CellCycles-Introduction

Theonionroottipandthewhitefishblastularemainasthestandardintroductiontothestudyofmitosis.Theonionhaseasilyobservablechromosomes,andthewhitefishhasoneoftheclearestviewsofthespindleapparatus.ThetestisofthegrasshopperandthedevelopingzygoteoftheroundwormAscarisarethetrADItionalmaterialsusedforviewingthevariousstagesofmeiosis.

Inasinglelongitudinalsectionofagrasshoppertestisonecanusuallyfindallofthestagesofmeioticdevelopment.Thestagesarealsoalignedfromonepoleofthetestis.Fewothermeioticsamplesareasconvenient.Formostmaterial,meiosisoccursinamorerandomlydistributedpatternthroughoutthetestis.

Ascarisisutilizedtoobservethefinalstagesofdevelopmentineggs(oogenesis).TheAscariseggliesdormantuntilfertilized.Itthencompletesmeiosisformingtwopolarbodieswhilethespermnucleusawaitsfushionwiththefemalenucleus.WhenthisphenomenoniscoupledtothelargeabundanceofeggsintheAscarisbody,itmakesanidealspecimenforobservingtheeventsoffertilization,polarbodyformation,fusionofpronucleiandthesubsequentdivisionofthecell(cytokinesis).

InterphaseG1-S-G2

Thestagesofmitosiswereoriginallydetailedaftercarefulanalysisoffixedcells.Morerecently,timelapsephotographycoupledwithphasecontrastmicroscopyhasallowedustovisualizetheprocessinitsentirety,revealingadynamicstateofflux.

Inearlywork,somuchemphasiswasplacedonthemovementofthechromosomesthatthecellwasconsideredtobe"atrest"whennotinmitosis.Assignificantasmitoticdivisionis,itrepresentsonlyasmallfractionofthelifespanofacell.Nonetheless,youmaystillcomeacrosstheterm"restingphase"insomeoldertexts.Thistermisrarelyusedtoday,andtheterminterphaseissufficientforallactivitiesbetweentwomitoticdivisions.Thecellishighlyactiveduringinterphaseandmostofthemetabolicandgeneticfunctionsofthecellarereducedduringthephysicaldivisionofthenuclearandcellularmaterials(mitosis).

Figure11.1presentsourcurrentviewofacellcycles.Notethatinterphaseisdividedintothreesub-phases,G1,SandG2.ThebasisforthisdivisionisthesynthesisofDNA.Notealsothatwhiletheentirecyclemaybeaslongas24hours,mitosisisnormallylessthanonehourinlength.

BecauseofthesynthesisofDNAininterphase,theamountofDNApernucleusisdifferentdependingonwhichsubphaseofinterphasethecellisin.DNAcanbemeasuredusingthefuelgenreactionandamicroscpectrophotometer.ThebasicamountofDNAinahaploidnucleusisgiventhevalueC.Adiploidnucleuswouldbe2C.Atriploidandtetraploidcellwouldbe3Cand4Crespectivesly.

However,whennucleiareactuallymeasured,diploidcellsininterphasecanbedividedintothreegroups;someare2C,someare4Cwhileafewareatintermediatevaluesbetween2Cand4C.Theconclusionisthatthegeneticmaterial(DNA)andpresumablythechromosomesmustduplicate.TheperiodwithininterphaseandduringwhichDNAissynthesizedistermedtheSphase(forSynthesis).TheperiodofinterphaseprecedingtheSphaseistheG1phase(for1stGrowthPhase),whiletheperiodsubsequenttotheSphaseistheG2phase(forthe2ndGrowthPhase).DuringtheG1period,thecellisgenerallyincreasinginsizeandproteincontent.DuringS,thecellreplicatesthechromosomesandsynthesizesDNA.DuringG2,itcontinuestoincreaseinsize,butalsobeginstobuildasignificantpoolofATPandotherhighenergyphosphates,whicharebelievedtobeasignificantpartofthetriggeringmechanismforthesubsequentkaryokineticandcytokineticeventsofmitosis.

Mitosisreturnsthecellstothe2Cstate.MeiosisreducestheamountofDNAevenfurther,to1C.Meanwhilethenumberofchromosomes(designatedwiththeletterN)isalsochanging.Foradiploidcell,thenumberofchromosomesistwicethatofahaploid,or2N.Duringmitosis,adiploidcellwouldgofromone2Ncelltotwo2Ncells.Sincethedaughtercellshavethesamechromosomenumberastheparent,mitosisisalsoreferredtoasequationaldivision.Ifadiploid(2N)cellundergoesmeiosis,itwillresultinfourhaploidcells,each1N.Thus,meiosisisalsoreferredtoasreductionaldivision.RefertoFigure11.2forcomparisonofCandNvaluesduringdivision.

Figure11.1CellCycle

ItispossIBLetovisualizetheprocessofDNAsynthesiswithineithernucleiorchromosomesbytheincorporationofaradioactiveprecursortoDNAintocellsandsubsequentdetectionbyautoradiography.Incorporationofthymidine,aDNAprecursor,willonlyoccurduringtheSphase,andnotduringG1norG2.Ifapulse(shortperiodofexposure)ofH-thymidineispresentedtocells,thosethatareintheSperiodwillincorporatethisradioactivesubstance,whileallotherswillnot.CarefulapplicationofthepulsewillallowthetimingoftheSphase.Byknowingthetimingfortheentirecycle(frommitosistomitosis),onecandeducetheG1andG2periods.

Figure11.2AmountofDNApresentduringdivision

Meioticdivisiondiffersfrommitosisinthattherearetwodivisioncyclesinsteadonone.Inthefirstcycle,interphaseisthesameasformitosis.ThatisthereisanSphasewithcorrespondingG1andG2.Duringthesecondinterphase,however,noDNAsynthesisoccurs.ConsequentlythereisnoG1orG2inthesecondinterphase.TheresultisthatchromosomesarereplicatedpriortoMeiosis,anddonotreplicateagainduringmeiosis.

ForthefollowingdetailsofMitosis,referto

Figure11.3StagesofMitosisinOnion

Prophase

Thefirstphaseofmitosisismarkedbytheearlycondensationofthechromosomesintovisiblestructures.Atfirst,thechromatidsarebarelyvisible,butastheycontinuetocoil,thechromosomesbecomethickerandshorter.Thenuclearenvelopeisstillpresentduringthisstage,asareanynucleolarstructures.Thecentriolesaremovingtothepolesofthecellandspindlefibersarejustbeginningtoform.

Metaphase

Duringthemiddlephaseofkaryokinesis,thechromosomeslineupinthecenterofthecell,andformametaphaseplate.Viewedonedge,thechromosomesappeartobealignedacrosstheentirecell,butviewedfrom90Degreestheyappeartobespreadthroughouttheentirecell(visualizeaplatefromitsedgeorfromabove).Eachchromosomehasaclearprimaryconstriction,thecentromere,andattachedtoeachisadefinitivespindlefiber.Thespindleapparatusiscompletelyformed,andthecentrioleshavereachedtheirrespectivepoles.Thenucleolusandthenuclearenvelopehavedisappeared.

Anaphase

Themovementphasebeginspreciselyasthetwohalvesofachromosome,thechromatids,separateandbeginmovingtotheoppositepoles.Thecentromerewillleadthewayinthisprocess,andthechromatidsformaVwiththecentromerespointingtowardtherespectivepoles.

Telophase

Thelastphaseisidentifiedbytheaggregationofthechromatids(nowknownaschromosomes)attherespectivepoles.Duringthisphase,thechromosomesuncoil,thenuclearenvelopeisresynthesized,thespindleapparatusisdismantledandthenucleolusbeginstoappear.

Meiosis

Formeiosis,thephasesprophase,metaphase,anaphaseandtelophaseareidentified,butbecausetherearetwodivisions,therearetwosets.ThesearedesignatedbyRomannumberals;thusProphaseI,MetaphaseI,AnaphaseI,TelophaseI,Interphase,ProphaseII,MetaphaseII,AnaphaseIIandTelophaseII.InterphaseisnormallynotdesignatedwithaRomannumberal.BecauseofthesignificanceofthechromosomepairingwhichoccursinProphaseI,itisfurthersuBDividedintostages.Forthefollowingdescriptionsofmeiosis,referto

ThephasesofProphaseIarenamedfortheappearanceofathread-likestructure,knownasnema.Leptonemameans"thinthread"andleptoteneistheadjectiveappliedtothetermstage,i.e.properterminologyistheleptotenestageofprophaseI.Thewordstageisoftenomitted.

ProphaseI:Leptotene1

Thisstageismarkedbythefirstappearanceofthechromosomeswhenthechromosomesareintheirmostextendedform(exceptforduringinterphase).Theyappeartobeastringwithbeads.Thebeadsareknownaschromomeres.Thechromatidshavealreadyreplicatedpriortothisphase,buttypically,thereplicatedchromatidscannotbeobservedduringtheleptotenestage.

ProphaseI:Zygotene

Zygosmeans"yoked,"andduringthisstage,thehomologouschromosomesareseenaspairedunits.Thechromosomesareshorterandthickerthaninleptotene,andinsomecellstheyremainattachedtothenuclearenvelopeatthepointsneartheaster.Thisgivesrisetoanimagetermedthe"bouquet."Thisattachmentisrareininvertebratesandabsentinplants,wherethechromosomesappeartobeatangledmass.

ProphaseI:Pachytene

Whenthepairingofzygoteneiscomplete,thechromosomesappearas"thick"strings,orpachynema.Thechromsomesareabout1/4thelengththeywereinleptotene,andthereareobviouslytwochromosomes,eachwithtwochromatidsineachbundle.Thetwochromosomesarereferredtoasa"bivalent,"whilethesamestructureviewedasfourchromatidsisknownasa"tetrad."

ProphaseI:Diplotene

Thisstageresultsasthegapbetweenthetwohomologouschromosomeswidens.Thehomologshavealreadypairedduringzygotene,recombinedduringpachyteneandarenowbeginningtorepeleachother.Duringthisstage,thechromosomesofsomespeciesuncoilsomewhat,reversingthenormaldirectiontypicalofprophase.Asthechromosomesseparate,theyareobservedtoremainattachedatpointsknownas"chiasmata."Thesearebelievedtobethelocationswheregeneticrecombinationofthegeneshastakenplace.

ProphaseI:Diakinesis

ProphaseIendsasthehomologscompletelyrepeleachother.Thechromosomeswillcontinuetocoiltightly(reversingtheslightuncoilingofthediplotene)andwillreachtheirgreateststateofcontraction.Asdiakinesisprogresses,chiasmataappeartomovetowardtheendsofthechromosomes,aprocessknownas"terminalization."Sincethisstageistheendofprophase,thenucleolususuallydisappears,alongwiththenuclearenvelope.

MetaphaseI

Thetetradsmovetowardthecenterandlineuponametaphaseplate.Thenuclearenvelopecompletelydisappears.Asthetetradsalignthemselvesinthemiddleofthecell,theyattachtospindlefibersinauniquemanner.Thecentromeresofagivenhomologwillattachtothespindlesfromonlyonepole.

AnaphaseI

Theuniqueeventoccurringatthisphaseistheseparationofthehomologs.Incontrasttomitoticanaphase,thecentromeresofagivenhomologdonotdivide,andconsequentlyeachhomologmovestowardoppositepoles.Thisresultsinahalvingofthenumberofchromosomes,andisthebasisofthereductiondivisionthatcharacterizesmeiosis.

TelophaseI:Interphase

Telophaseinmeiosisissimilartothatofmitosis,exceptthatinmanyspecies,thechromosomesdonotcompletelyuncoil.Ifthechromosomesdouncoilandenterabriefinterphase,thereisnoreplicationofthechromatids.RememberthatthechromatidshavealreadybeenreplicatedpriortoProphaseI.

ProphaseII-TelophaseII

Thesephasesareessentiallyidenticalinmeioticandmitoticdivision:theonlydistinctionisthatthechromosomenumberishalfofthenumberthecellhadpriortomeiosis.Eachchromosome(homolog)iscomposedoftwochromatids,andduringAnaphaseII,thetwochromatidsofeachchromosomemoveapartandbecomeseparatechromosomes.2

Thereisashiftintheterminologyappliedtotheseunits.Whilethetwochromatidsremainattachedatthecentromere,theyareknownaschromatids.Immediatelyuponseparating,eachchromatidbecomesknownasachromosomeandisnolongerreferredtoasachromatid.Thisisthereasonthatacellcandivideonechromosome(withtwochromatids)intotwocells,eachwithachromosome-thetermappliedtothechromatidischanged.

Damageinducedduringdivision

In1949,LevandevelopedwhatwastobecomeknownastheAlliumtestforchromosomedamage.Growingrootsfromonionbulbsweresoakedinvariousagentsandanalyzedfortheireffectonmitosis.Itwasdiscoveredthatcaffeine,forexample,causedcompleteinhibitionofmitosis,primarilythroughtheinhibitionofcellplateformation.

ThistestwaslaterusedextensivelybyB.A.Kihlmanandextendedtootherhigherplants.Kihlmanfoundthat1to24hourtreatmentsofcellswithcaffeineandrelatedoxypurinesnotonlyinhibitedmitosis,butalsoinducedsignficantchromosomealterations(aberrations).Specifically,thistreatmentinduced"stickiness"and"pseudochiasmata."Stickinessistheclumpingofchromosomesatmetaphaseandtheformationofchromatinbridgesatanaphase.Pseudochiasmataistheformationofside-armbridgesduringanaphase.Caffeinealsocausestheformationofotherchromosomeandchromatidbreaksandexchanges.

Colchicine,adrugwhichinhibitsspindlefiberformationduringmitosis,canbeaddedtothegrowingcellstohaltcelldivisionatmetaphase.Thisoftenwillresultinadoublingofthechromosomenumber,sincecolchicinetypicallyinhibitscytokinesis,butnotkaryokinesis.Thedoubledchromosomeswillfusewithinasinglenucleus,thusincreasingtheploidyvalueofthenucleus.

Moreover,methylatedoxypurines(caffeine,theophylline,8-ethoxycaffeine)areinhibitorsofcellplateformation.Treatmentwiththeseagentsfor0.5-1hour,withconcentrationsaslowas0.02-0.04%,resultsinthecell"sfailuretoundergocytokinesis;inaddition,thenucleidonotfuseintoasingleunit.Thus,treatmentwithanyoftheseagentsshouldresultinbinucleateormultinucleatecells.

Someantibiotics(azaserine,mitomycinCandstreptonigrin)havebeenknowntohavechromosome-breakingproperties,usuallyassociatedwithG1orG2ofInterphase.G1inhibitionwouldresultinchromosomebreaks,whileG2inhibitionwouldresultinchromatidbreaks.

Inaddition,alkylatingagentssuchas(Di(2-chloroethyl)methylamineornitrogenmustard,Di(2.3-epoxypropyl)ether(DEPE)and-Propiolactone(BPL)),Nitrosocompounds(N-Nitroso-N-methylurethan(NMU)),N-Methyl-phenyl-nitrosamine(MPNA),N-hydroxylphenylnitrosamine-ammonium(cupferron)and1-Methyl-3-nitrosoguanidine(MNNG)haveallbeenindicatedaspotentchromosome-breakingagents.Othercompoundshaveincludedsuchthingsasmaleichydrazide,potassiumcyanide,hydroxylamine,anddyessuchasacridineorangeinvisiblelight.

Thedamagesinvolveabnormalmetaphases,isochromatidbreaks,chromatidexchangesandanaphasebridgestonameafew.