B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 79
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As a 30-nmfiber, a typical human chromosome would still be 0.1 cm in length and able tospan the nucleus more than 100 times. Clearly, there must be a still higher levelof folding, even in interphase chromosomes. Although the molecular details arestill largely a mystery, this higher-order packaging almost certainly involves thefolding of the chromatin into a series of loops and coils. This chromatin packing isfluid, frequently changing in response to the needs of the cell.We begin this section by describing some unusual interphase chromosomesthat can be easily visualized.
Exceptional though they are, these special casesreveal features that are thought to be representative of all interphase chromosomes. Moreover, they provide ways to investigate some fundamental aspects ofchromatin structure that we have touched on in the previous section. Next, wedescribe how a typical interphase chromosome is arranged in the mammaliancell nucleus. Finally, we shall discuss the additional tenfold compaction that chromosomes undergo in the passage from interphase to mitosis.Chromosomes Are Folded into Large Loops of ChromatinInsight into the structure of the chromosomes in interphase cells has come fromstudies of the stiff and enormously extended chromosomes in growing amphibian oocytes (immature eggs).
These very unusual lampbrush chromosomes (thelargest chromosomes known), paired in preparation for meiosis, are clearly visible even in the light microscope, where they are seen to be organized into a seriesof large chromatin loops emanating from a linear chromosomal axis (Figure 4–46and Figure 4–47).In these chromosomes, a given loop always contains the same DNA sequencethat remains extended in the same manner as the oocyte grows. These chromosomes are producing large amounts of RNA for the oocyte, and most of the genesextended chromatinin looped domain10 µmsisterchromatidslesscondensedchromatinhighlycondensedchromatinFigure 4–46 A model for the chromatindomains in a lampbrush chromosome.Shown is a small portion of one pair ofMBoC6 n4.126/4.47sister chromatids.Here, two identical DNAdouble helices are aligned side by side,packaged into different types of chromatin.The set of lampbrush chromosomesin many amphibians contains a total ofabout 10,000 loops resembling thoseshown here.
The rest of the DNA in eachchromosome (the great majority) remainshighly condensed. Four copies of eachloop are present in the cell, since eachlampbrush chromosome consists of twoaligned sets of paired chromatids. Thisfour-stranded structure is characteristic ofthis stage of development of the oocyte,which has arrested at the diplotene stageof meiosis; see Figure 17–56.208Chapter 4: DNA, Chromosomes, and Genomespresent in the DNA loops are being actively expressed. The majority of the DNA,however, is not in loops but remains highly condensed on the chromosome axis,where genes are generally not expressed.It is thought that the interphase chromosomes of all eukaryotes are similarlyarranged in loops. Although these loops are normally too small and fragile to beeasily observed in a light microscope, other methods can be used to infer theirpresence.
For example, modern DNA technologies have made it possible to assessthe frequency with which any two loci along an interphase chromosome are heldtogether, thus revealing likely candidates for the sites on chromatin that form thebases of loop structures (Figure 4–48). These experiments and others suggest thatthe DNA in human chromosomes is likely to be organized into loops of variouslengths. A typical loop might contain between 50,000 and 200,000 nucleotidepairs of DNA, although loops of a million nucleotide pairs have also been suggested (Figure 4–49).Polytene Chromosomes Are Uniquely Useful for VisualizingChromatin StructuresFurther insight has come from another unusual class of cells—the polytene cells offlies, such as the fruit fly Drosophila.
Some types of cells, in many organisms, growabnormally large through multiple cycles of DNA synthesis without cell division.Such cells, containing increased numbers of standard chromosomes, are said tobe polyploid. In the salivary glands of fly larvae, this process is taken to an extremedegree, creating huge cells that contain hundreds or thousands of copies of the(A)(B)100 µm20 µmFigure 4–47 Lampbrush chromosomes.(A) A light micrograph of lampbrushchromosomes in an amphibian oocyte.Early in oocyte differentiation, eachchromosome replicates to beginmeiosis, and the homologous replicatedchromosomes pair to form this highlyextended structure containing a total offour replicated DNA double helices, orchromatids. The lampbrush chromosomestage persists for months or years, whilethe oocyte builds up a supply of materialsrequired for its ultimate development intoa new individual.
(B) An enlarged regionof a similar chromosome, stained with afluorescent reagent that makes the loopsactive in RNA synthesis clearly visible.(Courtesy of Joseph G. Gall.)THE GLOBAL STRUCTURE OF CHROMOSOMESDNA-bindingproteins209cross-linkformedDNA probes used for PCRTREATWITHFORMALDEHYDECUTWITHRESTRICTIONNUCLEASEDNALIGATIONREMOVECROSS-LINKSBY HEAT TREATMENTAND PROTEOLYSISgenome.
Moreover, in this case, all the copies of each chromosome are alignedside by side in exact register, like drinking straws in a box, to create giant polytenechromosomes. These allow features to be detected that are thought to be sharedwith ordinary interphase chromosomes, but are normally hard to see.When polytene chromosomes from a fly’s salivary glands are viewed in thelight microscope, distinct alternating dark bands and light interbands are visible(Figure 4–50), each formed from a thousand identical DNA sequences arrangedside by side in register.
About 95% of theMBoC6DNAm4.56/4.47in polytene chromosomes is inbands, and 5% is in interbands. A very thin band can contain 3000 nucleotidepairs, while a thick band may contain 200,000 nucleotide pairs in each of its chromatin strands. The chromatin in each band appears dark because the DNA is morecondensed than the DNA in interbands; it may also contain a higher concentration of proteins (Figure 4–51).
This banding pattern seems to reflect the same sortof organization detected in the amphibian lampbrush chromosomes describedearlier.There are approximately 3700 bands and 3700 interbands in the complete setof Drosophila polytene chromosomes. The bands can be recognized by their different thicknesses and spacings, and each one has been given a number to generate a chromosome “map” that has been indexed to the finished genome sequenceof this fly.The Drosophila polytene chromosomes provide a good starting point for examining how chromatin is organized on a large scale.
In the previous section, wesaw that there are many forms of chromatin, each of which contains nucleosomeswith a different combination of modified histones. Specific sets of non-histoneproteins assemble on these nucleosomes to affect biological function in different ways. Recruitment of some of these non-histone proteins can spread for longdistances along the DNA, imparting a similar chromatin structure to broad tractsfoldedchromatinfiberTEST FOR JOINEDSEGMENTS BYPCRDNA product is obtainedonly if proteins hold thetwo DNA sequences closetogether in the cellFigure 4–48 A method for determiningthe position of loops in interphasechromosomes. In this technique, knownas the chromosome conformationcapture (3C) method, cells are treatedwith formaldehyde to create the indicatedcovalent DNA–protein and DNA–DNAcross-links.
The DNA is then treated withan enzyme (a restriction nuclease) thatchops the DNA into many pieces, cuttingat strictly defined nucleotide sequencesand forming sets of identical “cohesiveends” (see Figure 8–28). The cohesiveends can be made to join through theircomplementary base-pairing. Importantly,prior to the ligation step shown, the DNAis diluted so that the fragments that havebeen kept in close proximity to each other(through cross-linking) are the ones mostlikely to join. Finally, the cross-links arereversed and the newly ligated fragmentsof DNA are identified and quantified byPCR (the polymerase chain reaction,described in Chapter 8). From the results,combined with DNA sequence information,one can derive models for the interphaseconformation of chromosomes.high-levelexpressionof genesin looplooped domainhistonemodifying enzymeschromatinremodeling complexesRNA polymeraseproteins forming chromosome scaffoldFigure 4–49 A model for the organization of an interphase chromosome.
A section of an interphase chromosome is shown folded into a seriesof looped domains, each containing perhaps 50,000–200,000 or more nucleotide pairs of double-helical DNA condensed into a chromatin fiber.The chromatin in each individual loop is further condensed throughpoorlyunderstood folding processes that are reversed when the cell requiresMBoC6m4.57/4.48direct access to the DNA packaged in the loop. Neither the composition of the postulated chromosomal axis nor how the folded chromatin fiber isanchored to it is clear. However, in mitotic chromosomes, the bases of the chromosomal loops are enriched both in condensins (discussed below)and in DNA topoisomerase II enzymes (discussed in Chapter 5), two proteins that may form much of the axis at metaphase.210Chapter 4: DNA, Chromosomes, and Genomesright arm ofchromosome 2normal mitoticchromosomes atsame scaleregionwhere twohomologouschromosomesare separatedleft arm ofchromosome 2X chromosomechromosome 4chromocenterleft arm ofchromosome 320 µmright arm ofchromosome 3of the genome (see Figure 4–40).
Such regions, where all of the chromatin hasa similar structure, are separated from neighboring domains by barrier proteins(see Figure 4–41). At low resolution, the interphase chromosome can thereforebe considered as a mosaic of chromatin structures, each containing particularnucleosome modifications associatedwith a particular set of non-histone proMBoC6 m4.58/4.49teins.
Polytene chromosomes allow us to see details of this mosaic of domains inthe light microscope, as well as to observe some of the changes associated withgene expression.Figure 4–50 The entire set of polytenechromosomes in one Drosophila salivarycell. In this drawing of a light micrograph,the giant chromosomes have beenspread out for viewing by squashing themagainst a microscope slide. Drosophilahas four chromosomes, and there are fourdifferent chromosome pairs present. Buteach chromosome is tightly paired withits homolog (so that each pair appearsas a single structure), which is not truein most nuclei (except in meiosis).
Eachchromosome has undergone multiplerounds of replication, and the homologsand all their duplicates have remained inexact register with each other, resultingin huge chromatin cables many DNAstrands thick.The four polytene chromosomesare normally linked together byheterochromatic regions near theircentromeres that aggregate to createa single large chromocenter (pinkregion). In this preparation, however, thechromocenter has been split into twohalves by the squashing procedure used.(Adapted from T.S. Painter, J. Hered.25:465–476, 1934. With permission fromOxford University Press.)There Are Multiple Forms of ChromatinBy staining Drosophila polytene chromosomes with antibodies, or by using amore recent technique called ChIP (chromatin immunoprecipitation) analysis(see Chapter 8), the locations of the histone and non-histone proteins in chromatin can be mapped across the entire DNA sequence of an organism’s genome.Such an analysis in Drosophila has thus far localized more than 50 different chromatin proteins and histone modifications.
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