19 an introduction to genetic analysis.docx
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19 an introduction to genetic analysis.docx
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19anintroductiontogeneticanalysis
Chapter19
MechanismsofRecombination
KeyConcepts
RecombinationoccursatregionsofhomologybetweenchromosomesthroughthebreakageandreunionofDNAmolecules.
Modelsforrecombination,suchastheHollidaymodel,involvethecreationofaheteroduplexbranch,orcrossbridge,thatcanmigrateandthesubsequentsplicingoftheintermediatestructuretoyielddifferenttypesofrecombinantDNAmolecules.
Recombinationmodelscanbeappliedtoexplaingeneticcrosses.
Manyoftheenzymesparticipatinginrecombinationinbacteriahavebeenidentified.
Introduction
Throughoutouranalysisoflinkage,westudiedtherecombinationofgenesbycrossing-over.Inthischapter,weconsidermolecularmechanismsforgeneratingrecombinationbycrossing-over.Figure19-1depictsabasiccrossoverevent,inwhichtwohomologousmoleculesarealignedandsubsequentlyundergorecombination.WhenBenzer'sworkandthatofothersrevealedthatrecombinationoccurswithingenes,itbecameevidentthatrecombinationhadtobeveryprecise,becauseevensingle-base-pairerrorscoulddisrupttheintegrityofthegene.Howcanrecognitionofhomologouschromosomesandrecombinationeventsbesoprecise?
Theanswerliesinthepowerofbase-paircomplementarity.Weshallseehowbase-paircomplementarityandtheformationofheteroduplexregionsbetweencomplementaryregionsofhomologouschromosomesleadtotherecombinationeventsthatwehavebeenstudying.
BreakageandreunionofDNAmolecules
TheexperimentsdiscussedinChapter5providegoodindirectevidenceinfavorofbreakageandreunion.Oneofthefirstdirectproofsthatchromosomes(inthiscase,viralchromosomes)canbreakandrejoincamefromexperimentsonλphagedonein1961byMatthewMeselsonandJeanWeigle.TheysimultaneouslyinfectedE.coliwithtwostrainsofλ.Onestrain,whichhadthegeneticmarkerscandmiatoneendofthechromosome,was“heavy”becausethephageswereproducedfromcellsgrowninheavyisotopesofcarbon(13C)andnitrogen(15N).Theotherstrainwasc+
14mi+forthemarkersandhad“light”DNAbecauseitwasharvestedfromcellsgrownonthenormallightisotopes12Cand14N.ThetwoDNAs(chromosomes)canberepresentedasshowninFigure19-2a.Themultiplyinfectedcellswerethenincubatedinalightmediumuntiltheylysed.
Theprogenyphagesreleasedfromthecellswerespuninacesiumchloridedensitygradient.Awidebandwasobtained,indicatingthattheviralDNAsrangedindensityfromtheheavyparentalvaluetothelightparentalvalue,withagreatmanyintermediatedensities(Figure19-2b).Interestingly,somerecombinantphageswererecoveredwithdensityvaluesveryclosetotheheavyparentalvalue.Theywereofgenotypec
14mi+,andtheymusthavearisenthroughanexchangeeventbetweenthetwomarkers(Figure19-2c).Theheavydensityofthechromosomewouldbeexpectedbecauseonlythesmalltipofthechromosomecarryingthemi+allelewouldcomefromthelightparentalchromosome.Inthereciprocalcrossofheavyc+mi+phagestolightcmi,theheavyrecombinantswerefoundtobec+mi,asexpected.Theseresultscanbeexplainedinonlyoneway:
therecombinationeventmusthaveoccurredthroughthephysicalbreakageandreunionofDNA.Althoughwehavetobecarefulaboutextrapolatingfromviraltoeukaryoticchromosomes,thisevidenceshowsthatthebreakageandreunionofDNAstrandsdoesoccur.
Chiasmata:
thecrossoverpoints
InChapter5,wemadethesimpleassumptionthatchiasmataaretheactualsitesofcrossovers.Mappinganalysisindirectlysupportsthisidea:
becauseanaverageofonecrossoverpermeiosisproduces50geneticmapunits,thereshouldbecorrelationbetweenthesizeofthegeneticmapofachromosomeandtheobservedmeannumberofchiasmatapermeiosis.Thecorrelationhasbeenmadeinwell-mappedorganisms.
However,theharlequinchromosome-stainingtechnique(seeChapter8)hasmadeitpossibletotesttheideadirectly.In1978,C.TeaseandG.H.Jonespreparedharlequinchromosomesinmeiosesofthelocust.Rememberthattheharlequintechniqueproducessisterchromatids:
onedarkandtheotherlight.Whenacrossoveroccurs,itcanbebetweentwodark,twolight,ornonsisterdarkandlightchromatids,asshowninFigure8-16.Thislastsituationiscrucialbecausemixed(partdarkandpartlight)crossoverchromatidsareproduced.TeaseandJonesfoundthatthedark–lighttransitionisrightatthechiasma—provingthatthechiasmataarethecrossoversitesandsettlingaquestionthathadbeenunresolvedsincetheearly1900s(Figure19-3).
Geneticresultsleadingtorecombinationmodels
Tetradanalysisinfilamentousfungi,suchasNeurosporacrassa,whereallfourproductsofasinglemeiosiscanberecoveredandexamined(seeChapter6),providedtheimpetusforthefirstmodelsofintragenicrecombination.Thesecrucialfindings,reviewedinthefollowinglist,weregeneconversion,evidenceforpostmeioticsegregationofgene-conversionevents,polarity,andtheassociationofgeneconversionwithcrossing-over.
1.Geneconversion.DeparturesfromthepredictedMendelian4:
4segregationratiosaredetectableinsomeasci(0.1–1.0percentinfilamentousfungi,butashighas4percentinyeast).Figure19-4givesthemostcommonaberrantratiosobtained.Itappearsasthoughsomeallelesinthecrosshavebeen“converted”intotheoppositealleles(Figure19-5).Theprocessthereforehasbecomeknownasgeneconversion;itcanoccuronlywherethereisheterozygosityfortwodifferentallelesofagene.Inasciwitha6:
2or2:
6ratio,oneentirechromatidofachromosomeseemstohaveconverted.Inasciwitha5:
3or3:
5ratio,onlyhalfachromatidseemstohaveconverted.Here,differentmembersofasporepairhavedifferentgenotypes.Recallthateachsporepairisproducedbymitosisfromasingleproductofmeiosis.The5:
3or3:
5ratioscanbeexplainedonlybythetwostrandsofthedoublehelixcarryinginformationfortwoMdifferentallelesattheconclusionofmeiosis.Thenextmitoticdivisionisthereforeapostmeioticsegregationofalleles.
Conversioncannotbeexplainedbymutation,becausetheallelethatisconvertedalwayschangesintotheotherspecificalleletakingpartinthecross,nottosomeotheralleleknownforthelocusbutnotapartofthecross.
2.Polarity.Ingenesforwhichaccurateallelemapsareavailable,wecancomparetheconversionfrequenciesofallelesatvariouspositionswithinthegene.Inalmosteverycase,thesitesclosertooneendshowhigherfrequenciesthandothesitesfartherawayfromthatend.Inotherwords,thereisagradient,orpolarity,ofconversionfrequenciesalongthegene(Figure19-6).
3.Conversionandcrossing-over.Inheteroalleliccrosseswherethelocusunderstudyiscloselyflankedbyothergeneticallymarkedloci,theconversioneventisveryoften(about50percentofthetime)accompaniedbyanexchangeinoneoftheflankingregions.Thisexchangenearlyalwaystakesplaceonthesidenearertheallelethathasconvertedandalmostalwaysincludesthechromatidinwhichconversionhasoccurred.
Forexample,considerthechromatidsdiagrammedinFigure19-6.Supposethatthepolarityissuchthattheallelestowardtheleftendofthechromatidconvertmoreoftenthanthosetowardtherightend.Thecrossdiagrammedhereisbetweena+m2b+andam1b,wherem1andm2aredifferentallelesofthemlocusandaandbrepresentcloselylinkedflankingmarkers.Ifwelookatasciinwhichconversionhasoccurredatthem1site(themostfrequentkindofconversioninthislocus),wefindthathalfoftheseasciwillalsohaveacrossoverinregionIandhalfwillhavenocrossover.Inthesmallernumberofascishowinggeneconversionatthem2site,halfwillalsohaveacrossoverinregionIIandhalfwillhavenocrossover.SucheventsaredetectedinascusgenotypesliketheoneshowninFigure19-7,whichcanbeinterpretedasaconversionofm1→+,accompaniedbyacrossoverinregionI.
Insomeasci,asingleconversioneventseemstoincludeseveralsitesatonce.Inaheteroalleliccross,thiseventiscalledaco-conversion(Figure19-8).Thefrequencyofco-conversionincreasesasthedistancebetweenallelesdecreases.
Hollidaymodel
OneofthefirstplausiblemodelstoaccountfortheprecedingobservationswasformulatedbyRobinHolliday.ThekeyfeaturesoftheHollidaymodelaretheformationofheteroduplexDNA;thecreationofacrossbridge;itsmigrationalongthetwoheteroduplexstrands,termedbranchmigration;theoccurrenceofmismatchrepair;andthesubsequentresolution,orsplicing,oftheintermediatestructuretoyielddifferenttypesofrecombinantmolecules.ThemodelisdepictedinFigure19-9.
EnzymaticcleavageandthecreationofheteroduplexDNA
LookingatFigure19-9a,wecanseethattwohomologousdoublehelicesarealigned,althoughnotethattheyhavebeenrotatedsothatthebottomstrandofthefirsthelixhasthesamepolarityasthetopstrandofthesecondhelix(5′→3′inthiscase).Thenanucleasecleavesthetwostrandsthathavethesamepolarity(Figure19-9b).Thefreeendsleavetheiroriginalcomplementarystrandsandundergohydrogenbondingwiththecomplementarystrandsinthehomologousdoublehelix(Figure19-9c).LigationproducesthestructureshowninFigure19-9d.Thispartiallyheteroduplexdoublehelixisacrucialintermediateinrecombination,andhasbeentermedtheHollidaystructure.
Branchmigration
TheHollidaystructurecreatesacrossbridge,orbranch,thatcanmove,ormigrate,alongtheheteroduplex(Figure19-9dande).ThisphenomenonofbranchmigrationisadistinctivepropertyoftheHollidaystructure.Figure1
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