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Clearly,there must be a still higher level of folding, even in interphase chromosomes.Aithough its molecular basis is still largely a mystery, this higher-order packaging almost certainly involves the folding of the 30-nm fiber into a seriesof loopsand coils. This chromatin packing is fluid, frequently changing in response tothe needs of the cell.We shall begin by describing some unusual interphase chromosomes thatcan be easily visualized, inasmuch as certain features of these exceptional casesare thought to be representativeof all interphase chromosomes.
Moreover, theyFigure4-53 Schematicillustrationofcell memory stored as chromatin-basedepigenetic information in the genesofeucaryotes.Genesin eucaryoticcellscanbe packagedinto a largevarietYofdifferentchromatinstructures,indicatedhere by differentcolors.At leastsomeofthesechromatinstructureshavea specialeffecton gene expressionthat can bedirectlyinheritedasePigeneticThisinformationwhen a celldivides.allowssomeof the generegulatoryproteinsthat createdifferentgene statesto act only once,inasmuchasthe statecan be rememberedafter the regulatoryprotein is gone.Epigeneticinformationcan alsobe storedin networksofthat controlgenesignalingmoleculesexpression(seeFigure7-86).234Chapter4: DNA,Chromosomes,and GenomesFigure 4-54 Lampbrushchromosomes.(A)A light micrographof lampbrushchromosomesin an amphibianoocyte.Earlyin oocytedifferentiation,eachchromosomereplicatesto beginmeiosis,and the homologousreplicatedchromosomespairto form this highlyextendedstructurecontaininga total offour replicatedDNA molecures,orchromatids.The lampbrushchromosomestagepersistsfor monthsor years,whilethe oocytebuildsup a supplyofmaterialsrequiredfor its ultimatedevelopmentinto a new individual.(B)An enlargedregionof a similarchromosome,stainedwith a fluorescentreagentthat makesthe loopsactiveinRNAsynthesisclearlyvisible.(CourtesyofJosephG.Gall.)1 0 0p m20pmprovide a unique means for investigating some fundamental aspects of chromatin structure raised in the previous section.
Next we describe how a typicalinterphase chromosome is arranged in the cell nucleus, focusing on humancells. Finally, we conclude by discussing the additional tenfold comlpaction thatinterphase chromosomes undergo during the process of mitosis.ChromosomesAreFoldedinto LargeLoopsof ChromatinInsight into the structure of the chromosomes in interphase cells has beenobtained from studies of the stiff and extended meiotically paired chromosomesin growing amphibian oocytes (immature eggs).These rr"ry unnsual lampbrushchromosomes (the largest chromosomes knor,r,n)are cleaily visible even in thelight microscope, where they are seen to be organized into a seriesof large chromatin loops emanating from a linear chromosomal axis (Figure +-s4).The organization of a lampbrush chromosome is illustrated in Figure 4-55.A gi_venloop always contains the same DNA sequence, and it remains extendedin the same manner as th9 ooclte grows.
Theie chromosomes are producinglarge amounts of RNA for the ooclte, and most of the genes present in the DNAloops are being actively expressed.The majority of ttrJ DNA, however, is not inloops but remains highly condensed in the chromomeres on the axis, wheregenes are generally not expressed.It is thought that the interphase chromosomes of all eucaryotesare similarlyarranged in loops. Although these loops are normally too smail and fragile to b!easily observed in a light microscope, other methods can be used to infer theirpresence. For example, it has become possible to assessthe frequencvwithi''",,,:,": :: ''f35'OFCHROMOSOMESTHEGLOBALSTRUCTUREFigure4-55 A model for the structureofa lampbrush chromosome.The set ofin manYlampbrushchromosomesamohibianscontainsa total of about10,000chromatinloops,althoughmostofthe DNAin eachchromosomeremainshighlycondensedin the chromomeres.to a particularDNAEachloop correspondsFourcopiesof eachloop aresequence.presentin eachcell,sinceeachofthe twoofmajorunitsshownat the top consiststwo closelyapposed,newly replicatedThisfour-strandedchromosomes.of this stageofstructureis characteristicdevelopmentof the oocyte,the diploteneseeFigure21-9.stageof meiosis;REGIONOFSINGLECHROMOSOMEextendedchromatinin loopOFS M A L LR E G I O NCHROMOSOMESHOWINGSISTERCHROMATIDSchromatinJornrngadjacentchromomerescnromomeref o r m e df r o mh i g h l yc o n d e n s e dchromatinwhich any two Ioci along an interphase chromosome are paired with eachother, thus revealing Iikely candidates for the sites on chromatin that form theclosely apposed bases of loop structures (Figure 4-56).
These experiments andothers suggestthat the DNA in human chromosomes is organized into loops ofDNA-bindingprorernSt-:, tiIIt-.+TREATWITHFORMALDEHYDEREMOVECROSS-LINKSBY HEATTREATMENTAND PROTEOLYSIST E S TF O RJ O I N E DS E G M E N TBSYPCRDNA product is obtainedonly if proteins hold thetwo DNA sequencesclosetogether in the cellIn thistechnique,knownasFigure4-56 A methodfor determiningthe positionof loopsin interphasechromosomes.the indicatedcovalentcreatetoformaldehydewith(:C)aretreatedcellsmethod,.uptur"the chromosomeconformationthat chopsthe DNAinto manyThe DNAisthen treatedwith a restrictionnucleaseDNA-proteinand DNA-DNAcross-links,ends"(seeFigure8-34)'Theand formingsetsof identical"cohesivepieces,cuttingat strictlydefinednucleotidesequenceslmportantly,priorto the ligationstepbase-pairin9.cohesiveendscan be madeto join throughtheir complementary(throughcrossshown,the DNAis dilutedso that the fragmentsthat havebeenkept in closeproximityto eachotherand the newlyligatedfragmentsof DNAarearereversedthe cross-linkslinking)arethe onesmost likelyto join.
Finally,describedin Chapter8).By combiningthe frequency-ofchainreaction,identiiiedand quantifiedby pCR(the polymeiasestructuralmodelscan beinformationgeneratedby the 3Ctechniquewith DNAsequenceinformation,associationof chromosomes.producedfor the interphaseconformation236Chapter4: DNA,Chromosomes,and Genomesl o o p e dd o m a i nfolded30-nmfiberIhistonem o d i f y i n ge n z y m e schromatinr e m o d e l i n gc o m p l e x e sRNA polymerasep r o t e i n sf o r m i n g c h r o m o s o m es c a f f o l dFigure4-57 A model for the organizationof an interphasechromosome.A sectionof an interphasechromosomeisshownfoldedinto a seriesof loopeddomains,eachcontainingperhaps50,000-2O0,OOOnucleotidepairsof double-helicalDNAcondensedinto a 30-nmfiber.Thechromatinin eachindividualloop isfurthercondensedthroughpoorlyunderstoodfoldingprocessesthat are reversedwhen the cellrequiresdirectaccessto the DNApackagedin the lJop.Neitherthecompositionof the postulatedchromosomalaxisnor how the folded30-nmfiber is anchoredto it is clear.However,rnmitoticchromosomesthe basesof the chromosomalloopsareenrichedboth in condensinsand in DNAtopoisomerasellenzymes,two proteinsthat mayform muchof the axisat metaphase(seeFigure4-74).different lengths.
A typical loop might contain between 50,000 and 200,000nucleotide pairs of DNA, although loops of a million nucleotide pairs have alsobeen suggested (Figure 4-SZ).PolyteneChromosomesAre UniquelyUsefulfor VisualizingChromatinStructurescertain giant insect cells have grown to their enormous size through multiplecycles of DNA slmthesis without cell division. such cells with more thin the nbr-chromosome.pairs, while a thick band may contain 200,000 nucleotide pairs in each of itschromatin strands. The chromatin in each band appears dark because the DNAis more condensed than the DNA in interbands; it may also contain a higher proportion of proteins (Figure 4-59).There are approximately 3700 bands and 3700 interbands in the completeset of Dro sophila p olltene chromosomes. The bands can be recognized by theirdifferent thicknesses and spacings, and each one has been given a number togenerate a chromosome "map" that has been indexed to the finished genomesequence of this fly.The Drosophila polytene chromosomes provide a good starting point forexamining how chromatin is organized on a large scale.In the previous section,we saw that there are many forms of chromatin, each of which contains nucleosomes with a different combination of modified histones.
By reading this histone code, specific sets of non-histone proteins assembleon tire nucleosomes to'.''THEGLOBALSTRUCTUREOFCHROMOSOMESFigure4-58 The entire set of polytenechromosomes in one Drosophilasalivarycell.In this drawingof a lightthe giantchromosomesmicrograph,havebeen spreadout for viewing bythem againsta microscopesquashingslide.Drosophilahasfour chromosomes,and there arefour differentchromosomepairspresent.Buteachchromosomeistightly pairedwith its homolog (sothateachpairappearsasa singlestructure),whichis not true in mostnuclei(exceptEachchromosomehasin meiosis).undergonemultipleroundsofand the homologuesand allreplication,theirduolicateshaveremainedin exactregisterwith eachother,resultinginhugechromatincablesmanyDNAstrandsthick.The four polytenechromosomesarenormallylinkedtogetherbyregionsneartheirheterochromaticcentromeresthat aggregateto createasinglelargechromocenter(pinkregion).In this preparation,however,thehasbeensplitinto twochromocenterhalvesby the squashingprocedureused.(Adaptedfrom T.S.Painter,J.
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