微生物学 双语教学Chapter 8 Microbial genetics.ppt

Microbialgenetics,Chapter8,8.1MutationsandMutants8.2GeneticRecombination8.3GeneticTransformation8.4Transduction8.5Conjugation8.6Plasmids8.7TransposonsandInsertionSequences8.8ComparativeProkaryoticGenomics8.9GeneticsinEukaryoticMicroorganisms,Microorganismsproviderelativelysimplesystemsforstudyinggeneticphenomenaandarethususefultoolsinattemptstodecipherthemechanismsunderlyingthegeneticsofallorganisms.Microorganismsareusedfortheisolationandduplicationofspecificgenesfromotherorganisms,atechniquecalledmolecularcloning.Inmolecularcloning,genesaremanipulatedandplacedinamicroorganismwheretheycanbeinducedtoincreaseinnumber.,Section1.Mutationandrecombination,Mutationisaninheritedchangeinthebasesequenceofthenucleicacidcomprisingthegenomeofanorganism.Mutationusuallybringsaboutonlyaverysmallamountofgeneticchangeinacell.,Geneticrecombinationistheprocessbywhichgeneticelementscontainedintwoseparategenomesarebroughttogetherinoneunit.Thismechanismmayenabletheorganismtocarryoutsomenewfunctionandresultinadaptationtochangingenvironments.Geneticrecombinationusuallyinvolvesmuchlargerchanges.Entiregenes,setsofgenes,orevenwholechromosomes,aretransferredbetweenorganisms.,Section2.Techniquesofbacterialgenetics:invivo,Invivo:manipulatethegeneticmaterialwithintheorganism,GeneticTransformationTransductionConjugation,Section3.Techniquesofbacterialgenetics:invitro,Invitro:operategeneticmaterialinthetesttube,RestrictionEnzymesMolecularCloningAmplifyingDNA:PCR,AuxotrophanorganismthathasdevelopedanutritionalrequirementthroughmutationCloningvectorgeneticelementintowhichgenescanberecombinedandreplicatedConjugationtransferofgenesfromoneprokaryoticcelltoanotherbyamechanisminvolvingcell-to-cellcontactandaplasmidDiploidaeukaryoticcellororganismcontainingtwosetsofchromosomesElectroporationtheuseofanelectricpulsetoinducecellstotakeupfreeDNAGenedisruptionuseofgenetictechniquestoinactivateagenebyinsertingwithinitaDNAfragmentcontaininganeasilyselectablemarker.Theinsertedfragmentiscalledacassette,andtheprocessofinsertion,cassettemutagenesis,WORKINGGLOSSARY,GeneticmapthearrangementofgenesonachromosomeGenomethetotalcomplementofgenesofacelloravirusGenotypetheprecisegeneticmakeupofanorganismHybridizationformationofaduplexnucleicacidmoleculewithstrandsderivedfromdifferentsourcesbycomplementarybasepairingMolecularcloningisolationandincorporationofafragmentofDNAintoavectorwhereitcanbereplicatedHaploidacellororganismthathasonlyonesetofchromosomesMutagensagentsthatcausemutationMutantanorganismwhosegenomecarriesamutationMutationaninheritablechangeinthebasesequenceofthegenomeofanorganism,NucleicacidprobeastrandofnucleicacidthatcanbelabeledandusedtohybridizetoacomplementarymoleculefrommixtureofothernucleicacidsPhenotypetheobservablecharacteristicsofanorganismPlasmidanextrachromosomalgeneticelementthathasnoextracellularformPointmutationamutationthatinvolvesoneoronlyaveryfewbasepairsPolymerasechainreaction(PCR)amethodusedtoamplifyaspecificDNAsequenceinvitrobyrepeatedcyclesofsynthesisusingspecificprimersandDNApolymeraseRecombinationtheprocessbywhichpartsoralloftheDNAmoleculesfromtwoseparatesourcesareexchangedorbroughttogetherintoasingleunit.,Restrictionenzymeanenzymethatrecognizesandmakesdouble-strandedbreaksatspecificDNAsequencesShotguncloningmakingagenelibrarybyclosingrandomDNAfragmentsSite-directedmutagenesisatechniquewherebyagenewithaspecificmutationcanbeconstructedinvitroSyntheticDNAaDNAmoleculemadebyachemicalprocessinalaboratoryTransductiontransferofhostgenesfromonecelltoanotherbyavirusTransformationtransferofbacterialgenesinvolvingfreeDNATransposonatypeoftransposableelementthatcarriesgenesinadditiontothoseinvolvedintransposition,GeneticRecombination,Geneticrecombinationinvolvesthephysicalexchangeofgeneticmaterialbetweengeneticelements.,HomologousrecombinationresultsingeneticexchangebetweenhomologousDNAsequencesfromtwodifferentsources.Thistypeofrecombinationisextremelyimportanttoallorganisms.However,itisalsoverycomplex.EveninthebacteriumEscherichiacolithereareatleast25genesinvolved.,Asimplifiedversionofonemolecularmechanismofrecombination.HomologousDNAmoleculespairandexchangeDNAsegments.Themechanisminvolvesbreakageandreunionofpairedsegments.Twooftheproteinsinvolved,asingle-strandedbinding(SSB)proteinandtheRecAprotein.Notethattherearetwopossibleoutcomes,dependingonwhichstrandsarecutduringtheresolutionprocess.Inoneoutcometherecombinantmoleculeshavepatches,whereasintheotherthetwoparentalmoleculesappeartohavebeencutandthensplicedtogether.,InordertodetectphysicalexchangeofDNAsegments,thecellsresultingfromrecombinationmustbephenotypicallydifferentfromtheparents.,DetectionofRecombination,Strainsthatlacksomeselectablecharacteristicthattherecombinantswillpossess.Forinstance,therecipientmaynotbeabletogrowonaparticularmedium,andgeneticrecombinantsareselectedthatcan.,Variouskindsofselectableandnonselectablemarkers(suchasdrugresistance,nutritionalrequirements,andsoon)maybeused.,Complementation,Thiscanbedeterminedbyatypeofexperimentcalledacomplementationtest.Complementationwasfirstusedindiploideukaryoticorganisms,InprokaryotesgeneticrecombinationisobservedbecausefragmentsofhomologousDNAfromadonorchromosomearetransferredtoarecipientcellbyoneofthreeprocesses:transformation,whichinvolvesdonorDNAfreeintheenvironmenttransduction,inwhichthedonorDNAtransferismediatedbyavirusconjugation,inwhichthetransferinvolvescell-to-cellcontact,GeneticTransformation,(1)Transformation,whichinvolvesdonorDNAfreeintheenvironment,(2)Transduction,inwhichthedonorDNAtransferismediatedbyavirus,(3)Conjugation,inwhichthetransferinvolvescell-to-cellcontactandaconjugativeplasmidinthedonorcell,ThreemainprocessesofgeneticrecombinationinprokaryotesfragmentsofhomologousDNAfromadonorchromosomearetransferredtoarecipientcell,Conjugation,Transformation,Transduction,CompetenceAcellthatisabletotakeupamoleculeofDNAandbetransformedissaidtobecompetent.,Transformation,BindingoffreeDNAbyamembrane-boundDNAbindingprotein.(b)Passageofoneofthetwostrandsintothecellwhilenucleaseactivitydegradestheotherstrand.(c)Thesinglestrandinthecellisboundbyspecificproteins,andrecombinationwithhomologousregionsofthebacterialchromosomemediatedbyRecAproteinoccurs.,TheintroductionofDNAintocellsbymixingtheDNAandthecell,Transformedcell,Themechanismofbacterialtransformation,Transductioninvolvestransferofhostgenesfromonebacteriumtoanotherbyviruses.Ingeneralizedtransduction,defectivevirusparticlesrandomlyincorporatefragmentsofthecellschromosomalDNA;virtuallyanygeneofthedonorcanbetransferred,buttheefficiencyislow.Inspecializedtransduction,theDNAofatemperatevirusexcisesincorrectlyandbringsadjacenthostgenesalongwithit;onlygenesclosetotheintegrationpointofthevirusaretransferred,buttheefficiencymaybehigh.,Concept,Transduction,Intransduction,DNAistransferredfromcelltocellthroughtheagencyofviruses.Genetictransferofhostgenesbyvirusescanoccurintwoways.,GeneralizedtransductionAndSpecializedtransduction,Specializedtransduction:occursonlyinsometemperateviruses;DNAfromaspecificregionofthehostchromosomeisintegrateddirectlyintothevirusgenome-usuallyreplacingsomeofthevirusgenes.,Generalizedtransduction:hostDNAderivedfromvirtuallyanyportionofthehostgenomebecomesapartoftheDNAofthematurevirusparticleinplaceofthevirusgenome.,Generalizedtransduction,Ingeneralizedtransduction,virtuallyanygeneticmarkercanbetransferredfromdonortorecipient,Duringalyticinfection,theenzymesresponsibleforpackagingviralDNAintothebacteriophagesometimesaccidentallypackagehostDNA.ThisDNAcannotreplicate,itcanundergogeneticrecombinationwiththeDNAofthenewhost.,theDNAoflambdaisinsertedintothehostDNAatthesiteadjacenttothegalactosegenes,Oninduction,Underrareconditions,thephagegenomeisexcisedincorrectly,AportionofhostDNAisexchangedforphageDNA,calledlambdadgal(dgalmeansdefectivegalactose“),Phagesynthesisiscompleted,Celllysesandreleasesdefectivephagecapableoftransducinggalactosegenes,SpecializedTransduction,Geneticrecombinationinvolvesthephysicalexchangeofgeneticmaterialbetweengeneticelements.ItresultsingeneticexchangebetweenhomologousDNAsequencesfromtwodifferentsources.,Homologousrecombinationisextremelyimportanttoallorganisms.However,itisalsoverycomplex.,GeneticRecombination,Directcontactbetweentwoconjugatingbacteriaisfirstmadeviaapilus.ThecellsarethendrawntogetherfortheactualtransferofDNA.NotetheF-specificbacteriophagesonthepilus,Bacterialconjugation(mating)isaprocessofgenetictransferthatinvolvescell-to-cellcontact.,conjugation,Conjugationinvolvesadonorcell,whichcontainsaparticulartypeofconjugativeplasmid,andarecipientcell,whichdoesnot.,Thegenesthatcontrolconjugationarecontainedinthetraregionoftheplasmid(seeSection9.8inyourtext).Manygenesinthetraregionhavetodowiththesynthesisofasurfacestructure,thesexpilus.Onlydonorcellshavethesepili,Thepilimakespecificcontactwithareceptorontherecipientandthenretract,pullingthetwocellstogether.Thecontactsbetweenthedonorandrecipientcellsthenbecomestabilized,probablyfromfusionoftheoutermembranes,andtheDNAisthentransferredfromonecelltoanother.,MechanismofDNATransferDuringConjugation,AmechanismofDNAsynthesisincertainbacteriophages,calledrollingcirclereplication,waspresentedheretoexplainsDNAtransferduringconjugation.,iftheDNAofthedonorislabeled,somelabeledDNAistransferredtotherecipientbutonlyasinglelabeledstrandistransferred.Therefore,attheendoftheprocess,bothdonorandrecipientpossesscompletelyformedplasmids.,DNATransferinBacteria,transformation,transduction,conjugation,(1)Transformation,whichinvolvesdonorDNAfreeintheenvironment,(2)Transduction,inwhichthedonorDNAtransferismediatedbyavirus,(3)Conjugation,inwhichthetransferinvolvescell-to-cellcontactandaconjugativeplasmidinthedonorcell,ThreemainprocessesofgeneticrecombinationinprokaryotesfragmentsofhomologousDNAfromadonorchromosomearetransferredtoarecipientcell,Conjugation,Transformation,Transduction,Transformation,Anumberofprokaryoteshavebeenfoundtobenaturallytransformable,includingcertainspeciesofbothgram-negativeandgram-positiveBacteriaandsomespeciesofArchaea.However,evenwithintransformablegenera,onlycertainstrainsorspeciesaretransformable,CompetenceAcellthatisabletotakeupamoleculeofDNAandbetransformedissaidtobecompetent.Competenceinmostnaturallytransformablebacteriaisregulated,andspecialproteinsplayaroleintheuptakeandprocessingofDNA.Thesecompetence-specificproteinsmayincludeamembrane-associatedDNAbindingprotein,acellwallautolysin,andvariousnucleases.,CompetentcellsbindmuchmoreDNAthandononcompetentcellsasmuchas1000timesmore,BindingoffreeDNAbyamembrane-boundDNAbindingprotein.(b)Passageofoneofthetwostrandsintothecellwhilenucleaseactivitydegradestheotherstrand.(c)Thesinglestrandinthecellisboundbyspecificproteins,andrecombinationwithhomologousregionsofthebacterialchromosomemediatedbyRecAproteinoccurs.,TheintroductionofDNAintocellsbymixingtheDNAandthecell,Transformedcell,Themechanismofbacterialtransformation,ArtificiallyInducedCompetenceHighefficiencynaturaltransformationisfoundonlyinafewbacteria;Azotobacter,Bacillus,Streptococcus,forexample,areeasilytransformed.Manyprokaryotesaretransformedonlypoorlyornotatallundernaturalconditions.Determinationofhowtoinducecompetenceinsuchbacteriamayinvolveconsiderableempiricalstudy,withvariationinculturemedium,temperature,andotherfactors,whenE.coliistreatedwithhighconcentrationsofcalciumionsandthenstoredinthecold,thetransformationbyplasmidDNAisrelativelyefficient.,DNATransferbyElectroporationforartificialinductionofcompetencearebeingsupplantedbyanewmethodtermedelectroporation.Smallporesareproducedinthemembranesofcellsexposedtopulsedelectricfields.WhenDNAmoleculesarepresentoutsidethecellsduringtheelectricpulse,theycanthenenterthecellsthroughthesepores.Thisprocessiscalledelectroporation.,TransfectionBacteriacanbetransformedwithDNAextractedfromabacterialvirusratherthanfromanotherbacterium,aprocessknownastransfection.,Intransduction,DNAistransferredfromcelltocellthroughtheagencyofviruses.Genetictransferofhostgenesbyvirusescanoccurintwoways.,GeneralizedtransductionAndSpecializedtransduction,Transduction,Transductioninvolvestransferofhostgenesfromonebacteriumtoanotherbyviruses.Ingeneralizedtransduction,defectivevirusparticlesrandomlyincorporatefragmentsofthecellschromosomalDNA;virtuallyanygeneofthedonorcanbetransferred,buttheefficiencyislow.Inspecializedtransduction,theDNAofatemperatevirusexcisesincorrectlyandbringsadjacenthostgenesalongwithit;onlygenesclosetotheintegrationpointofthevirusaretransferred,buttheefficiencymaybehigh.,Concept:,Specializedtransduction:occursonlyinsometemperateviruses;DNAfromaspecificregionofthehostchromosomeisintegrateddirectlyintothevirusgenome-usuallyreplacingsomeofthevirusgenes.,Generalizedtransduction:hostDNAderivedfromvirtuallyanyportionofthehostgenomebecomesapartoftheDNAofthematurevirusparticleinplaceofthevirusgenome.,Transductionhasbeenfoundtooccurinavarietyofprokaryotes,includingcertainspeciesoftheBacteria:Desulfovibrio,Escherichia,Pseudomonas,Rhodococcus,Rhodobacter,Salmonella,Staphylococcus,andXanthobacter,aswellasthearchaeanMethanobacteriumthermoautotrophicum.,Notallphagescanbetransducerandnotallbacteriaaretransducible,Generalizedtransduction,Ingeneralizedtransduction,virtuallyanygeneticmarkercanbetransferredfromdonortorecipient,Duringalyticinfection,theenzymesresponsibleforpackagingviralDNAintothebacteriophagesometimesaccidentallypackagehostDNA.ThisDNAcannotreplicate,itcanundergogeneticrecombinationwiththeDNAofthenewhost.,Generalizedtransduction,theDNAoflambdaisinsertedintothehostDNAatthesiteadjacenttothegalactosegenes,Oninduction,Underrareconditions,thephagegenomeisexcisedincorrectly,AportionofhostDNAisexchangedforphageDNA,calledlambdadg