Does DNA Replication Follow the Conservative, Semiconservative, Or Dispersive Model?

Does DNA Replication Follow the Conservative, Semiconservative, or Dispersive Model?

Introduction—TheArticleandPhenomenonUnderStudy

WhenWatsonandCrickpublishedtheirdouble-helixmodelofDNAstructurein1953,theynotedthatthecomplementarypairingofnitrogenousbasessuggested,intheory,apossiblemechanismforreplication.However,inpractice,itwasdifficulttotesttheiridea.IninnovativeandcarefulresearchwithE.colibacteria,investigatorsMeselsonandStahlusedanovelwaytolabelDNAandwereabletoconfirmthemechanismofDNAreplication.Thisresearch,fromthefollowingpaper,isthesubjectofthis week’s discussion:

M.MeselsonandF.W.Stahl,ThereplicationofDNAinEscherichiacoli,ProceedingsoftheNationalAcademyofSciencesUSA44:671–682(1958).

A.AbouttheArticle

1.Statethelastnamesoftheauthorsofthearticleandthenameoftheuniversityatwhichtheyworked.

2.Whatisthenameofthescientificjournalinwhichthearticlewaspublished?Inwhatyearwasitpublished?

3.Thisresearchwasaidedbygrantsfromwhatorganizations?(Seethenotesattheendofthearticle,page682,beforethereferences.)

4.Specializedvocabulary:Writeabriefdefinitionofeachterm.

density-gradientcentrifugation

generationtime

isotope

B.AbouttheStudy

5.Theinvestigators“anticipatedthatalabelwhichimpartstotheDNAmoleculeanincreaseddensity”(Introduction,paragraph1)mighthelprevealhowatomsinparentalDNAaredistributedtoprogenyDNAmolecules.Whatlabeldidtheinvestigatorsusetobeabletodetectsmalldensitydifferencesamongmolecules?

6.Whenbacteriacellsarelysed(brokenopen)andthecontentsareplacedinacesiumchloridemixtureandspuninacentrifuge,thecontentsofthesolutionwillbesortedbydensity.Figure2apresentstheresultsofseparationofDNAcomposedofN14fromDNAcomposedofN15bydensity-gradientcentrifugation.Theleftsideofthephotographisthepartofthecentrifugetubeclosesttotheaxisofrotation(topofthetube),andtherightsideiswherematerialhasspunfarthestfromthecenterofrotationbycentrifugalforce(bottomofthetube).Whichwouldhavegreaterdensity,DNAcomposedofN14orDNAcomposedofN15?Therefore,inaphotographsuchasinFigure2a,whichisthelocationofthehigher-densitymaterialineachsample—totheleftortotheright?

7.DNAabsorbsultravioletlight,soUVlightphotographycanbeusedtomakeDNAvisible.Figure1presents13images,eachaphotographofultravioletabsorption,indicatingthepresenceofDNAatcertainlocationsinthesampleinacentrifugetube.Attime0,beforecentrifugation,thedarkbandcoversnearlytheentirelengthofthesampleinthetube,fromlefttoright.Thismeansthatbeforecentrifugation,theDNAmoleculesarescatteredthroughoutthemixtureofcellcontentsinthecentrifugetubeandhavenotyetbeensortedaccordingtodensity.(a)After10.7hoursofcentrifugation,thedarkbandislimitedtoaboutwhatfractionofthelengthofthecentrifugetube?(b)Afterwhatamountoftime(hours)inthecentrifugedidthebandbegintonarrow?(c)After36.5hoursofcentrifugation,thedarkbandislocatedaboutwhereonthephotograph—attheleft,middle,orright?

8.Figure2showstwoviewsofthesameresults.WhatistherelationshipbetweentheUVabsorptionbandsshowninFigure2aandthelinewithtwopeaksinFigure2b?

9.Intheirexperiment,howdidtheinvestigatorscausethebacteriatoswitchfromincorporatingN15intotheirDNAtogrowingwithonlylightnitrogenisotopes,N14?

10.Basedonthesecondparagraphonp.676,howdoestherelativepositionofabandindicatehowmuchoftheDNAwaslabeledwithN15?

11.(a)FromthelegendofFigure3,whatwastheaveragebacterialgenerationtime(inhours)fortheseexperiments?(b)Abouthowlongwouldittakeforfourgenerations?(c)InFigure3,whatwastheexperimentalchangemadeattime0?

12.ExaminetheresultsofFigure4,inwhichhigherdensityDNAistotheright.(a)WhatisthedensitydifferencebetweentheinitialDNA(0generations)andtheDNAafter1.0generations?(b)HowdidtheDNAdensitydistributionafter1generationcomparetothatafter2.5generations?(c)DNAathighdensity(thefarthestrightband)ispresentatgenerationtimes0,0.3,and0.7.Atwhatothergenerationsisthehigh-densityDNApresent?

13.ThesecondpointoftheDiscussionsection(p.676)statesthateachdaughtermoleculereceivesonesubunitfromtheparentDNA.Whatpatternintheresultssupportsthisstatement?

14.IntheDiscussionsection,theauthorsdrawconnectionsbetweentheirowndensityresultsandtheWatson-CrickproposalforapossibleDNAduplicationmechanism.Inorderforyoutoseetheconnectionsclearly,drawaquicksketchofthreefiguresshowninthispaperthatrepresenttheDNAafter1generation:(a)thesketchfromFigure4,atracingofthepeakafter1.0generation;(b)theschematicrepresentationofthefirst-generationdaughtermoleculesfromFigure5;and(c)thefirst-generationdaughtermoleculesfromFigure6(illustrationofmechanismproposedbyWatsonandCrick).

15.Examineyoursketchesfor(b)and(c)inthepreviousquestion.Ineachsketch,thetwoimagesarethesame.Iftheyweredifferentfromeachother,howwouldtheimageinsketch(a)bedifferent?

16.Summarizethe“structurallyplausiblehypothesis”ofWatsonandCrickfortheduplicationoftheDNAmolecule,asexplainedonp.677.

C.GeneralConclusionsandExtensionsoftheWork

17.Attheendofthepaper,theauthorsstatethatthenitrogenofaDNAmoleculeisdividedequallybetweentwosubunitsandeachdaughtermoleculereceivesoneofthese.(a)WhichcomponentofaDNAnucleotidecontainsnitrogen?(b)HowcouldyoulabelanotherpartoftheDNAandrepeatthesetestswithanothercomponent?

18.Intheintroduction,theauthorspointoutthathypothesesaboutDNAreplicationmakedifferentpredictionsaboutthedistributionofparentalatomsintodaughtermolecules.Today,weoftenrefertothemodelpresentedhereasa“semi-conservative”modelforreplication,becausecomponentsofeachstrandstaytogether(thatis,thestrandisconserved)butthetwostrandsoftheparentmoleculedonotstaytogether.WhatwouldhappeninthedistributionofparentalatomsintodaughtermoleculesifDNAreplicationfolloweda“conservative”modelinwhichtheentireparentDNAmoleculewasconserved?

19.TheMeselson-Stahlexperimentsandthispaperareconsideredimportant,groundbreakingstudiesinmolecularbiology.Decadeslater,theexperimentsarestilladmired,and“thepaperisstillheldaloftforitsclarity”[T.H.Davis,MeselsonandStahl:TheartofDNAreplication,PNAS101:17895–17896(2004).].SomeoftheclarityofthecentrifugationresultswasduetothefactthattheDNAwasfragmentedduringhandling(althoughtheexperimentersdidnotknowitatthetime).AsquotedinDavis(2004),StahlsaidthatpipettingDNAwaslike“throwingspaghettioverNiagaraFalls.”WhenDNAbreaksintofragments,thesugar-phosphatebackboneisbroken,formingmanyshortdoublehelixes.WhydidthefragmentedDNAstillshowthesamepatternofnitrogendensitybanding?

20.Imaginethatyouwereamemberofthisresearchteamandinvolvedintheseexperiments.Whatcouldbeapossiblefollow-uptestthatextendsthiswork?

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