Recombination and Genetic Engineering

8/7/2019 Recombination and Genetic Engineering

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Recombination andRecombination and

Genetic EngineeringGenetic Engineering

MicrobiologyMicrobiology


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Eucaryotic recombinationEucaryotic recombination

RecombinationRecombination process in which one orprocess in which one or

more nucleicacidmore nucleicacidmolecules aremolecules are

rearrangedorcombinedrearrangedorcombinedtoproduce anewtoproduce anewnucleotide sequencenucleotide sequence

In eucaryotes, usuallyIn eucaryotes, usuallyoccurs as the resultofoccurs as the resultofcrossingcrossing--overover duringduringmeiosismeiosis

Figure 13.1


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Bacterial Recombination:Bacterial Recombination:

General PrinciplesGeneral Principles Severaltypes ofrecombinationSeveraltypes ofrecombination

GeneralrecombinationGeneralrecombination canbe reciprocalornonreciprocalcanbe reciprocalornonreciprocal SiteSite--specificrecombinationspecificrecombination Replicative recombinationReplicative recombination


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Reciprocal generalReciprocal general

recombinationrecombination Mostcommontype ofrecombinationMostcommontype ofrecombination

Areciprocal exchange betweenpairAreciprocal exchange betweenpairof homologous chromosomesof homologous chromosomes Results from DNA strandbreakageResults from DNA strandbreakage

andreunion, leadingtocrossingandreunion, leadingtocrossing--overover


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Figure 13.2

ReciprocalReciprocal

generalgeneralrecomrecom--

binationbination


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Figure 13.2


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Nonreciprocal generalNonreciprocal general

recombinationrecombination IncorporationofIncorporationof

single strandofsingle strandofDNAintoDNAintochromosome,chromosome,forminga stretchforminga stretchofofheteroduplexheteroduplexDNADNA

ProposedtooccurProposedtooccurduringbacterialduringbacterialtransformationtransformation

Figure 13.3


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SiteSite--specificspecific

recombinationrecombination Insertionofnonhomologous DNAInsertionofnonhomologous DNA

intoachromosomeintoachromosome oftenoccurs duringviralgenomeoftenoccurs duringviralgenome

integrationinto hostchromosomeintegrationinto hostchromosome enzymes responsible are specificforenzymes responsible are specificfor

virus andits hostvirus andits host


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Site SpecificSite Specific

RecombinationRecombination

Ifthe two sites undergoingIfthe two sites undergoing

recombinationare orientedintherecombinationare orientedinthesame direction, this mayresultinasame direction, this mayresultinadeletiondeletion


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InversionsInversions

Recombinationatinvertedrepeats causesRecombinationatinvertedrepeats causesandinversionandinversion


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Replicative recombinationReplicative recombination

Accompanies replicationofgeneticAccompanies replicationofgeneticmaterialmaterial

Usedbygenetic elements thatmoveUsedbygenetic elements thatmoveaboutthe genomeaboutthe genome


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Horizontal gene transferHorizontal gene transfer

Transferofgenes fromone mature,Transferofgenes fromone mature,independentorganism (donor)toanotherindependentorganism (donor)toanother(recipient)(recipient)

ExogenoteExogenote DNAthatis transferredtorecipientDNAthatis transferredtorecipient EndogenoteEndogenote

genome ofrecipientgenome ofrecipient

MerozyogoteMerozyogote recipientcellthatis temporarilydiploidasrecipientcellthatis temporarilydiploidasresultoftransferprocessresultoftransferprocess


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Bacterial PlasmidsBacterial Plasmids

Small, doubleSmall, double--stranded, usuallycircularstranded, usuallycircularDNAmoleculesDNAmolecules

AreAre repliconsreplicons have theirownoriginofreplicationhave theirownoriginofreplication can existas single copies oras multiple copiescan existas single copies oras multiple copies

CuringCuring eliminationofplasmideliminationofplasmid canbe spontaneous orinducedbytreatmentscanbe spontaneous orinducedbytreatments

thatinhibitplasmidreplicationbutnot hostcellthatinhibitplasmidreplicationbutnot hostcellreproductionreproduction


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Bacterial plasmidsBacterial plasmids

EpisomesEpisomes plasmids thatcan exist either with orplasmids thatcan exist either with or

withoutintegratingintochromosomewithoutintegratingintochromosome Conjugative plasmidsConjugative plasmids

have genes forpilihave genes forpili

cantransfercopies ofthemselves tocantransfercopies ofthemselves tootherbacteriaduringconjugationotherbacteriaduringconjugation


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Fertility FactorsFertility Factors

conjugativeconjugativeplasmidsplasmids

e.g., F factore.g., F factorofof E. coliE. coli

manyare alsomanyare alsoepisomesepisomes

Figure 13.5


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Figure 13.7

mediated by

insertion sequences

(IS)

F plasmid integrationF plasmid integration


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Resistance FactorsResistance Factors

R factors (plasmids)R factors (plasmids) Have genes forresistance toHave genes forresistance to

antibioticsantibiotics Some are conjugativeSome are conjugative usuallydonotintegrate intousuallydonotintegrate into

chromosomechromosome


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Col plasmidsCol plasmids

Encode colicinEncode colicin killskills E. coliE. coli atype ofatype ofbacteriocinbacteriocin

proteinthatdestroys otherbacteria,proteinthatdestroys otherbacteria,usuallycloselyrelated speciesusuallycloselyrelated species

Some are conjugativeSome are conjugative Some carryresistance genesSome carryresistance genes


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Other Types of PlasmidsOther Types of Plasmids

Virulence plasmidsVirulence plasmids carryvirulence genescarryvirulence genes

e.g., genes thatconferresistance to hoste.g., genes thatconferresistance to hostdefense mechanismsdefense mechanisms

e.g., genes that encode toxinse.g., genes that encode toxins MetabolicplasmidsMetabolicplasmids

carrygenes formetabolicprocessescarrygenes formetabolicprocesses e.g., genes encodingdegradative enzymes fore.g., genes encodingdegradative enzymes for

pesticidespesticides e.g., genes fornitrogenfixatione.g., genes fornitrogenfixation


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Transposable ElementsTransposable Elements

TranspositionTransposition the movementofpieces of DNAaroundthe movementofpieces of DNAaround

the genomethe genome Transposable elements (transposons)Transposable elements (transposons)

segments of DNAthatcarrygenes forsegments of DNAthatcarrygenes for

transpositiontransposition Widespreadinbacteria, eucaryotesWidespreadinbacteria, eucaryotes

andarchaeaandarchaea


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Types of transposableTypes of transposable

elementselements Insertion sequencesInsertion sequences (IS elements)(IS elements)

Containonlygenes encoding enzymes requiredContainonlygenes encoding enzymes required

fortranspositionfortransposition TransposaseTransposase Composite transposons( Tn)Composite transposons( Tn)

Carrygenes inadditiontothose neededforCarrygenes inadditiontothose neededfortranspositiontransposition

Conjugative transposonsConjugative transposons Carrytransfergenes inadditiontotranspositionCarrytransfergenes inadditiontotransposition

genesgenes


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IS sequencesIS sequences

Insertion elements areInsertion elements are mobile genetic elementsmobile genetic elements thatthatoccasionallyinsertintochromosomal sequences, oftenoccasionallyinsertintochromosomal sequences, oftendisruptinggenesdisruptinggenes ..

Insertion elements are characterizedbyInsertion elements are characterizedbyinvertedterminalinvertedterminalrepeatsrepeats . These terminalrepeats likelyare recognition. These terminalrepeats likelyare recognitionsites foran enzyme responsible forthe insertion.sites foran enzyme responsible forthe insertion.

Mobilityofthe elementdepends onlyonthe elementMobilityofthe elementdepends onlyonthe elementitself;itis anitself;itis anautonomousautonomous element. Thus, itmustcarrytheelement. Thus, itmustcarrythecodingabilityforthe transposase recognizingthe invertedcodingabilityforthe transposase recognizingthe invertedterminalrepeats.terminalrepeats.

The directrepeats externallyflankingthe invertedThe directrepeats externallyflankingthe invertedrepeats are notpartofthe insertion sequence. Instead,repeats are notpartofthe insertion sequence. Instead,theyare chromosomal sequences thatbecome duplicatedtheyare chromosomal sequences thatbecome duplicateduponinsertion, with one copyat each end;this is calleduponinsertion, with one copyat each end;this is calledtargettarget--site duplicationsite duplication..


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Characteristics of ISCharacteristics of IS

elementselements The majorityof IS elements are between 0.7 andl.8 kbin sizeThe majorityof IS elements are between 0.7 andl.8 kbin size

andthe terminitendtobe l0 to 40 base pairs inlength withandthe terminitendtobe l0 to 40 base pairs inlength withperfectornearlyperfectperfectornearlyperfectrepeatrepeats.s.

These sequences alsotendto have RNAtermination signals asThese sequences alsotendto have RNAtermination signals as

wellas nonsense codons inallthree readingframes andarewellas nonsense codons inallthree readingframes andaretherefore polar.therefore polar. Typicallythey encode one large openreadingframe of 300 toTypicallythey encode one large openreadingframe of 300 to

400 aminoacids andbydefinitionthe protein encodedbythis400 aminoacids andbydefinitionthe protein encodedbythisreadingframe is involvedinthe transposition event.readingframe is involvedinthe transposition event.

Two exceptions tothe size range givenabove shouldbe noted:Two exceptions tothe size range givenabove shouldbe noted:The first, };is 5.7 kbandthe other, IS101, is a scant 0.2 kbinThe first, };is 5.7 kbandthe other, IS101, is a scant 0.2 kbin

size. Although there are exceptions, insertion sequences tendtosize. Although there are exceptions, insertion sequences tendtobe presentina smallnumberofcopies inthe genome.be presentina smallnumberofcopies inthe genome. For example, IS1is presentin 6 tol0 copies inFor example, IS1is presentin 6 tol0 copies in E. coliE. coli

chromosome while IS2 and 3 are typicallypresentinaboutfivechromosome while IS2 and 3 are typicallypresentinaboutfivecopies.copies.


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IS actionsIS actions

Insertion sequences mediate avarietyof DNAInsertion sequences mediate avarietyof DNArearrangements. One ofthe firstrecognitions ofrearrangements. One ofthe firstrecognitions ofthis fact was the involvementofinsertionthis fact was the involvementofinsertion

sequences inthe integrationof F and R plasmidssequences inthe integrationof F and R plasmidsintothe hostchromosome. This eventgives riseintothe hostchromosome. This eventgives risetoHfr strains.toHfr strains.

The initial DNArearrangementmediatedby ISThe initial DNArearrangementmediatedby ISelements is the "insertionalduplication" thatelements is the "insertionalduplication" thattheytendtogenerate atthe site ofinsertion.theytendtogenerate atthe site ofinsertion.

IS1generates an 8 or 9 base pairduplicationIS1generates an 8 or 9 base pairduplicationwhile IS2 generates a 5 base pairduplication.while IS2 generates a 5 base pairduplication.


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TransposonsTransposons As definedabove, aAs definedabove, atransposontransposon is amobile geneticis amobile genetic

ele

me

ntcontaining

additional

ge

nes

unre

late

dto

ele

me

ntcontaining

additional

ge

nes

unre

late

dto

transpositionfunctions. Ingeneral, there are knowntobetranspositionfunctions. Ingeneral, there are knowntobetwogeneralclasses:twogeneralclasses:

Class lor "compoundTns" encode drugresistance genesClass lor "compoundTns" encode drugresistance genesflankedbycopies ofan ISinadirectorindirectrepeat.flankedbycopies ofan ISinadirectorindirectrepeat.Adirectrepeat exists whenthe two sequences at eitherAdirectrepeat exists whenthe two sequences at either

endare orientedinthe same direction while anindirectendare orientedinthe same direction while anindirect(orinverted)repeat exists whentheyare inopposite(orinverted)repeat exists whentheyare inoppositedirections. Inthis class oftransposons, the IS sequencedirections. Inthis class oftransposons, the IS sequencesupplies the transpositionfunction.supplies the transpositionfunction.

The secondclass oftransposons are knownas "complex"The secondclass oftransposons are knownas "complex"

or Class 2. With these, the elementis flankedby shortor Class 2. With these, the elementis flankedby short(30(30--40 bp)indirectrepeats with the genes fordrug40 bp)indirectrepeats with the genes fordrugresistance andtransposition encodedinthe middle (seeresistance andtransposition encodedinthe middle (seefigure ofTn3 belowfigure ofTn3 below).).


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Preferential sites forPreferential sites for

transpositiontransposition Class 1Class 1 GCTNAGCGCTNAGC -- NotATrichNotATrich Sites foundapproximately every100Sites foundapproximately every100

bases inthe E. coligenomebases inthe E. coligenome

Class 2Class 2 ATrich regions are preferable sitesATrich regions are preferable sites Homologyat ends ofregionHomologyat ends ofregion


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The transposition eventThe transposition event

Usuallytransposonreplicated,Usuallytransposonreplicated,remaininginoriginal site, whileremaininginoriginal site, whileduplicate inserts atanother siteduplicate inserts atanother site

Insertiongenerates directrepeatsInsertiongenerates directrepeatsofflanking host DNAofflanking host DNA


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Figure 13.8

IR = inverted repeats


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TnTn33

transtrans--positionposition

Class 2 Transpoison

Complex Transposon


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GenerationGeneration

of directof directrepeatsrepeats


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Effects of transpositionEffects of transposition

MutationincodingregionMutationincodingregion--deletionofgeneticmaterialdeletionofgeneticmaterial

ArrestoftranslationorArrestoftranslationortranscriptiontranscription

ActivationofgenesActivationofgenes Generationofnew plasmidsGenerationofnew plasmids

resistance plasmidsresistance plasmids


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Figure 13.13

The U-tube experiment

demonstrated that

direct cell to cell

contact was

necessary

after incubation,

bacteria plated onminimal media

no prototrophs


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R1plasmid

RTF = resistancetransferfactor

aconjugativeplasmid

sources ofresistance genesare transposons


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Bacterial ConjugationBacterial Conjugation

transferoftransferofDNAbydirectDNAbydirect

celltocellcelltocellcontactcontact discovereddiscovered1946 by1946 by

LederbergandLederbergandTatumTatum


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FF++ xxFF MatingMating

FF++ = donor= donor contains F factorcontains F factor

FF

= recipient= recipient does notcontain F factordoes notcontain F factor F factorreplicatedbyrollingF factorreplicatedbyrolling--circlecircle

mechanismandduplicate is transferredmechanismandduplicate is transferred recipients usuallybecome Frecipients usuallybecome F++ donorremains Fdonorremains F++


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F factorF factor

The F factorcan existinthree different states:The F factorcan existinthree different states: F+ refers toafactorinanautonomous, extrachromosomalF+ refers toafactorinanautonomous, extrachromosomal

state containingonlythe geneticinformationdescribedstate containingonlythe geneticinformationdescribedabove.above.

The "Hfr" (which refers to "high frequencyThe "Hfr" (which refers to "high frequencyrecombination") state describes the situation whentherecombination") state describes the situation whenthefactor has integrateditselfintothe chromosomefactor has integrateditselfintothe chromosomepresumablydue toits various insertion sequences.presumablydue toits various insertion sequences.

The F' or (F prime) state refers tothe factor whenitThe F' or (F prime) state refers tothe factor whenitexists as an extrachromosomal element, but with theexists as an extrachromosomal element, but with the

additionalrequirementthatitcontain some sectionofadditionalrequirementthatitcontain some sectionofchromosomal DNAcovalentlyattachedtoit. A strainchromosomal DNAcovalentlyattachedtoit. A straincontainingno F factoris saidtobe "Fcontainingno F factoris saidtobe "F--".".


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FF++ xxFF matingmating

Inits extrachromosomalInits extrachromosomalstate the factor has astate the factor has amolecular weightofmolecular weightofapproximately 62 kbandapproximately 62 kbandencodes atleast 20 traencodes atleast 20 tragenes. Italsocontainsgenes. Italsocontainsthree copies of IS3, onethree copies of IS3, onecopyof IS2, andone copycopyof IS2, andone copyofa sequence as wellasofa sequence as wellasgenes forincompatibilitygenes forincompatibilityandreplication.andreplication.


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Hfr ConjugationHfr Conjugation

Hfr strainHfr strain donor having F factorintegratedintodonor having F factorintegratedinto

its chromosomeits chromosome both plasmidgenes andchromosomalboth plasmidgenes andchromosomal

genes are transferredgenes are transferred


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Figure 13.14b

HfrHfr xxFF matingmating


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FFdd ConjugationConjugation

FFdd plasmidplasmid formedbyformedby

incorrectincorrectexcisionfromexcisionfromchromosomechromosome

containscontains uu 11genes fromgenes fromchromosomechromosome

FFdd cellcancellcantransfertransfer FFdd

plasmidtoplasmidtorecipientrecipientFigure 13.15a

integrated F factor

chromosomal gene


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FFdd xxFF matingmating


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Tra YTra Y

Characterization of theCharacterization of theEscherichia coli F factor traY geneEscherichia coli F factor traY gene

product and its binding sitesproduct and its binding sites WCNelson, BSMorton, EE LahueWCNelson, BSMorton, EE Lahueand SW Matsonand SW Matson

Departmentof Biology, UniversityofDepartmentof Biology, UniversityofNorth Carolina, ChapelHill 27599.North Carolina, ChapelHill 27599.


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Tra GenesTra Genes

Tra YTra Y gene codes forthe proteinbinds togene codes forthe proteinbinds tothe OriTthe OriT

Initiates the transferofplasmidacrossInitiates the transferofplasmidacrossthe bridge betweenthe twocellsthe bridge betweenthe twocells

Tra ITra I Gene is a helicase responsible forGene is a helicase responsible for

the conjugationthe conjugation strandstrand--specifictransesterificationspecifictransesterification

(relaxase)(relaxase)


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Conjugative ProteinsConjugative Proteins

Keyplayers are the proteins thatKeyplayers are the proteins thatinitiate the physicaltransferofinitiate the physicaltransferof

ssDNA, the conjugative initiatorssDNA, the conjugative initiatorproteinsproteins

Theynickthe DNAandopenittoTheynickthe DNAandopenitto

beginthe transferbeginthe transfer Workinginconjunction with theWorkinginconjunction with the

helicases theyfacilitate thehelicases theyfacilitate the

transferof ss RNAtothe Ftransferof ss RNAtothe F-- cellcell


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DNA TransformationDNA Transformation

Uptake ofnaked DNAmolecule fromUptake ofnaked DNAmolecule fromthe environmentandincorporationthe environmentandincorporationintorecipientina heritable formintorecipientina heritable form

CompetentcellCompetentcell capable oftakingup DNAcapable oftakingup DNA

Maybe importantroute ofgeneticMaybe importantroute ofgeneticexchange innatureexchange innature


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DNA binding

protein

nuclease nicks and degrades one

strand

competence-specific

protein

Streptococcus pneumoniae


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Artificial transformationArtificial transformation

Transformationdone inlaboratoryTransformationdone inlaboratorywith species thatare notnormallywith species thatare notnormallycompetent (competent (E. coliE. coli))

Varietyoftechniques usedtomakeVarietyoftechniques usedtomakecells temporarilycompetentcells temporarilycompetent

calciumchloride treatmentcalciumchloride treatment makes cells more permeable to DNAmakes cells more permeable to DNA


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LysogenicPhage


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LambdaLambda

Inorderforthe lambdaprophage toInorderforthe lambdaprophage toexistina hostexistina host E.E. colicoli cell, itmustcell, itmustintegrate intothe host chromosomeintegrate intothe host chromosomewhich itdoes bymeans ofawhich itdoes bymeans ofasitesite--specific recombination reactionspecific recombination reaction..


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Attachment siteAttachment site

TheThe E.E. colicoli chromosome contains one site at which lambdachromosome contains one site at which lambdaintegrates. The site, locatedbetweentheintegrates. The site, locatedbetweenthe galgalandandbiobiooperons, is calledtheoperons, is calledthe attachment siteattachment site and is designatedand is designatedattBattBsince it is the attachment site onthesince it is the attachment site onthe bbacterialacterial

chromosome.chromosome. The site is only 30 bpin size andcontains aconservedThe site is only 30 bpin size andcontains aconservedcentral15 bpregion where the recombinationreaction willcentral15 bpregion where the recombinationreaction willtake place.take place.

he structure ofthe recombination site was determinedhe structure ofthe recombination site was determinedoriginallybygeneticanalyses andis usuallyrepresentedasoriginallybygeneticanalyses andis usuallyrepresentedasBOB'BOB', where, where BB andandB'B' representthe bacterial DNAonrepresentthe bacterial DNAoneither side ofthe conservedcentral elementeither side ofthe conservedcentral element


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Recombination siteRecombination site

The bacterioThe bacteriopphage recombination sitehage recombination site --attPattP -- is more complex. Itcontains theis more complex. Itcontains theidenticalcentral15 bpregionasidenticalcentral15 bpregionas attBattB..

The overall structure canbe representedThe overall structure canbe representedasas POP'POP'. However, the flanking sequences. However, the flanking sequenceson either side ofon either side ofattPattP are veryimportantare veryimportantsince theycontainthe binding sites forasince theycontainthe binding sites foranumberofotherproteins which arenumberofotherproteins which arerequiredforthe recombinationreaction.requiredforthe recombinationreaction.TheThe PP armis 150 bpinlength andthearmis 150 bpinlength andthe P'P'armis 90 bpinlength.armis 90 bpinlength.


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IntegrationIntegration

Integrationofbacteriophage lambdarequiresIntegrationofbacteriophage lambdarequiresone phageone phage--encodedproteinencodedprotein-- IntInt, which is the, which is theintegraseintegrase -- andone bacterialproteinandone bacterialprotein-- IHFIHF,,wh

ich

iswh

ich

is I

ntegrationI

ntegrationH

ostH

ostF

actorF

actor..

Both ofthese proteins bindto sites ontheBoth ofthese proteins bindto sites onthe PP andandP'P' arms ofarms ofattPattP toformacomplex in which thetoformacomplex in which thecentralconserved15 bp elements ofcentralconserved15 bp elements ofattPattP andandattBattBare properlyaligned.are properlyaligned.

The integrase enzyme carries outalloftheThe integrase enzyme carries outallofthesteps ofthe recombinationreaction, whichsteps ofthe recombinationreaction, whichincludes a short 7 bpbranch migration.includes a short 7 bpbranch migration.


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Enzymes andEnzymes and

RecombinationRecombination There are twomajorgroups of enzymes thatcarryout siteThere are twomajorgroups of enzymes thatcarryout site--

specificrecombinationreactions;one groupspecificrecombinationreactions;one group-- knownas theknownas thetyrosine recombinase familytyrosine recombinase family -- consists ofover140 proteins.consists ofover140 proteins.

These proteins are 300These proteins are 300--400 aminoacids in size, theycontaintwo400 aminoacids in size, theycontaintwoconserved structuraldomains, andtheycarryoutrecombinationconserved structuraldomains, andtheycarryoutrecombinationreactions usingacommonmechanisminvolvingathe formationofreactions usingacommonmechanisminvolvingathe formationofacovalentbond with anactive site tyrosine residue.acovalentbond with anactive site tyrosine residue.

The strand exchange reactioninvolves staggeredcuts thatare 6The strand exchange reactioninvolves staggeredcuts thatare 6to 8 bpapart withinthe recognition sequence.to 8 bpapart withinthe recognition sequence.

Allofthe strandcleavage andreAllofthe strandcleavage andre--joiningreactions proceedjoiningreactions proceedthrough a series oftransesterificationreactions like thosethrough a series oftransesterificationreactions like thosemediatedbytype I topoisomerases.mediatedbytype I topoisomerases.


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Generalized TransductionGeneralized Transduction

Anypartofbacterialgenome canbeAnypartofbacterialgenome canbetransferredtransferred

Occurs duringlyticcycleOccurs duringlyticcycle Duringviralassembly, fragments ofDuringviralassembly, fragments of

host DNAmistakenlypackagedintohost DNAmistakenlypackagedintophage headphage head generalizedtransducingparticlegeneralizedtransducingparticle


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Generalized transductionGeneralized transduction


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Specialized TransductionSpecialized Transduction

alsocalledalsocalledrestrictedtransductionrestrictedtransduction carriedoutonlybytemperate phagescarriedoutonlybytemperate phages

that have establishedthat have establishedlysogenylysogeny only specificportionofbacterialonly specificportionofbacterialgenome is transferredgenome is transferred occurs whenprophage is incorrectlyoccurs whenprophage is incorrectly

excisedexcised


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Figure 13.20

SpecializedSpecialized

transductiontransduction


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Mapping the GenomeMapping the Genome

locatinggenes onanorganismslocatinggenes onanorganismschromosomeschromosomes

mappingbacterialgenesmappingbacterialgenesaccomplishedusingallthree modesaccomplishedusingallthree modesofgene transferofgene transfer


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Hfr mappingHfr mapping

usedtomaprelative locationofbacterialusedtomaprelative locationofbacterialgenesgenes

basedonobservationthatchromosomebasedonobservationthatchromosome

transferoccurs atconstantratetransferoccurs atconstantrate interruptedmating experimentinterruptedmating experiment

Hfr x FHfr x F-- matinginterruptedatvarious intervalsmatinginterruptedatvarious intervals orderandtimingofgene transferdeterminedorderandtimingofgene transferdetermined


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Figure 13.22a

Interrupted matingInterrupted mating


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Figure 13.22b


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E. coliE. coli genetic mapgenetic map

gene locationsgene locationsexpressedinexpressedin

minutes,minutes,reflectingtimereflectingtimetransferredtransferred

made usingmade using

numerous Hfrnumerous Hfrstrainsstrains

Figure 13.23


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Transformation mappingTransformation mapping

usedto establish gene linkageusedto establish gene linkage expressedas frequencyofexpressedas frequencyof

cotransformationcotransformation iftwogenes close together, greateriftwogenes close together, greater

likelihood willbe transferredonlikelihood willbe transferredon

single DNAfragmentsingle DNAfragment


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Generalized transductionGeneralized transduction

mappingmapping usedto establish gene linkageusedto establish gene linkage expressedas frequencyofexpressedas frequencyof

cotransductioncotransduction iftwogenes close together, greateriftwogenes close together, greater

likelihood willbe carriedon singlelikelihood willbe carriedon single

DNAfragmentintransducingDNAfragmentintransducingparticleparticle


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Recombination and GenomeRecombination and Genome

Mapping in VirusesMapping in Viruses

viralgenomes canalsoundergoviralgenomes canalsoundergo

recombination eventsrecombination events viralgenomes canbe mappedbyviralgenomes canbe mappedby

determiningrecombinationfrequenciesdeterminingrecombinationfrequencies physicalmaps ofviralgenomes canalsobephysicalmaps ofviralgenomes canalsobe

constructedusingothertechniquesconstructedusingothertechniques


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Recombination mappingRecombination mapping

recombinationrecombinationfrequencyfrequencydetermineddeterminedwhencellswhencellsinfectedinfectedsimultaneouslysimultaneouslywith twowith twodifferentdifferentvirusesviruses

Figure 13.24


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Physical mapsPhysical maps

heteroduplex mapsheteroduplex maps genomes oftwodifferentvirusesgenomes oftwodifferentviruses

denatured, mixedandallowedtoannealdenatured, mixedandallowedtoanneal regions thatare notidentical, donotregions thatare notidentical, donot

reannealreanneal

allows forlocalizationofmutantallelesallows forlocalizationofmutantalleles


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Physical mapsPhysical maps

restriction endonuclease mappingrestriction endonuclease mapping compare DNAfragments fromtwocompare DNAfragments fromtwo

differentviral strains interms ofdifferentviral strains interms ofelectrophoreticmobilityelectrophoreticmobility sequence mappingsequence mapping

determine nucleotide sequence ofviraldetermine nucleotide sequence ofviral

genomegenome identifycodingregions, mutations, etc.identifycodingregions, mutations, etc.

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Recombination and Genetic Engineering