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We will see in Chapter 6 that the initiation of transcription-the firststep in gene expression-requires the assembly of over 100 proteins, and itmakes sensethat this would occur most rapidly in regions of the nucleus particularly rich in these proteins.More generally,it is clear that the nucleus is very heterogeneous,with functionally different regions to which portions of chromosomes can move as theyare subjected to different biochemical processes-such as when their geneexpression changes (Figure 4-66). There is evidence that some of these nuclearregions are marked with different inositol phospholipids, reminiscent of the waythat the same lipids are used to distinguish different membranes in the cytoplasm (seeFigure 13-11).
But what these lipids are attached to in the interior ofthe nucleus is a mystery as the onlyknown lipid-rich environments are the lipidbilayers of the nuclear envelope.5[m-r'-ffin u c l e a re n v e l o p ehomologouschromosomesdetected by hybridizationtechniques(B)G E N EO F F.-G E N EO NFigure4-65 An effect of high levelsofgene expressionon the intranuclearlocation of chromatin.(A) Fluorescencemicrographsof humannucleishowinghow the positionof a genechangeswhen it becomeshighlytranscribed.Theregionof the chromosomeadjacenttothe gene (red)is seento leaveitschromosomalterritory(green)only whenit is highlyactive.(B)Schematicrepresentationof a largeloop ofchromatinthat expandswhen the geneison,and contractswhen the geneis offOthergenesthat arelessactivelyexpressedcan be shownby the samemethodsto remaininsidetheirchromosomalterritorywhen transcribed.(FromJ.R.Chubb and W.A.Bickmore,Cel/112:403-406,2003.With permissionfromElsevier.)241THEGLOBALSTRUCTUREOFCHROMOSOMESnuclearneighborhoodf o r g e n es i l e n c i n gnuclearneighborhoodf o r g e n ee x p r e s s i o nCELLCHANGESIN RESPONSETO SIGNALSn u c l e a renverop"envelopenucrearn",#;J:il:t:l:"t;ff:l!fl%,"'i'llli'"Ti"T$I l""lil"':,."ffFigure4-66 The movement of genestodifferent regionsof the nucleuswhentheir expressionchanges.The interiorofandthe nucleusis very heterogeneous,aredifferentnuclearneighborhoodsknownto havedistincteffectson geneMovementssuchasthoseexoression.reflectindicatedherepresumablythat thechangesin the bindingaffinitieschromatinand RNAmoleculesa genehavefor differentsurroundinglt is thoughtthatnuclearneighborhoods.the movementis drivenby diffusionanddoesnot reouirea directedmovementprocess,inasmuchas eachregionof achromosomecan be seento undergoconstantrandommotionwhen markedin a way that allowsits positionto befollowedin a livingcell.Forma Setof DistinctBiochemicalNetworksof Macromoleculesi n si d eth e N u cl e u sEn v i r o n m e ntsIn Chapter 6, we describe the function of a variety of subcompartments that arepresent within the nucleus.
The largest and most obvious of these is the nucleoIus, a structure well known to microscopists even in the 19th century (seeFigure4-9). Nucleolar regions consist of networks of RNAs and proteins surroundingtranscribing ribosomal RNA genes, often existing as multiple nucleoli. Thenucleolus is the cell's site of ribosome assembly and maturation, as well as theplace where many other specialized reactions occur.A variety of less obvious organelles are also present inside the nucleus.
Forexample, spherical structures called Cajal bodies and interchromatin granuleclusters are present in most plant and animal cells (Figure 4-67). Like the nucleolus, these organellesare composed of selectedprotein and RNA molecules thatbind together to create networks that are highly permeable to other protein andRNA molecules in the surrounding nucleoplasm (Figure 4-68).Structures such as these can create distinct biochemical environments byimmobilizing select groups of macromolecules, as can other networks of proteins and RNA molecules associatedwith nuclear pores and with the nuclearenvelope. In principle, this allows the molecules that enter these spaces to beprocessedwith great efficiency through complex reaction pathways.
Highly permeable, fibrous networks of this sort can thereby impart many of the kineticadvantagesof compartmentalization (seep. 186) to reactions that take place inthe nucleus (Figure 4-69A). However, unlike the membrane-bound compartments in the cltoplasm (discussedin Chapter 12), these nuclear subcompartments-lacking a lipid bilayer membrane-can neither concentrate nor excludespecific small molecules.The cell has a remarkable ability to construct distinct biochemical environments inside the nucleus. Those thus far mentioned facilitate various aspectsofgene expressionto be discussedin Chapter 6 (seeFigure 6-49).
Like the nucleolus, these subcompartments appear to form only as needed, and they create ahigh local concentration of the many different enzlrnes and RNA moleculesneeded for a particular process.In an analogous way, when DNA is damaged byirradiation, the set of enzymes needed to carry out DNA repair are observed tocongregate in discrete foci inside the nucleus, creating "repair factories" (seeFigure 5-60). And nuclei often contain hundreds of discrete foci representingfactories for DNA or RNA synthesis.It seemslikely that all of these entities make use of the type of tethering illustrated in Figure 4-698, where Iong flexible lengths of pollpeptide chain (or someother polymer) are interspersed with binding sites that concentrate the multipleproteins and/or RNA molecules that are needed to catalyzea particular process.Not surprisingly, tethers are similarly used to help to speed biological processesfrFigure 4-67 Electronmicrographshowingtwo very common fibrous nuclearThe largesphereheresubcompartments.is a Cajalbody.Thesmallerdarkersphereisgranulecluster,alsoan interchromatinknownasa spreckle(seealsoFigure6-49).arefrom theorganelles"These"subnuclearnucleusof aXenopusoocYte.(FromK.E.Handwergerand J.G.Gall,TrendsCellWith permissionfromBiol.16:19-26,2006.Elsevier.)242Chapter4: DNA,Chromosomes,and Genomesm o l e c u l awr e i g h t o f f l u o r e s c e ndt e x t r a ni n n u c l e u s70,000500,0001 0p min the cytoplasm, increasing specific reaction rates (for example, see Figurel6-38).Is there also is an intranuclear framework, analogous to the cltoskeleton, onwhich chromosomes and other components of the nucleus are organized? Thenuclear matrix, or scaffold, has been defined as the insoluble material left in thenucleus after a series of biochemical extraction steps.
Many of the proteins andRNA molecules that form this insoluble material are likely to be derived from theflbrous subcompartments of the nucleus just discussed, while others seem to beproteins that help to form the base of chromosomal loops or to attach chromosomes to other structures in the nucleus. Whether or not the nucleus also containsFigure4-69 Effectivecompartmentalizationwithout a bilayer membrane.(A)Schematicillustrationof the organizationof a sphericalsubnuclearorganelle(/eft)and of a postulatedsimilarlyorganizedsubcompartmentjust beneaththe nuclearenvelope(rlght).In both cases,RNAsand/or proteins(groy)associateto form highly porous,gel-likestructuresthat containbinding sitesfor other specificproteinsand RNAmolecules(coloredobjects).(B)How tnetetheringof a selectedsetof proteinsand RNAmoleculesto long flexiblepolymerchains,as inA, could create"stagingareas"that greatlyspeedthe ratesof reactionsin subcompartmentsofthe nucleus.Thereactionscatalyzedwill dependon the particularmacromoleculesthat arelocalizedby the tethering.The sametype of rateaccelerationsare of courseexpectedfor similarsubcompartmentsestablishedelsewherein the cell(seealsoFigure3-g0C).Figure4-68 An experiment showingthat the subnuclearorganellesarehighly permeableto macromolecules.Inthesemicrographsof a living oocytenucleus,the top row comparesthefluorescenceof the interiorsof nucleoli,Cajalbodies,and sprecklesto thefluorescenceof the surroundingnucleoplasm,12 hoursafterfluorescentdextransof the indicatedmolecularweight had been injectedinto thenucleoplasm.The brightnessof eachorganellereflectsits permeability,withthe mostpermeableorganellebeingthebrightest.Forcomparison,the bottomrow presentsnormallight micrographsofthe samemicroscopefields,with thenucleolusin eachfieldof view markedbrownto distinguishit.
Cajalbodiescanbe seento be more permeablethanquantitationshowsnucleoli.However,that a great deal of dextranenterseachorganelle,evenfor the largestdextrantested.(FromK.E.Handwerger,J.A.Corderoand J.G.Gall,Mol. Biol.Cell'16.'202-2'11,2005.With permissionfromAmericanSocietyof CellBiology.)243THEGLOBALSTRUCTUREOFCHROMOSOMESFigure4-70 A typicalmitoticchromosomeEachsisterat metaphase.chromatidcontainsoneof two identicaldaughterDNAmolecules(seealsoFiguregenerated17-26).earlierin thecellcycleby DNAreplicationcnromosomelong filaments that form organized tracks on which nuclear components canmove, analogous to some of the filaments in the cytoplasm, is still disputed.MitoticChromosomesAre Formedfrom Chromatinin lts MostCondensedStateHaving discussed the dynamic structure of interphase chromosomes, we nowturn to mitotic chromosomes.The chromosomes from nearly all eucaryotic cellsbecome readily visible by light microscopy during mitosis, when they coil up toform highly condensed structures.
This condensation reduces the length of atJ,?ical interphase chromosome only about tenfold, but it produces a dramaticchange in chromosome appearance.Figure 4-70 depicts a typical mitotic chromosome at the metaphase stageof mitosis (for the stages of mitosis, see Figure 17-3). The two daughter DNAmolecules produced by DNA replication during interphase of the cell-divisioncycle are separately folded to produce two sister chromosomes, or sister chromatids, held together at their centromeres (see also Figure 4-50). These chromosomes are normally covered with a variety of molecules, including largeamounts of RNA-protein complexes.Once this covering has been stripped away,each chromatid can be seen in electron micrographs to be organized into loopsof chromatin emanating from a central scaffolding (Figure 4-71), Experimentsusing DNA hybridization to detect specific DNA sequencesdemonstrate that theorder of visible features along a mitotic chromosome at least roughly reflects theorder of genes along the DNA molecule.
Mitotic chromosome condensation canthus be thought of as the final level in the hierarchy of chromosome packaging(Figure 4-72).The compaction of chromosomes during mitosis is a highly organized anddynamic process that serves at least two important purposes. First, when condensation is complete (in metaphase), sister chromatids have been disentangled from each other and lie side by side. Thus, the sister chromatids can easilyseparate when the mitotic apparatus begins pulling them apart. Second, thecompaction of chromosomes protects the relatively fragile DNA molecules frombeing broken as they are pulled to separatedaughter cells.The condensation of interphase chromosomes into mitotic chromosomesbegins in early M phase, and it is intimately connected with the progression ofthe cell cycle, as discussedin detail in Chapter 17.
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