Gene-X-ppt(Gene10-基因十)--Chapter05

Chapter5TheContentoftheGenome5.1Introduction•genome–Thecompletesetofsequencesinthegeneticmaterialofanorganism.–ItincludesthesequenceofeachchromosomeplusanyDNAinorganelles.•transcriptome–ThecompletesetofRNAspresentinacell,tissue,ororganism.–ItscomplexityisduemostlytomRNAs,butitalsoincludesnoncodingRNAs.5.1Introduction•proteome–Thecompletesetofproteinsthatisexpressedbytheentiregenome.–Thetermissometimesusedtodescribethecomplementofproteinsexpressedbyacellatanyonetime.•interactome–Thecompletesetofproteincomplexes/protein–proteininteractionspresentinacell,tissue,ororganism.5.2GenomesCanBeMappedatSeveralLevelsofResolution•Linkage(genetic)mapsarebasedonthefrequencyofrecombinationbetweengeneticmarkers•Restrictionmapsarebasedonthephysicaldistancesbetweenmarkers.•Molecularcharacterizationofmutationscanbeusedtoreconcilelinkagemapswithphysicalrestrictionmaps.5.3IndividualGenomesShowExtensiveVariation•Polymorphismmaybedetectedatthephenotypiclevelwhenasequenceaffectsgenefunction,attherestrictionfragmentlevelwhenitaffectsarestrictionenzymetargetsite,andatthesequencelevelbydirectanalysisofDNA.•Theallelesofageneshowextensivepolymorphismatthesequencelevel,butmanysequencechangesdonotaffectfunction.FIGURE01:Apointmutationthataffectsarestrictionsiteisdetectedbyadifferenceinrestrictionfragments5.3IndividualGenomesShowExtensiveVariation•restrictionfragmentlengthpolymorphism(RFLP)–Inheriteddifferencesinsitesforrestrictionenzymes(forexample,causedbybasechangesinthetargetsite)thatresultindifferencesinthelengthsofthefragmentsproducedbycleavagewiththerelevantrestrictionenzyme.–Theyareusedforgeneticmappingtolinkthegenomedirectlytoaconventionalgeneticmarker.5.3IndividualGenomesShowExtensiveVariationFIGURE03:Arestrictionpolymorphismcanbeusedasageneticmarkertomeasurerecombinationdistancefromaphenotypicmarker(suchaseyecolor)FIGURE04:Ifarestrictionmarkerisassociatedwithaphenotypiccharacteristic,therestrictionsitemustbelocatednearthegeneresponsibleforthephenotype5.3IndividualGenomesShowExtensiveVariation•singlenucleotidepolymorphism(SNP)–Apolymorphism(variationinsequencebetweenindividuals)causedbyachangeinasinglenucleotide.–SNPsareresponsibleformostofthegeneticvariationbetweenindividuals.5.4RFLPsandSNPsCanBeUsedforGeneticMapping•RFLPsandSNPscanbethebasisforlinkagemapsandareusefulforestablishingparent–offspringrelationships.•haplotype–Theparticularcombinationofallelesinadefinedregionofsomechromosome;ineffect,thegenotypeinminiature.–Originallyusedtodescribecombinationsofmajorhistocompatibilitycomplex(MHC)alleles,itnowmaybeusedtodescribeparticularcombinationsofRFLPs,SNPs,orothermarkers.5.4RFLPsandSNPsCanBeUsedforGeneticMapping•DNAfingerprinting–Atechniqueforanalyzingthedifferencesbetweenindividualsofthefragmentsgeneratedbyusingrestrictionenzymestocleaveregionsthatcontainshortrepeatedsequences,orbyPCR.–Thelengthsoftherepeatedregionsareuniquetoeveryindividual.–Thepresenceofaparticularsubsetinanytwoindividualscanbeusedtodefinetheircommoninheritance(e.g.,aparent–childrelationship).5.5EukaryoticGenomesContainBothNonrepetitiveandRepetitiveDNASequences•ThekineticsofDNAreassociationafteragenomehasbeendenatureddistinguishsequencesbytheirfrequencyofrepetitioninthegenome.•PolypeptidesaregenerallycodedbysequencesinnonrepetitiveDNA.•Largergenomeswithinataxonomicgroupdonotcontainmoregenes,buthavelargeamountsofrepetitiveDNA.5.5EukaryoticGenomesContainBothNonrepetitiveandRepetitiveDNASequences•AlargepartofrepetitiveDNAmaybemadeupoftransposons.FIGURE05:Theproportionsofdifferentsequencecomponentsvaryineukaryoticgenomes5.6EukaryoticProtein-CodingGenesCanBeIdentifiedbytheConservationofExons•Conservationofexonscanbeusedasthebasisforidentifyingcodingregionsbyidentifyingfragmentswhosesequencesarepresentinmultipleorganisms.•zooblot–TheuseofSouthernblottingtotesttheabilityofaDNAprobefromonespeciestohybridizewiththeDNAfromthegenomesofavarietyofotherspecies.FIGURE07:CharacterizingtheDuchennemusculardystrophygene5.6EukaryoticProtein-CodingGenesCanBeIdentifiedbytheConservationofExons•HumandiseasegenesareidentifiedbymappingandsequencingDNAofpatientstofinddifferencesfromnormalDNAthataregeneticallylinkedtothedisease.•chromosomalwalk–Atechniqueforlocatingagenebyusingthemostcloselylinkedmarkersasaprobeforageneticlibrary.FIGURE06:IdentifyingthegeneinvolvedinDuchennemusculardystrophy5.6EukaryoticProtein-CodingGenesCanBeIdentifiedbytheConservationofExons•exontrapping–Insertingagenomicfragmentintoavectorwhosefunctiondependsontheprovisionofsplicingjunctionsbythefragment.5.7TheConservationofGenomeOrganizationHelpstoIdentifyGenes•Methodsforidentifyingactivegenesarenotperfectandmanycorrectionsmustbemadetopreliminaryestimates.•Pseudogenesmustbedistinguishedfromactivegenes.FIGURE09:Aseriesofexonsmustgenerateanopenreadingframewithappropriateinitiationandterminationcodons5.7TheConservationofGenomeOrganizationHelpstoIdentifyGenes•reversetranscriptionpolymerasechainreaction(RT-PCR)–AtechniqueforthedetectionandquantificationofexpressionofagenebyreversetranscriptionandamplificationofRNAsfroma