基因的功能，包括研究基因的表达及其调控模式，这就是功能基因组学（functional genomics）。2011-10-25向光盛长江大学医学院主要具体内容包括以下方面（一）鉴定DNA序列中的基因（二）同源搜索设计基因功能（三）实验性设计基因功能（四）描述基因表达模式 2011-10-25向光盛长江大学医学院功能基因组学研究策略及主要内容2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25

5、向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2.Recombination2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院1stmechanismforgeneticdiversity:independentassortmentofchromosomesMendelsLawsimplyindependentassortment.Thatis,genesonthesamechromosomeareinheritedtogether

6、;genesondifferentchromosomesareinheritedindependently.With23humanchromosomes,thereisapossible223=8.4x106distinctgametes.Somaticcell2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院diploid1.Crossing-over(recombination)2ndMeioticdivisionCompletionof1stMeioticdivision2.NoDNAreplicationbetween1stand2nddivisions2

7、011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院Iftwogenesarerecombinedx%ofthetime,theyaresaidtobeseparatedbyageneticmapdistanceofxcentimorgans(cM).Quantitativemeasureofrecombinationphenotype(Greek,toshow)2011-10-25向光盛长江大学医学院Thegeneticmapandthephysicalmaparecolinear,butnotquiteproportional.2011-10-25向光盛长江大学医

8、学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医

9、学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医

10、学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院HumanMitochondrialGenomeSmall(16.5kb)circularDNArRNA,tRNAandproteinencodinggenes(37)1gene/0.45kbVeryfewrepeatsNointrons93%coding;Genesaret

11、ranscribedasmultimerictranscriptsRecombinationnotevidentMaternalinheritance2011-10-25向光盛长江大学医学院HstrandenrichedinGLstrandenrichedinC7SDNAshortrepetitivesegmentofHstrandattachedtoLstrand(abortivereplication)ElementoftripleDNAstandstructure2011-10-25向光盛长江大学医学院Whatarethemitochondrialgenes?24of37genesare

12、RNAcoding22mttRNA2mitribosomalRNA(23S,16S)13of37genesareproteincoding(synthethizedonribosomesinsidemitochondria)somesubunitsofrespiratorycomplexesandoxidativephosphorylationenzymes2011-10-25向光盛长江大学医学院LimitedautonomyofmitochondriamtencodednuclearNADHdehydrogenase 7subunits41subunitsSuccinateCoQreduct

13、ase0subunits4subunitsCytochromeb-c1comp1subunit10subunitsCytochromeCoxidase3subunits10subunitsATPsynthasecomplex2subunits14subunitstRNAcomponents 22tRNAsnonerRNAcomponents2componentsnoneRibosomalproteinsnone80Othermtproteins nonemtDNApolRNApoletc.2011-10-25向光盛长江大学医学院Twooverlappinggenesencodedbysames

14、trandofmtDNA(uniqueexample)TwoindependentAUGlocatedinFrame-shifttoeachother,secondstopcodonisderivedfromTA+A(frompoly-A)2011-10-25向光盛长江大学医学院Mitochondrialcodontable22tRNAcoverfor60positionsviathirdbasewobble2011-10-25向光盛长江大学医学院HumanNuclearGenome3200Mb23(XX)or24(XY)linearchromosomes30-35,000genes1gene

15、/100kbIntronsinthemostofthegenes1,5%ofDNAiscodingGenesaretranscribedindividuallyRepetitiveDNAsequences(45%)Recombinationatleastonceforeachchrom.Mendelianinheritance(X+auto,paternalY)2011-10-25向光盛长江大学医学院Human Genome OrganizationHUMANGENOMEGenesandgene-relatedsequencesExtragenicDNANucleargenome3000Mb6

16、5-80000genesMitochondrialgenome16.6kb37genesCodingDNANoncodingDNAUniqueorlowcopynumberModeratetohighlyrepetitivePseudogenesGenefragmentsIntrons,untranslatedsequences,etc.TandemlyrepeatedorclusteredrepeatsInterspersedrepeatsUniqueormoderatelyrepetitiveTworRNAgenes22tRNAgenes13polypeptide-encodinggene

17、s30%70%10%90%80%20%From:Dr Finbarr Hayes lec2011-10-25向光盛长江大学医学院HumannucleargenomeEuchromaticportion3000MbConstitutiveheterochromatine200MbHeterochromatinisdistributedbetweenchromosomesunevenlychrTotalDNAHeteroDNA127930225131610415178832145112011-10-25向光盛长江大学医学院Gene-poorchromosomes(Withextraheteroch

18、romatin)Shortarmsofacrocentricchromosomes13,14,15,21,22Partoflongarmsofchr1,9,16LongarmofchromosomeY2011-10-25向光盛长江大学医学院Humangenomebasecontent41%CGinaverage38%CGforchromosomes4and1349%forchromosome19RegionswithwideswingsinGCcontent(e.g.from33,1%to59,3%)GCcontentiscorrelatedwithGiemsastaining;Genesco

19、rrelatedtoo.GenedensitycorrelateswithhigherGCcontent2011-10-25向光盛长江大学医学院CpGdinucleotideconspiciousdepletionExpectedfrequencyis0,042(4,2%)ObservedfrequencyisfivetimeslowerIthappensduetomethylation-dependentmutationbasedCpGdepletionCpGislandsintheregulatoryareasofhumangenes2011-10-25向光盛长江大学医学院Location

20、ofCpGislandsinthegeneCpGislandsdoNOThaveadeficitofCpGdinucelotides2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院REPEATS!2011-10-25向光盛长江大学医学院3MainComponentsinEukaryoticGenomesDNApurifiedfromahuman,donotself-annealasasimplesigmoidalcurve.Insteadweseeacurvewhichisthesumofthereannealingsofmanydifferentcompone

21、ntsC0=theinitialconcentrationofnucleotides,TtimeinsecondsCoTcurveisameasureofsequencecomplexityREPEATSNOREPEATS2011-10-25向光盛长江大学医学院humanCoTDNA(commercialpreparate)ThisishumanDNAwhichhasbeendenaturedandallowedtoreannealtoaC0tvalueof1.Thedoublestrandedcomponentisthenpurifiedfromthesinglestrandedcompon

22、entandissuppliedcommercially.ItcontainsmostofthehumanrepetitiveDNAbutverylittlesinglecopyDNA(uniquegenes).Usedtosuppressbackgroundhybridizationofcompleprobes2011-10-25向光盛长江大学医学院SatelliteDNAisrepetitiveDNAthatcouldbeseparatedbybuoyantdensityEquilibriumdensitygradientcentrifugationShearedDNAinCesiumCh

23、loridegradient2011-10-25向光盛长江大学医学院SatelliteDNAAlphasatellite(CentromereDNA)MicrosatellitesMinisatellitesAreyoustillrememberwhatitis?Ifnotpleaserefertopreviouslecturesandtothebook2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院RepetitiveDNAModeratelyrepeatedDNATandemlyrepeatedrRNA,tRNAandhistonegenes(genepro

24、ductsneededinhighamounts)LargeduplicatedgenefamiliesMobileDNA(transposons)tobediscussedsoonSimple-sequenceDNATandemlyrepeatedshortsequencesFoundincentromeresandtelomeres(andothers)=(MINIandMICROSATELLITES)2011-10-25向光盛长江大学医学院HumanMobileDNA(transposons)MoveswithingenomeLINE(Longinterspersednuclearele

25、ments)L1,L2,L3LINEis21%ofhumanDNA(1,00,000copies)SINE(Shortinterspersednuclearelements)Aluis10,7%ofhumanDNA(1,200,000copies)MIR,MIR3is3%ofhumDNA(500,000copies)LTRelements(LongTerminalRepeats)ERVandMalRare8%ofhumanDNA(500,000copies)TransposonsMER1(Charlie),MER2(Tigger),others(350,000copies),2,8%ofhum

26、anDNATOTAL:approx;45%ofhumanDNA2011-10-25向光盛长江大学医学院LINEsandERVs2011-10-25向光盛长江大学医学院Longinterspersednuclearelements(LINEs)20%ofgenomeLINE1active(Alsomanytruncatedinactivesequences)Line2inactiveLine3inactiveRNAbindingalsoendonucleaseLINEspreferAT-richeuchromaticbandsInternalpromoterINeveryonesgenome60

27、-100copiesofLINE1arestillcapableoftransposing,andmayoccasionallycausethediseasebygenedisruption2011-10-25向光盛长江大学医学院MechanismofLINErepeatjumpsFulllengthLINEtranscriptisgeneratedfrom5-UTR-basedpromoterORF1andORF2translatedintoproteinsthatstayboundtoLINEmRNAORF1/ORF2/mRNAcomplexmovesbackintothenucleus5

28、353orf1orf253orf1orf2533535ProductofORF2cutdsDNAFreed3servesasaprimerforLINEreversetranscriptionfrom3UTR2011-10-25向光盛长江大学医学院ORF2andORF1functionORF1keepsORF2andLINEmRNAboundtogetherandretractedintonucleusORF2(endonuclease)cutdsDNAtoprovidefree3endasaprimertoLINE3UTRORF2(reversetranscriptase)makescDNA

29、copyofLINEmRNA,whichbecomesintegratedintochromosomalDNA(asitboundtoitbyformer3freedend)TTTTAisORF1cleavagesite,thatiswhyintegrationprefersATrichregions2011-10-25向光盛长江大学医学院LINEreplicationisnotveryefficientprocessReversetranscriptaseofLINEelementsisa“weak”enzyme(havealowprocessivity)Manyinsertionsaret

30、runcational(copiesarenotabletocopyitselffurther)Mostinsertionsareonly900bp(insteadof6.1kb),only1of100insertionsissuccessful2011-10-25向光盛长江大学医学院Illustrationtofull-sizeLINEsandtheirfossilderivates2011-10-25向光盛长江大学医学院Shortinterspersednuclearelements(SINE)13%ofgenomeNon-autonomous(norevertase)100-400bpl

31、ong;NoopenreadingframesDerivedfromtRNA(transcribedwithRNApolIII,leavinginternalpromoter)Sharesequenceswith3endsofLINEsDependonLINEmachineryforitsmovement2011-10-25向光盛长江大学医学院AluI-elementsDerivedfromsignalrecognitionparticle7SLDoesnotshareits3endwithaLINEInternalpromoterisactive,butrequireappropriatef

32、lankingsequenceforactivationsoitsactiveonlyifluckywithitsintegrationsiteIntegratesinGCrichsequencesOnlyactiveSINEinthehumangenome2011-10-25向光盛长江大学医学院AsALUrepeatsdonothaveopenreadingframes,ALUshavetouseRTenzymeandendonucleaseprovidedbyLINErepeatsorothertransposons2011-10-25向光盛长江大学医学院AfterintegrationA

33、lucopiesrapidlymutateatsitesoftheir24CpGsAlignmentofAlu-subfamilyconsensussequences.2011-10-25向光盛长江大学医学院TheexpansionofAlu-elementsinprimatelineage2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院PotentialAlu-mediateddamagetohumangenomeInsertionalmutagenesisALU-mediatedunevenrecombination2011-10-25向光盛长江大学医学院D

34、iseasesthatsometimescausedbydenovoAlu-integrationNeurofibromatosis(Shwanncelltumors),haemophilia,breastcancer,Apertsyndrome(distortionsoftheheadandfaceandwebbingofthehandsandfeet),cholinesterasedeficiency(congenitalmyasthenicsyndrome)complementdeficiency(hereditaryangioedema)2011-10-25向光盛长江大学医学院Dise

35、asethatsometimescausedbyAlu-mediatedunevenrecombinationinsulin-resistantdiabetestypeII(InsReceptor)LeschNyhansyndrome(overproductionofuricacidleadingtoneurologicsyndrome),TaySachsdisease,complementcomponentC3deficiency,Familialhypercholesterolaemia-thalassaemiaSeveraltypesofcancer,includingEwingsarc

36、oma,breastcancer,acutemyelogenousleukaemia2011-10-25向光盛长江大学医学院PositiveroleofAlurepeatsinevolutionAluAluAluInsertionsoftherepeatneargenemaychangeitsexpressionpattern,genestructure,orleadstoalternativelysplicedmRNAisoformsLTRscontainpromoters,ALUsrepeatscontainTFbindingsites2011-10-25向光盛长江大学医学院Humanre

37、peatdistributiondependsonGCcontentofintegrationsites2011-10-25向光盛长江大学医学院AluparadoxAlurepeatsarefoundinGC-rich(generich)regionsmoreoftenthaninATrich;DenovointegrationofALU-repeatshappensinAT-richareas(astheyhijackedORF2productofLINE)ALUsaresubjectofpositiveselection(astheyCREATEnewgenes)bysupplyingge

38、nomesegmentsreadytobecomegeneswithpromoterlikeelementsandexonic-likeboundaries.AlsotheyareGCrichthemselves,sotheytransformAT-richregionsintoGCrich2011-10-25向光盛长江大学医学院LTRtransposonsAnytrasposonflankedbyLongTerminalRepeats;DNAbasestransposonsandRetrotransposons;ContainTransposase;Alreadysilentinthehum

39、angenomeFossils(CharlieandTiggertypes)EndogenousRetroviralSequences(ERVs)ContainGagandPolgenesOnlyHERV-KlookstillOKformoving2011-10-25向光盛长江大学医学院DNAtransposonsandretrotransposonsKazazian,Science,Vol303,Issue5664LINESINE2011-10-25向光盛长江大学医学院HumanRNAgenes(non-codingRNAtranscripts)3000RNAgenesinhumangeno

40、me(rough)rRNAtRNASmallnuclearRNASmallnucleolarRNASRPRNAMicroRNAAntisenseRNANon-codinggenemRNAisoforms;RNAsformtranscribedpseudogenesTHISISNOTTRUE,MYOPINIONISCLOSETO100,0002011-10-25向光盛长江大学医学院miRNAandantisenseRNAareunderestimated;“othernon-codingRNA”arenotrepresented2011-10-25向光盛长江大学医学院rRNAgenes(1200

41、genes)18S,5.8Sand28Sareencodedbysingletranscriptionunits;Locatedin5clusters:Chr.13,14,15,21,225Sisintandemarrays,largestisonChr.1q41-42Allthisistoincreaseagenedosage2011-10-25向光盛长江大学医学院tRNAgenes(497nucleargenes+324putativepseudogenes)HumanshavefewertRNAgenesthattheworm(584),butmorethanthefly(284);Fr

42、ogX.laevishavethousandsoftRNAgenes;NumberoftRNAgenescorrelateswithsizeoftheoocytes;Inlargeoocyteslotsofproteinneedstobesythethizedsimultaneously.2011-10-25向光盛长江大学医学院49familiesaccordingtocodonrecognition;(Shouldby61foreverycodingtriplet)ParadoxiseliminatedbycodonwobblingVeryroughcorrelationbetweentRN

43、Agenenumberandaminoacidfrequencyintheprotein280outof497genesareonChr.6,mostareclusteredinthesame4Mbregion;otherarealsomoreorlessclustered(Chr.1and7)AllchromosomesstillcarryatleastonetRNAgenechr.22andYareexclusionstRNAgenes(497nucleargenes+324putativepseudogenes)2011-10-25向光盛长江大学医学院Representationofam

44、inoacidsbyhumantRNA(examples)AminoAcidFrequencyNumberoftRNAsAlanine7,06%40Leucine9,95%35Tryptophan1,30%7Valine6,12%44Aspartate4,78%10Cysteine2,25%30Histidine2,56%12Selenocysteinef=5000/3x109settobeonsafeside i.e.,99.9%ofthegenomeisrepresentedatleastonceor,anyparticulargenesegmentispresentwith99.9%pr

45、obabilityThen,N=4.1x106plasmidclonesrequired2011-10-25向光盛长江大学医学院Youllneedlessclones,ifyouraveragesizeofinsertwillbelarger2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院PartialrestrictionwithSau3A4-bpsequenceGATCwithstickyendsPartialdigestionofthisregionofDNAwouldyieldavarietyofoverlappingfragments(blue)20k

46、blong.UseofsuchoverlappingfragmentsincreasestheprobabilitythatallsequencesinthegenomicDNAwillberepresentedinaLambdalibrary.2011-10-25向光盛长江大学医学院Griddedlibraryina96wellplate2011-10-25向光盛长江大学医学院Forhumangenomesequencing:Underlyingmap:YACmapYACsaretoolargeforsequencing,oftenarerearrangedanddifficulttohan

47、dle);Subjecttosequencingmap:BAC/PACmapMostimportantandinterestingregions:weremappedbycosmid-basedmaps2011-10-25向光盛长江大学医学院Yeastartificialchromosome(YAC)YACvectorscapacityis150kb-2Mb.(inyeastcells)TheYACvectorscontain:1)yeastcentromere(CEN)andyeasttelomeres(TEL)2)autonomouslyreplicatingsequence(ARS)=o

48、riginsofreplication3)URA3geneinvolvedinuracilsynthesisforpositiveselectionofyeastcellswithYAC4)Topropagateemptyvectorabacterialreplicationorigin(ORI)andbacterialselectionmarker(Amp)(inURA-hoststrain)ARSURA2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院BAC(BacterialArtificialChromosome)capacity=100-200kb201

49、1-10-25向光盛长江大学医学院CosmidvectorsCosmid=plasmidthatcontainscossite(packagingsignal)ofthelambdaphageVectorsize=5kbVectorcapacity=42-45kbPhage-derivedadvantages:Sizeselection,largecapacityPlasmid-derivedadvantages:Plasmidminipreps,growingasacolony2011-10-25向光盛长江大学医学院2011-10-25向光盛长江大学医学院CosmidmapinPDE4ahu

50、manregion2011-10-25向光盛长江大学医学院Forhumangenomesequencing:YAC,BAC/PACandcosmidmapwereverifiedbySTS-maps:STS-mapsincluded:1)LotsofpolymorphicSTRmarkers(geneticmaps)2)LotsofshortsequencesmappedonRHhybridpanelsoronsomaticcellhybrids3)LotsofESTs(mostlyrepresentinggenes)2011-10-25向光盛长江大学医学院Forhumangenomesequ