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Hered.6, 1934.With permissionfrom25:465-47OxfordUniversityPress.)t:\'.,.N"right arm ofc h r o m o s o m e3237X chromosomen o r m a lm i t o t i cchromosomesats a m eS c a l eleft arm ofc h r o m o s o m e3,o lrmv, ,!,.i11,-.1{.interbandsDanos(A)2pm1 ilmFigure4-59 Micrographsof polytenechromosomesfrom Drosophilo salivaryglands.(A) Light micrographof a portionDNAhasbeenofa chromosome.Thestainedwith a fluorescentdye, but areverseimage is presentedherethatrendersthe DNA black rather than white;the bandsareclearlyseento be regionsThisof increasedDNAconcentration.chromosomehasbeen ProcessedbY ahigh pressuretreatmentso asto show itsdistinctpatternof bandsand interbandsmore clearly.(B)An electronmicrographof a smallsectionof a Droso7hilapolytenechromosomeseenin thinsection.Bandsof very differentthicknessseparatedcan be readilydistinguished,whichcontainlessbv interbands,condensedchromatin.(A,adaptedfromD.V.Novikov l.
Kireevand A.S.Belmont,WithNat.Methods4:483-485,2007.permissionfrom MacmillanPublishersLtd.B,courtesyofVeikkoSorsa.)238Chapter4: DNA,Chromosomes,and Genomes1 0p maffect biological function in different ways. some of these non-histone proteinscan spread for long distances along the DNA, imparting a similar chromatinstructure to contiguous regions of the genome (seeFigure 4-46). Thus, in someregions, all of the chromatin has a similar structure and is separatedfrom neighboring domains by barrier proteins (see Figure 4-47). ft low resolution, theinterphase chromosome can therefore be considered as a mosaic of chromatinstructures, each containing particular nucleosome modifications associatedwith a particular set of non-histone proteins.
(At a higher level of resolution onewould also emphasize the many sequence-specificDNA-binding proteins thatwill be described in Chapter 7).This view of an interphase chromosome helps us to interpret the resultsobtained from studies of polltene chromosomes. By staining with highly specific antibodies, one can show that differently modified histones (Figure 4-60),as well as distinct sets of non-histone proteins, are located on different polytenechromosome bands. This suggests a powerful general strategy. By employingcombinations of antibodies that bind tightly to each of the many different histone modifications that create the histone code (seeFigure 4-39), it may be possible to determine which combinations of modifications specify particular typesof chromatin domains. And by carrying out similar experiments with antibodiesthat recognize each of the hundreds of different non-histone proteins in chromatin, one can attempt to decipher the many different meanings encoded inhistone modifications.ThereAreMultipleFormsof HeterochromatinMolecular studies have led to a reevaluation of our view of heterochromatin.
Formany decades,heterochromatin was thought to be a single entity defined by itshighly condensed structure and its ability ro silence genes permanently. But ifwe define heterochromatin as a form of compact chromatin that can silencegenes, be epigenetically inherited, and spread along chromosomes to causeposition effect variegation (seeFigure 4-36), it is clear that different types ofheterochromatin exist. In fact, we have already considered three of these types indiscussing the human centromere (seeFigure 4-50).Each domain of heterochromatin is thought to be formed by the cooperativeassemblyof a set of non-histone proteins.
For example,classicalpericentromericheterochromatin contains more than six such proteins, including heterochromatin protein I (HPI), whereas the so-called polycomb form of heterochromatincontains a similar number of proteins in a non-overlapping set (pcc proteins).There are hundreds of small blocks of heterochromatin spread across the armsof Drosophilapolytene chromosomes, as identified by their late replication (discussedin chapter 5). Antibody staining of these regions of heterochromatin suggests that the knor,tm forms of heterochromatin can account for no more thanhalf of the heterchromatic polytene bands (Figure 4--61).Thus, other rypes ofheterochromatin must exist whose protein composition is not knolrm.
rt is titetvFigure4-60 The pattern of histonemodificationson Drosophilapolytenechromosomes.Antibodiesthatspecificallyrecognizedifferenthistonemodificationscan revealwhere eachmodificationis found with referencetothe manybandsand interbandson thesechromosomes.In the two preparationsshownhere,the positionsof twodifferentmarkingson histoneH3 tailsarecompared.In both cases,the antibodylabelingthe modifiedhistoneis green,and the DNAis stainedred.Onlya smallregionsurroundingeachchromocenterisshown.(A) DimethylLys9 (green)is ahistonemodificationassociatedwith thepericentricheterochromatin.lt is seentobe associatedwith the chromocenter.(B)Acetylated Lys9 (green)is amodificationthat is concentratedinhistonesassociatedwith activegenes.ltis seento be presentin numerousbandson the chromosomearms,but not in theheterochromaticchromocenter.Similarexperimentscan be carriedout topositionmanyothermodifiedhistones,proteins(see,aswell asthe non-histonefor example,Figure 22-45 forchromosomesstainedfor Polycomb).(Adaptedfrom A.
Ebert,S.Lein,G. Schottaand G. Reuter,ChromosomeRes.14:377-392,2006.With permissionfrom Springer.)i o n l y P c Gp r o t e i n'g/rtb o t h H P l a n d P c Gp r o t e i nneitherHP1nor PcGprotein5075100percentof heterochromatinsitesFigure4-61 Evidencefor multipleformsof heterochromatin,In this study,240late-replicatingsiteson the Drosophilapolytenechromosomearmswereexaminedfor the presenceof two nonhistoneproteins.Theseproteinsareknownto help compacttwo differentforms of heterochromatin(seetext).Asindicated,antibodystainingsuggeststhat roughlyhalfof the sitesarepackagedinto forms of heterochromatinthat are differentfrom eitherof thesetwo. Experimentssuchasthesedemonstratethat we havea greatdealmoreto learnaboutthe packagingofDNA in eucaryotes.(Datafroml.F.Zhimulevand E.5.Belyaeva,BioEssays25:1040-1051, 2003.With permissionfrom JohnWiley& Sons.)239THEGLOBALSTRUCTUREOF CHROMOSOMESthat each of these types of heterochromatin is differently regulated and has different roles in the cell.The chromatin structure in each domain ultimately depends on the proteinsthat bind to specific DNA sequences,and these are kno'o,nto vary depending onthe cell type and its stageof development in a multicellular organism.
Thus, boththe pattern of chromatin domains and their individual compositions (nucleosome modifications plus non-histone proteins) can vary between tissues.Thesedifferences make different genes accessible for genetic readout, helping toexplain the cell diversification that accompanies embryonic development(describedin Chapter 22).Comparisons of the polyene chromosomes in two different tissues of a fly lend support to this general idea: although the patterns ofbands and interbands are largely the same,there are reproducible differences.Whenthe GenesWithinThemAreChromatinLoopsDecondenseExpressed\Mhen an insect progressesfrom one developmental stageto another, distinctivechromosome puffs arise and old puffs recede in its polltene chromosomes asnew genes become expressedand old ones are turned off (Figure 4-62).
Frominspection of each puff when it is relatively small and the banding pattern is stilldiscernible, it seems that most puffs arise from the decondensation of a singlechromosome band.The individual chromatin fibers that make up a puff can be visualized withan electron microscope. In favorable cases, loops are seen, much like thoseobserved in the amphibian lampbrush chromosomes discussed above' \A4rennot expressed,the loop of DNA assumesa thickened structure, possibly a folded30-nm fiber, but when gene expression is occurring, the loop becomes moreextended. In electron micrographs, the chromatin located on either side of thedecondensed loop appears considerably more compact, suggestingthat a loopconstitutes a distinct functional domain of chromatin structure.Observations made in human cells also suggestthat highly folded Ioops ofchromatin expand to occupy an increased volume when a gene within them isexpressed.For example, quiescent chromosome regions from 0.4 to 2 millionnucleotide pairs in length appear as compact dots in an interphase nucleuswhen visualized by fluorescence microscopy using FISH or other technologies.However, the same DNA is seen to occupy a Iarger territory when its genes areexpressed,with elongated, punctate structures replacing the original dot.1 0p tFigure 4-62 RNAsynthesisin polytenechromosomepuffs.An autoradiographof a singlepuff in a polytenefrom the salivaryglandsofchromosomethe freshwatermidge C.tentans.Asoutlinedin Chapter1 and describedindetailin Chapter6, the firststepin geneof an RNAis the synthesisexpressionmoleculeusingthe DNAasa template.portionoftheThedecondensedchromosomeis undergoingRNAand hasbecomelabeledwithsynthesis3H-uridine(seep.
603),an RNAprecursorintomoleculethat is incorporatedof Jos6growingRNAchains.(CourtesyBonner.)SitesWithinthe Nucleusto AlterChromatinCanMoveto SpecificGeneExpressionNew ways of visualizing individual chromosomes have shonm that each of the 46interphase chromosomes in a human cell tends to occupy its ovrmdiscrete territory within the nucleus (Figure tl-63). However, pictures such as these presentonly an average view of the DNA in each chromosome. Experiments that specifically localize the heterochromatic regions of a chromosome reveal that they areoften closely associated with the nuclear lamina, regardless of the chromosomeexamined. And DNA probes that preferentially stain gene-rich regions of human1 0p m1'l19910t3of the chromosometerritoriesvisualizationFigure4-63 Simultaneousfor all of the human chromosomesin a singleinterphasenucleus.A FISHfor markingthe DNAofanalysisusinga differentmixtureof fluorochromesdetectedwith sevencolorchannelsin a fluoresenceeachchromosome,in threeto be distinguishedmicroscope,allowseachchromosomeeachchromosomeisBelowthe micrograph,reconstructions.dimensionalof the actualimage.Notethat the twoidentifiedin a schematic(e.g.,the two copiesof chromosome9),arenothomologouschromosomes(FromM.R.SpeicherNat'Rev.Genet.and N.P.Carter,in generalco-located.Ltd.)from MacmillanPublishersWith permission6:782-792,2005.5812240Chapter4: DNA,Chromosomes,and GenomesFigure4-64Thedistributionof gene-richregionsof the humangenomein an interphasenucleus.Gene-richregionshavebeenvisualizedwithafluorescentprobethathybridizesto theAluinterspersedrepeat,whichispresentin morethana millioncopiesin thehumangenome(seeFigure5-75).Forunknownreasons,thesesequencesclusterin chromosomalregionsrichin genes.Inthisrepresentation,regionsenrichedfortheAlusequenceategreen,regionsdepletedfor thesesequencesarered,whiletheaverageregionsareyel/or,v.Thegene-richregionsareseento bedepletedin the DNAnearthe nuclear(FromA.
Bolzeret al.,pLoSenvelope.Biol.3:826-842,2005.WithpermissionfrompublicLibrarvof Science.)chromosomes produce a striking picture of the interphase nucleus that presumably reflects different average positions for active and inactive genes (Figure4-Sq.A variety of different types of experiments have led to the conclusion thatthe position of a gene in the interior of the nucleus changes when it becomeshighly expressed.Thus, a region that becomes very actively transcribed is oftenfound to extend out of its chromosome territory, as if in an extended loop (Figure 4-65).
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