细菌遗传学5单元

chapter13chapteroutline主讲教师：靳全文•13.1Theenormousdiversityofbacteria•13.2Bacterialgenomes•13.3Bacteriaasexperimentalorganisms•13.4Genetransferinbacteria-conjugation•13.5Bacterialgeneticanalysis13.4.2Inconjugation,adonortransfersDNAdirectlytoarecipientTheFplasmidandconjugationConjugationaltransferofchromosomalgenesMappinggenesbygenetransferduringconjugationTheuseofF'episomesforcomplementationstudiesThediscoveryofconjugation(接合)1946,JoshuaLederbergandEdwardTatum.StrainAmet–bio–thr+leu+thi+StrainBmet+bio+thr–leu–thi–met+bio+thr+leu+thi+cellsgrowintocoloniesThediscoveryofconjugation(接合)NogrowthMixtureofAandBForeachsample,washandspread~108cellsontominimalmedium.NogrowthConjugationrequirescell-to-cellcontactMediummovedthroughfilterusingpressureStrainAStrainBAfterincubation,cellsplatedontominimalmedium1950,B.D.DavisTheFplasmidandconjugationConjugationismediatedbyF(fertilityfactor)plasmidindonorstrains.Fplasmid:mediateconjugationandtransfergenes.F+(donor)andF(recipient,lacksFplasmid)strains.Exconjugant(接合后个体)–recipientcellinconjugation.OriginoftransferF+bacteriumFfactor:100kbDNATheprocessofconjugationFpilusbindstoF–cellwallPilusretractsandcellsaredrawntogetherGenetransferAfterconjugation,bothcellsareF+F+F+F+F–F+F+F–F–TheFplasmidoccasionallyintegratesintotheE.colichromosomeHfr(highfrequencyofrecombination)strain:containsanintegratedFplasmidonchromosomesandcantransferhostgenestoarecipientstrainwithhighefficiency.Episomes(附加体):Plasmidsthatcanintegrateintochromosome.FplasmidChromosomeinF+cellIS3IS3ChromosomeinHfrcellTransferintoF–cellIS3IS3IS3xOriginoftransferOriginoftransferDifferentHfrchromosomesRecombinationcanoccurbetweenanyISontheFplasmidandanycorrespondingISonthechromosome.RecombinationcanproduceHfrstrainsthattransfergenesclockwiseorcounterclockwise,dependingontheinitialorientationoftheISelementonthechromosome.F+recipientchromosomeInsertionofFplasmidResultingHfrcellclockwisecounter-clockwiseGenetransferinamatingbetweenHfrdonorsandF-recipientsFpilusconnectswithF–cellSinglestrandofintegratedFplasmidiscut.Hfrchromosomereplicatesitselfastransferproceeds.FplasmidfollowedbychromosomalDNApassesintorecipientcell.DonorDNAisreplicatedinhostcell.F-cellF-cellHfrcellHfrcellGenetransferinamatingbetweenHfrdonorsandF-recipientsCellsseparateCrossoversbetweenhomologousregionsondonorandrecipientDNASomeoftransferredDNAmaybeincorporatedintorecipient'schromosomebyrecombinationRecipientcontainspartofFplasmidandsomeofHfrgenomeplusitsownF–genome.CellremainsF–DNAfragmentsfromdonorandrecipientcellsaredigestedbynucleases.HfrcellHfrcellF-cellF-cellHfrHstrain(strsthr+azirtonrlac+gal+)Fstrain(strrthrazistonslacgal)1956,Wollman-Jacobinterrupted-matingexperimentMappinggenesbygenetransferduringconjugationnonselectivemediumseparateconjugatingcellseveryminwithstreptomycinselectingforstrrwithoutthreonineAzideLactoseGalactoseBacteriophageT1azirlac+gal+tonr半乳糖耐受乳糖耐受叠氮化物不敏感能合成苏氨酸不敏感MappinggenesbygenetransferduringconjugationTimeofgenetransferMapbasedonmatingresultsMinutespriortointerruptionofconjugationMinutesaftermating%cellswithHfrgeneticmarkersamongthr+strrrecombinantsFplasmid:FplasmidthatcarriesapieceofchromosomalDNA.FplasmidformationIntegratedFfactorArarerecombinationeventbetweenregionsoflimitedsequencesimilaritypermitsout-loopingofFfactorincludingtrp+locus.SeparationofFcreatesF'trp+plasmidandachromosomedeletedforthetrpgenes.Hfrchromosome+Fplasmidtransfercangenerateamerodiploid(部分二倍体)F'trp+plasmidtrp+merodiploid(部分二倍体)Althoughbacteriaaremonoploid(单倍体),F’plasmidscarryingbacterialgenescancreatespecificregionsofpartialdiploidy(二倍体).Complementationtesting(互补测验)tellsuswhethertwomutationsareinthesameordifferentgenes.ComplementationNocomplementationm1andm2inthesamegenem1andm2inthedifferentgenesGGRRx♀♂ComplementationtestingComplementationtestingusingF'plasmidsF'trp-xF–trp-yrecA-Merodiploid(部分二倍体)xToanalyzemutationsaffectingtryptophanbiosynthesisFivedifferenttrpgenesinE.coli:A,B,C,D,andE,eachonecorrespondingtooneofthefiveenzymesrequiredfortryptophanbiosynthesis.主讲教师：靳全文