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PreparationofHumanChromosomeSpreads  

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Introduction: The46chromosomeslocatedineachsomatic cellcontainallthegeneticmaterialinherited bythatindividual. Locatedinthenucleus,these23pairofhomologouschromosomesare comprisedof22pairofautosomes(nonsexchromosomes)and1pairofsexchromosomes (XXorXY). Thegeneticmaterial,DNA,existswithinthechromosomesandcontainsthe entiregeneticblueprintforthedevelopmentofanindividual. Mostnormalhuman cells containidenticalnumbersandtypesofchromosomes .Theanalysisofhuman chromosomeshasallowedresearchers toidentifythecauseofspecificgeneticdiseasesand abnormalities. Eachchromosomepairco ntain sunique physicalatt ributes,whichdistinguishes themfromthe22otherpairs.Thethreemaincriteriausedtoidentifyindividual chromosomesinclude: 1.thelengthofthechromosome. 2.thepositionofthecentromere. 3.bandingpatterns onthechromosomewhichappearafter stain ing. Usingthesecriteria,geneticistshavesetupaclassificationsystem,whichlabelseach chromosomebynumber.Inakaryotype,chromosomesareplacedinhomologouspairsand arrangedinorderofdescendingsize.AtameetinginDenverin1960,itwasagreedthatthe standardhumankaryotypewouldcontain7groups(A‐‐‐G)basedonchromosomesizeand shape.Theshapeofachromosomeisdefinedprimarilybythepositionofitscentromere: acrocentricchromosomeshavethecentromerenearoneendofthechromatids, metacentricchromosomeshavetheircentromereabouthalfwaydownthechromatids,and submetacentricchromosomeshavetheircentromeresaboutathirdofthewaydownthe chromatids.Thecentromerepositioncanbedefinedmoreexactlybyreferringtothe centromereindexofthechromosome.Thecentromeredividesthesisterchromatidsintoa shortarm(calledtheparm)andalongarm(calledtheqarm),andthecentromereindexis calculatedbydividingthelengthoftheparmbythetotallengthofthechromosometimes 100orC.I.=[p/(p+q)]x100.Thusthecentromereindexforametacentricchromosomeis about45‐‐‐50;forasubmetacentric,about15‐‐‐44;andforanacrocentric,around14and below.Inaddition,acrocentricchromosomesofhumansmayhave,visibleontheendsof theirparms,anarrowstalk(calledasecondaryconstriction)and ablobofchromatin (calledasate llite). Thecomposi tionsof thehumankaryotypegroupsarea sfoll ows:  Group A B C D E F G

Chromosome #'s 1-3 4-5 6-12 and X 13-15 16-18 19-20 21, 22 and Y

Size and Shape Largest, metacentric and submetacentric Large, submetacentric Medium, submetacentric Medium, acrocentric Small, submetacentric Small, metacentric Smallest, acrocentric

 G ‐‐‐bandingisatechnique forproducinglightanddarkbandingpatternson chromosomes.Bandsareproducedbystainingwith Giemsastaina fter pre‐‐‐treatingthe

chromosomeswithtrypsin.EachhomologouschromosomepairhasauniquepatternofG‐‐‐ bands,enablingrecognitionofparticularchromosomessothatkaryotypescanbe interpreted.Figure1showsactualG‐‐‐bandedkaryotypesfromahumanmale(Fig.1a)and female(Fig.1b)andapictorialrepresentationofahumanG‐‐‐bandedkaryotype(Fig.1c). Manygeneticdisordershavebeenassociatedwithalterationsofthechromosomesan individualpossesses. Insomeinstances,piecesofchromosomesmaybetransferred (translocation). Onotheroccasions,piecesofchromosomesmaybreakoffandbelost entirely(deletion).Severalkindsofcancerareassociatedwithchromosomalabnormalities. Anotherpossibilityisthatentirechromosomesmaybelostoraddedtoanindividual’s chromosomearrangement. Someexamplesofgeneticdiseasesandtheirrespective chromosomalaberrationsare: 1.Down’sSyndrome‐‐‐ characterizedbyanextrachromosome#21(trisomy21). 2.CriduChat‐‐‐ characterizedbyadeletionoftheshortarmofchromosome#5. 3.Turner’sSyndrome‐‐‐ characterizedbytheabsenceofoneXchromosome(oneofthe sexchromosomes);thesefemalesonlyhave45chromosomes. Ontheotherhand,therearemanygeneticdiseases,whichresultfromadefectwithina particulargene.Theabnormalgenotypemayresultinanabnormalphenotype.Such defectsmaybemoresubtleandmoredifficulttoanalyze.Recentadvancesinrecombinant DNAtechnologyandgenetics,however,haveallowedresearcherstoidentifyspecific locationsofgenesonchromosomes(Fig.2).Thisinformationisusefulforresearchersfrom aroundtheworldwhoareconstructingageneticmapofthehumangenome.Continued advancementinthisfieldmayultimatelyleadtotheeradicationofdiseasessuchas diabetes ,musculardystrophy,cysticfibrosis,andhundredsmore. Analyzinganindividual’schromosomebyperformingakaryotypecanidentify chromosomalabnormalities,butnotdefectsinthegenes.Akaryotypepreparationallowsa geneticisttoeasilyobservethechromosomesanindividualhasinthenucleiofhis cells.This  isaccomplishedbyusingachemicalcalledcolchicinetostopcellmitosisinthemetaphase stage. Itisduringthisstageofnucleardivisionthatthechromosomesaremostcondensed and,asaresult,visiblewithalightmicroscope. Afterthecellshavebeenarrestedinthis stage,theyarethenplacedintoahypotonicsolution,whichcauseswatertoenterand enlargethecells.Thecellsarethenplacedintoachemicalfixativetomaintain thiscondition. Followingthisprocedure,thecellscanbe“splatted”ontomicroscopeslides, stained,andviewedmicroscopically. Thefinalstepwouldbetophotographthe chromosomesfromonecell,enlargethephotograph,andthencutthechromosomesfrom thephotographandarrangethemonapaperbasedontheirsize,centromerelocation,and bandingpatterns. Theresultingarrangementofthechromosomesiscalledakaryotype. Onepracticalapplicationofkaryotypeanalysisistheearlydetectionofgenetic defectsbyremovingamnioticfluidsurroundingafetusandanalyzingthechromosomal make‐‐‐upoftheunbornchild. Karyotypespreparedonolderindividualsusuallyinvolve theanalysisofchromosomesinlymphocytes. Inthisexercise,ahumantumorcellline,HeLa,isused.Tumorcellsdifferfromtheir normalcounterpartsinmanyrespects:thelackofcontactinhibition(thecontinuetodivide) anddifferentcellstructure(morphology).Theyoftenhavedifferentcell ‐‐‐to‐‐‐cellinteractions, membraneproperties,cytoskeletalstructure,proteinsecretion,andgeneexpressionthat normalnon‐‐‐cancerouscells.TheHeLacelllineoriginatedintheearly1950’sfromthecervical cancercellsofawomannamedHenriettaLacks. Becausethecellsareoftumororigin,they havecontinuallydividedandwilldosoinlabculturesforanindefiniteperiodoftime.

a.MaleG‐‐bandedkaryotype 

b.FemaleG‐‐bandedkaryotype

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c.Pictoralrepresentationof G‐‐bandedkaryotype

Figure1.G‐‐‐bandedkaryotypeofa(a)maleand(b)female.(c)Pictoralrepresentaion ofG‐‐‐band edkaryotype.

Furthermore,thesecellsdonotcontainthenormaldiploidnumberofchromosomesofhuman beings(46). Insteadtheywillpossessachromosomenumbergreaterthanthediploid number,whichisreferredtoasbeinganeuploid.TheHeLa,cellsweregrownincultureand treatedwithcolchicinetoallowforthemicroscopicexaminationofthechromosomes. Colchicine,aplantalkaloid,arreststhecellsinmetaphaseofmitosisbyinterferingwith formationofthemitoticspindlewhichisneededforthemovementofchromosomeduringthe metaphasetoanaphaseprogression.Thisblockageincreasesthefrequencyofmetaphase cells.Metaphasechromosomesaremostreadilyobservedwiththelightmicroscopeand variouschromosomalfeaturessuchassisterchromatidsandcentromeresareevident.In summary,theprocedureforchromosomevisualization(andkaryotyping)entailsarrestinga fractionofalogphasepopulationinmetaphase,treatingthecellswithahypotonicsaline solutiontoswellthecellsandincreasetheirfragility,fixationwithacetica cid‐‐‐me thanol, splatteringontoslides,andstaining.Thisisfollowedbyasearchforidealchromosome spreadsforthestudyofchromosomenumberandstructure. YouwillperformakaryotypeofHeLacells.TheHeLacellshavebeencultured, blockedinmetaphasewithcolchicine,swolleninhypotonicsolution,andfixedinaceticaci d‐ ‐‐methanol(thefixativeinactivatesthehumanpapillomaviruswhichispresentinthecells– thereforethesecellsareharmless).YouwillprepareachromosomespreadfromtheHeLa cellsandthenlocatecomplete,non ‐‐‐over lappingmetaphasechromosomespreadsontheir HeLaslidessoyoucantakedigitizedimagesofthespreadsforanalysis.BecauseHeLacells areextremelyaneuploidandtheirchromosomeshaveundergonemanyrearrangements duringyearsofculture,theirchromosomesarenotsuitableforconstructingakaryotype (whenG‐‐‐bandedtheyarenotrecognizablyhuman).YouwillcompareyourHeLa chromosomespreadwiththenormalhumanchromosomespreadtoidentifythetotal numberofchromosomesandnumbersofmetacentric,submetacentric,andacrocentric chromosomes. 

Safety Notes: Avoidcontactwithstain#1andstain#2duringthelabprocedure(wear gloves).

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Materials: ฀ ฀ ฀ ฀ ฀ ฀ ฀

microscopewithoilimmersionlens transfer pipettes dH 2O microscopeslides andcoverglasses stainingjarscontainingstain#1andstain#2 nylongloves metaphaseblockedcancercells ‐‐‐  CellServkit#4willcontainthecellsinsuspension fixedinanaceticacid‐‐‐methanolfixative

Procedure: 1.Obtainatubecontainingthefixedcells,anduseyourtransferpipettetogently resuspendthem.Removeasmallsampleofthesuspensionwiththepipette.

 2.Positionseveralslidesnexttoeachotherata45degreeangleonthedisposable diaperonyourbench.

 3.Holdthepipette3–4feetabovetheslideand“splat”onedropontotheslideabout 3/4inchfromtheupperendoftheslides. Carefullyapply 10‐‐‐12moredropsfrom ontothesameregionoftheslide.*Itisimportanttoholdthecellsfarfromthe slide.Ifyoudropfromtooclosethecellswillnotlyse(breakopen).

 4.Gentlyblowacrosstheslidefor2‐‐‐3seconds. Thiswillhelpspreadchromosomes fromtherupturedcells. Allowtheslidetoairdrycompletely.

 5.Diptheslideintostain#1foronesecond. Repeatthisone‐twomoretimes.

 6.Removeexcessstainbyblottingtheslideonapapertowel. Diptheslideintostain #2foronesecond. Repeatthisonetotwomoretimes.

 7.Rinsetheslideindistilledwaterandthenallowtheslidetoairdry.

 8.Place1dropofwaterovertheareaoftheslidewhereyoubelievethecellsare located.Nowapplyacoverslipoverthewaterdrop.

 9.Observeyourslideunderlowandhighpowerwithyourmicroscope.

 10.Uselowpoweronyourmicroscopetofindagoodchromosomespreadonyourslide (chromosomesthatappeardistinctandseparate). Addadropofimmersionoilto theslideandswitchtotheoilimmersionlens(100x). Remembertoadjustthelight (morelightisneeded)onthemicroscopewhenusingthehighermagnification.Take apictureofyourchromosomespreadusingyourcellphonecamera.

 11.Trytocountthenumberofchromosomespresentfrom5differentcellsontheslide. Rememberthatthiscelllineisaneuploidandeachcellwillprobablycontaina differentnumberofchromosomes,eachgreaterthanthediploidnumberof46. 

12.Takeapreparedslideofanormalhumanchromosomes.Takeaphotoofanormal humankaryotypefromthisslide(notewhetheritismaleorfemale).Countthe numberofchromosomespresentfromawildtypecell.  13.Inyourlabnotebook,compareyourchromosomespreadfromtheHeLacellstoa chromosomespreadfromanormalhumancell.Thechromosomesaresmall,but therearesomeveryobviousdifferencesbetweenthenormalandHeLacell karyotypes.Somesuggestionsforanalysis:comparetotalnumberofchromosomes, centromereposition(doyouseeanytelocentricchromosomes?).Youcanalsolook upsomepicturesofHeLacellkaryotypesonlinetogetsomeotherinformation.    

Reference‐‐‐ CellServe‐‐‐Preparationofhumanchromosomesspreadskit#4,printed backgroundmaterial,andproceduralinformationandglossaryofterms,references,and furtherreadingsection. 1989CATCMB/TheCatholicUniversityofAmerica.   

Glossary

 acrocentricchromosomes‐‐‐ centromerenearoneendofthechromatids, aneuploid‐‐‐ chromosomenumbergreaterthanthediploidnumber autosomes‐‐‐ nonsexchromosomes colchicine‐‐‐ chemicalusedtostopmitosisinthemetaphasestage.

 karyotype–pictureofanindividualschromosomesorganizedbysize,shape(centromere position)andbandingpattern

 metacentricchromosomes‐‐‐ centromereabouthalfwaydownthechromatids

 sexchromosomes–chromosomesthatdeterminesexofindividual,XandYinhumans

 submetacentricchromosomes‐‐‐ centromeresaboutathirdofthewaydownthe chromatids....