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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

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Molecular Biology of the Cell. 4th edition.

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The Global Structure of Chromosomes

Having discussed the DNA and protein molecules from which the 30-nm chromatin fiber is made, we now turn to the organization of the chromosome on a more global scale. As a 30-nm fiber, the typical human chromosome would still be 0.1 cm in length and able to span the nucleus more than 100 times. Clearly, there must be a still higher level of folding, even in interphase chromosomes. This higher-order packaging is one of the most fascinating—but also one of the most poorly understood—aspects of chromosome structure. Although its molecular basis is still largely a mystery, it almost certainly involves the folding of the 30-nm fiber into a series of loops and coils, as we see below. Our discussion of this higher-order packing continues an important theme in chromosome architecture: interphase chromatin structure is fluid, exposing at any given moment the DNA sequences directly needed by the cell.

We first describe several rare cases in which the overall structure and organization of interphase chromosomes can be easily visualized, and we explain that certain features of these exceptional cases may be representative of the structures of all interphase chromosomes. Next we describe the different forms of chromatin that make up a typical interphase chromosome. Finally we discuss the additional compaction that interphase chromosomes undergo during the process of mitosis.

Lampbrush Chromosomes Contain Loops of Decondensed Chromatin

Most chromosomes in interphase cells are too fine and too tangled to be visualized clearly. In a few exceptional cases, however, interphase chromosomes can be seen to have a precisely defined higher-order structure, and it is thought that certain characteristics of these higher-order structures are representative of all interphase chromosomes. The meiotically paired chromosomes in growing amphibian oocytes (immature eggs), for example, are highly active in gene expression, and they form unusually stiff and extended chromatin loops. These so-called lampbrush chromosomes (the largest chromosomes known) are clearly visible even in the light microscope, where they are seen to be organized into a series of large chromatin loops emanating from a linear chromosomal axis (Figure 4-36).

Figure 4-36. Lampbrush chromosomes.

Figure 4-36

Lampbrush chromosomes. (A) A light micrograph of lampbrush chromosomes in an amphibian oocyte. Early in oocyte differentiation, each chromosome replicates to begin meiosis, and the homologous replicated chromosomes pair to form this highly extended structure (more...)

The organization of a lampbrush chromosome is illustrated in Figure 4-37. A given loop always contains the same DNA sequence, and it remains extended in the same manner as the oocyte grows. Other experiments demonstrate that most of the genes present in the DNA loops are being actively expressed (see Figure 4-36B). Most of the DNA, however, is not in loops but remains highly condensed in the chromomeres on the axis, which are generally not expressed. Lampbrush chromosomes illustrate a recurrent theme of this chapter—when the DNA in a region of chromatin is in use (in this case, for gene expression), that part of the chromatin has an extended structure; otherwise, the chromatin is condensed. In lampbrush chromosomes, the structural units of this regulation are large, precisely defined loops.

Figure 4-37. A model for the structure of a lampbrush chromosome.

Figure 4-37

A model for the structure of a lampbrush chromosome. The set of lampbrush chromosomes in many amphibians contains a total of about 10,000 chromatin loops, although most of the DNA in each chromosome remains highly condensed in the chromomeres. Each loop (more...)

Relatively few species undergo the specialization that produces lampbrush chromosomes. However, when injected into amphibian oocytes, the DNA from organisms that normally do not produce lampbrush chromosomes (e.g., DNA from a fish) is packaged into lampbrush chromosomes. On the basis of this type of experiment, it has been proposed that the interphase chromosomes of all eucaryotes are arranged in loops that are normally too small and fragile to be easily observed. It may be possible in the future to coax the DNA from a mammal such as a mouse to form lampbrush chromosomes by introducing it into amphibian oocytes. This could allow a detailed correlation of loop structure, gene arrangement, and DNA sequence, and we could begin to learn how the packaging into loops reflects the sequence content of our DNA.

Drosophila Polytene Chromosomes Are Arranged in Alternating Bands and Interbands

Certain insect cells also have specialized interphase chromosomes that are readily visible, although this type of specialization differs from that of lampbrush chromosomes. For example, many of the cells of certain fly larvae grow to an enormous size through multiple cycles of DNA synthesis without cell division. The resulting giant cells contain as much as several thousand times the normal DNA complement. Cells with more than the normal DNA complement are said to be polyploid when they contain increased numbers of standard chromosomes. In several types of secretory cells of fly larvae, however, all the homologous chromosome copies are held side by side, like drinking straws in a box, creating a single polytene chromosome. The fact that, in some large insect cells, polytene chromosomes can disperse to form a conventional polyploid cell demonstrates that these two chromosomal states are closely related, and that the basic structure of a polytene chromosome must be similar to that of a normal chromosome.

Polytene chromosomes are often easy to see in the light microscope because they are so large and because the precisely aligned side-by-side adherence of individual chromatin strands greatly elongates the chromosome axis and prevents tangling. Polyteny has been most studied in the salivary gland cells of Drosophila larvae, in which the DNA in each of the four Drosophila chromosomes has been replicated through 10 cycles without separation of the daughter chromosomes, so that 1024 (210) identical strands of chromatin are lined up side by side (Figure 4-38).

Figure 4-38. The entire set of polytene chromosomes in one Drosophila salivary cell.

Figure 4-38

The entire set of polytene chromosomes in one Drosophila salivary cell. These chromosomes have been spread out for viewing by squashing them against a microscope slide. Drosophila has four chromosomes, and there are four different chromosome pairs present. (more...)

When polytene chromosomes are viewed in the light microscope, distinct alternating dark bands and light interbands are visible (Figure 4-39). Each band and interband represents a set of 1024 identical DNA sequences arranged in register. About 95% of the DNA in polytene chromosomes is in bands, and 5% is in interbands. The chromatin in each band appears dark, either because it is much more condensed than the chromatin in the interbands, or because it contains a higher proportion of proteins, or both (Figure 4-40). Depending on their size, individual bands are estimated to contain 3000–300,000 nucleotide pairs in a chromatin strand. The bands of Drosophila polytene chromosomes can be recognized by their different thicknesses and spacings, and each one has been given a number to generate a chromosome “map.” There are approximately 5000 bands and 5000 interbands in the complete set of Drosophila polytene chromosomes.

Figure 4-39. A light micrograph of a portion of a polytene chromosome from Drosophila salivary glands.

Figure 4-39

A light micrograph of a portion of a polytene chromosome from Drosophila salivary glands. The distinct pattern produced by bands and interbands is readily seen. The bands are regions of increased chromatin concentration that occur in interphase chromosomes. (more...)

Figure 4-40. An electron micrograph of a small section of a Drosophila polytene chromosome seen in thin section.

Figure 4-40

An electron micrograph of a small section of a Drosophila polytene chromosome seen in thin section. Bands of very different thickness can be readily distinguished, separated by interbands, which contain less condensed chromatin. (Courtesy of Veikko Sorsa.) (more...)

Both Bands and Interbands in Polytene Chromosomes Contain Genes

The reproducible pattern of bands and interbands seen in Drosophila polytene chromosomes means that these interphase chromosomes are highly organized. Since the 1930s, scientists have debated the nature of this organization, and we still do not have a clear answer. Because the number of bands in Drosophila chromosomes was once thought to be roughly equal to the number of genes in the genome, it was initially thought that each band might correspond to a single gene; however, we now know this simple idea is incorrect. There are nearly three times more genes in Drosophila than chromosome bands, and genes are found in both band and interband regions. Moreover, some bands contain multiple genes, and some bands seem to lack genes altogether.

It seems likely that the band-interband pattern reflects different levels of gene expression and chromatin structure along the chromosome, with genes in the less compact interbands being expressed more highly than those in the more compact bands. In any case, the remarkable appearance of fly polytene chromosomes is thought to reflect the heterogeneous nature of the chromatin compaction found along all interphase chromosomes. In the next section we see how the appearance of a band can change dramatically when the gene or genes within it become highly expressed.

Individual Polytene Chromosome Bands Can Unfold and Refold as a Unit

A major factor controlling gene expression in the polytene chromosomes of Drosophila is the insect steroid hormone ecdysone, the levels of which rise and fall periodically during larval development. When ecdysone concentrations rise, they induce the expression of genes coding for proteins that the larva requires for each molt and for pupation. As the organism progresses from one developmental stage to another, distinctive chromosome puffs arise and old puffs recede as new genes become expressed and old ones are turned off (Figure 4-41). From inspection of each puff when it is relatively small and the banding pattern is still discernible, it seems that most puffs arise from the decondensation of a single chromosome band.

Figure 4-41. Chromosome puffs.

Figure 4-41

Chromosome puffs. This series of time-lapse photographs shows how puffs arise and recede in the polytene chromosomes of Drosophila during larval development. A region of the left arm of chromosome 3 is shown. It exhibits five very large puffs in salivary (more...)

The individual chromatin fibers that make up a puff can be visualized with an electron microscope. For technical reasons, this is easier in the polytene chromosomes from a different insect, Chironomus tentans, a midge. Electron micrographs of certain puffs, called Balbiani rings, of Chironomus salivary gland polytene chromosomes show the chromatin arranged in loops (Figures 4-42 and 4-43), much like those observed in the amphibian lampbrush chromosomes discussed earlier. Additional experiments suggest that each loop contains a single gene. When not expressed, the loop of DNA assumes a thickened structure, possibly a folded 30-nm fiber, but when gene expression is occurring, the loop becomes more extended. Both types of loops contain the four core histones and histone H1.

Figure 4-42. RNA synthesis in chromosome puffs.

Figure 4-42

RNA synthesis in chromosome puffs. (A) Polytene chromosomes from the salivary glands of the insect C. tentans. As outlined in Chapter 1 and described in detail in Chapter 6, the first step in gene expression is the synthesis of an RNA molecule using the (more...)

Figure 4-43. Polytene chromosomes from C. tentans.

Figure 4-43

Polytene chromosomes from C. tentans. The electron micrograph shows a thin section of the chromatin in a Balbiani ring, a chromosome puff very active in gene expression. The Balbiani ring gene codes for secretory proteins the larvae uses to spin a protective (more...)

It seems likely that the default loop structure is a folded 30-nm fiber and that the histone modifying enzymes, chromatin remodeling complexes, and other proteins required for gene expression all help to convert it to a more extended form whenever a gene is expressed. In electron micrographs, the chromatin located on either side of the decondensed loop appears considerably more compact, which is consistent with the idea that a loop constitutes an independent functional domain of chromatin structure.

Although controversial, it has been proposed that all of the DNA in polytene chromosomes is arranged in loops that condense and decondense according to when the genes within them are expressed. It may be that all interphase chromosomes from all eucaryotes are also packaged into an orderly series of looped domains, each containing a small number of genes whose expression is regulated in a coordinated way (Figure 4-44). We shall return to this issue in Chapter 7 when we discuss the ways in which gene expression is regulated by the cell.

Figure 4-44. A model for the structure of an interphase chromosome.

Figure 4-44

A model for the structure of an interphase chromosome. A section of an interphase chromosome is shown folded into a series of looped domains, each containing 20,000–100,000 nucleotide pairs of double-helical DNA condensed into a 30-nm fiber. Individual (more...)

Heterochromatin Is Highly Organized and Usually Resistant to Gene Expression

Having described some features of interphase chromosomes inferred from a few rare cases, we now turn to characteristics of interphase chromosomes that can be observed in a wide variety of organisms. Light-microscope studies in the 1930s distinguished between two types of chromatin in the interphase nuclei of many higher eucaryotic cells: a highly condensed form, called heterochromatin, and all the rest, which is less condensed, called euchromatin. Euchromatin is composed of the types of chromosomal structures—30-nm fibers and looped domains—that we have discussed so far. Heterochromatin, in contrast, includes additional proteins and probably represents more compact levels of organization that are just beginning to be understood. In a typical mammalian cell, approximately 10% of the genome is packaged into heterochromatin. Although present in many locations along chromosomes, it is concentrated in specific regions, including the centromeres and telomeres.

Most DNA that is folded into heterochromatin does not contain genes. However, genes that do become packaged into heterochromatin are usually resistant to being expressed, because heterochromatin is unusually compact. This does not mean that heterochromatin is useless or deleterious to the cell; as we see below, regions of heterochromatin are responsible for the proper functioning of telomeres and centromeres (which lack genes), and its formation may even help protect the genome from being overtaken by “parasitic” mobile elements of DNA. Moreover, a few genes require location in heterochromatin regions if they are to be expressed. In fact, the term heterochromatin (which was first defined cytologically) is likely to encompass several distinct types of chromatin structures whose common feature is an especially high degree of organization. Thus, heterochromatin should not be thought of as encapsulating “dead” DNA, but rather as creating different types of compact chromatin with distinct features and roles.

Heterochromatin's resistance to gene expression makes it amenable to study even in organisms in which it cannot be directly observed. When a gene that is normally expressed in euchromatin is experimentally relocated into a region of heterochromatin, it ceases to be expressed, and the gene is said to be silenced. These differences in gene expression are examples of position effects, in which the activity of a gene depends on its position along a chromosome. First recognized in Drosophila, position effects have now been observed in many organisms and they are thought to reflect an influence of the different states of chromatin structure along chromosomes on gene expression. Thus, chromosomes can be considered as mosaics of distinct forms of chromatin, each of which has a special effect on the ability of the DNA it contains to be addressed by the cell.

Many position effects exhibit an additional feature called position effect variegation, which is responsible for the mottled appearance of the fly eye and the sectoring of the yeast colony in the examples shown in Figure 4-45. These patterns can result from patches of cells in which a silenced gene has become reactivated; once reactivated, the gene is inherited stably in this form in daughter cells. Alternatively, a gene can start out in euchromatin early in development, and then be selected more or less randomly for packaging into heterochromatin, causing its inactivation in a cell and all of its daughters.

Figure 4-45. Position effects on gene expression in two different eucaryotic organisms.

Figure 4-45

Position effects on gene expression in two different eucaryotic organisms. (A) The yeast ADE2 gene at its normal chromosomal location is expressed in all cells. When moved near the end of a yeast chromosome, which is inferred to be folded into a form (more...)

The study of position effect variegation has revealed two important characteristics of heterochromatin. First, heterochromatin is dynamic; it can “spread” into a region and later “retract” from it at low but observable frequencies. Second, the state of chromatin—whether heterochromatin or euchromatin—tends to be inherited from a cell to its progeny. These two features are responsible for position effect variegation, as explained in Figure 4-46. In the next section, we discuss several models to account for the self-sustaining nature of heterochromatin, once it has been formed.

Figure 4-46. The cause of position effect variegation in Drosophila.

Figure 4-46

The cause of position effect variegation in Drosophila. (A) Heterochromatin (blue) is normally prevented from spreading into adjacent regions of euchromatin (green) by special boundary DNA sequences, which we discuss in Chapter 7. In flies that inherit (more...)

The Ends of Chromosomes Have a Special Form of Heterochromatin

Unlike the nucleosome and the 30-nm fiber, heterochromatin is not well understood structurally. It almost certainly involves an additional level of folding of 30-nm fiber and requires many proteins in addition to the histones. Although its chromosomes are too small to be seen under the light microscope, the molecular nature of heterochromatin is probably best understood in the simple yeast S. cerevisiae. Many experiments with yeast cells have shown that the chromatin extending inward roughly 5000 nucleotide pairs from each chromosome end is resistant to gene expression, and probably has a structure that corresponds to at least one type of heterochromatin in the chromosomes of more complex organisms. Extensive genetic analysis has led to the identification of many of the yeast proteins required for this type of gene silencing.

Mutations in any one of a set of yeast Silent information regulator (Sir) proteins prevent the silencing of genes located near telomeres, thereby allowing these genes to be expressed. Analysis of these proteins has led to the discovery of a telomere-bound Sir protein complex that recognizes underacetylated N-terminal tails of selected histones (Figure 4-47A). One of the proteins in this complex is a highly conserved histone deacetylase known as Sir2, which has homologs in diverse organisms, including humans, and presumably has a major role in creating a pattern of histone underacetylation unique to heterochromatin. As discussed earlier in this chapter, deacetylation of the histone tails is thought to allow nucleosomes to pack together into tighter arrays and may also render them less susceptible to some chromatin remodeling complexes. In addition, heterochromatin-specific patterns of histone tail modification are likely to attract additional proteins involved in forming and maintaining heterochromatin (see Figure 4-35).

Figure 4-47. Speculative model for the heterochromatin at the ends of yeast chromosomes.

Figure 4-47

Speculative model for the heterochromatin at the ends of yeast chromosomes. (A) Heterochromatin is generally underacetylated, and underacetylated tails of histone H4 are proposed to interact with a complex of Sir proteins, thus stabilizing the association (more...)

But how is the Sir2 protein delivered to the ends of chromosomes in the first place? Another series of experiments has suggested the model shown in Figure 4-47B. A DNA-binding protein that recognizes specific DNA sequences in yeast telomeres also binds to one of the Sir proteins, causing the entire Sir protein complex to assemble on the telomeric DNA. The Sir complex then spreads along the chromosome from this site, modifying the N-terminal tails of adjacent histones to create the nucleosome-binding sites that the complex prefers. This “spreading effect” is thought to be driven by the cooperative binding of adjacent Sir protein complexes, as well as by the folding back of the chromosome on itself to promote Sir binding in nearby regions (see Figure 4-47B). In addition, the formation of heterochromatin probably requires the action of chromatin remodeling complexes to readjust the positions of nucleosomes as they are packed together.

Unlike most deacetylases, Sir2 requires NAD+ as a cofactor (see Figure 2-60). The NAD+ levels in the cell fluctuate with the nutritional health of the cell, increasing as cells become nutritionally deprived. This feature might cause the telomeric heterochromatin to spread in response to starvation (perhaps to silence the expression of genes that are not absolutely required for survival) and then to retract when conditions improve.

The properties of the yeast heterochromatin just described may resemble features of heterochromatin in more complex organisms. Certainly, the spreading of yeast heterochromatin from telomeres is similar in principle to the movement of heterochromatin that causes position effect variegation in animals (see Figure 4-46). Moreover, these properties can be used to explain the heritability of heterochromatin, as outlined in Figure 4-48. Whatever the precise mechanism of heterochromatin formation, it has become clear that covalent modifications of the nucleosome core histones have a critical role in this process. Of special importance in many organisms are the histone methyl transferases, enzymes that methylate specific lysines on histones including lysine 9 of histone H3 (see Figure 4-35). This modification is “read” by heterochromatin components (including HP1 in Drosophila) that specifically bind this modified form of histone H3 to induce the assembly of heterochromatin. It is likely that a spectrum of different histone modifications is used by the cell to distinguish heterochromatin from euchromatin (see Figure 4-35).

Figure 4-48. Two speculative models for how the tight packaging of DNA in heterochromatin can be inherited during chromosome replication.

Figure 4-48

Two speculative models for how the tight packaging of DNA in heterochromatin can be inherited during chromosome replication. In both cases, half of the specialized heterochromatin components have been distributed to each daughter chromosome after DNA (more...)

Having the ends of chromosomes packaged into heterochromatin provides several advantages to the cell: it helps to protect the ends of chromosomes from being recognized as broken chromosomes by the cellular repair machinery, it may help to regulate telomere length, and it may assist in the accurate pairing and segregation of chromosomes during mitosis. In Chapter 5 we see that telomeres have additional structural features that distinguish them from other parts of chromosomes.

Centromeres Are Also Packaged into Heterochromatin

Heterochromatin is also observed around centromeres, the DNA sequences that direct the movement of each chromosome into daughter cells every time a cell divides (see Figure 4-22). In many complex organisms, including humans, each centromere seems to be embedded in a very large stretch of heterochromatin that persists throughout interphase, even though the centromere-directed movement of DNA occurs only during mitosis. The structure and biochemical properties of this so-called centric heterochromatin are not well understood, but, like other forms of heterochromatin, it silences the expression of genes that are experimentally placed into it. It contains, in addition to histones (which are typically underacetylated and methylated in heterochromatin), several additional structural proteins that compact the nucleosomes into particularly dense arrangements.

As with telomeres, our best understanding of the chromatin structure of a centromere comes from studies of the much simpler centromeres of the yeast S. cerevisiae. Earlier in this chapter we saw that a simple DNA sequence of approximately 125 nucleotide pairs was sufficient to serve as a centromere in this organism. Despite its small size, more than a dozen different proteins assemble on this DNA sequence; the proteins include a histone H3 variant that, along with the other core histones, is believed to form a centromere-specific nucleosome (Figure 4-49A). We do not yet understand what properties this variant type of nucleosome provides to the cell, but similar specialized nucleosomes seem to be present in all eucaryotic centromeres (Figure 4-49B). The additional proteins at the yeast centromere attach it to the spindle microtubules and provide signals that ensure that this attachment is complete before the later stages of mitosis are allowed to proceed (discussed in Chapters 17 and 18).

Figure 4-49. The specialized nucleosome formed on centromeres.

Figure 4-49

The specialized nucleosome formed on centromeres. (A) A model for the proteins that assemble on a yeast centromere. The specialized nucleosome contains an H3 variant (called CENP-A in most organisms), along with core histones H2A, H2B, and H4. The folding (more...)

The centromeres in more complex organisms are considerably larger than those in budding yeasts. For example, fly and human centromeres extend over hundreds of thousands of nucleotide pairs and do not seem to contain a centromere-specific DNA sequence. Rather, most consist largely of short, repeated DNA sequences, known as alpha satellite DNA in humans (Figure 4-50). But the same repeat sequences are also found at other (noncentromeric) positions on chromosomes, and how they specify a centromere is poorly understood. Somehow the formation of the inner plate of a kinetochore is “seeded,” followed by the cooperative assembly of the entire group of special proteins that form the kinetochore (Figure 4-50B). It seems that centromeres in complex organisms are defined more by an assembly of proteins than by a specific DNA sequence.

Figure 4-50. The structure of a human centromere.

Figure 4-50

The structure of a human centromere. (A) The organization of the alpha satellite DNA sequences, which are repeated many thousands of times at a centromere. (B) An entire chromosome. The alpha satellite DNA sequences (red) are AT-rich and consist of a (more...)

There are some striking similarities between the formation and maintenance of centromeres and the formation and maintenance of other regions of heterochromatin. The entire centromere forms as an all-or-none entity, suggesting a highly cooperative addition of proteins after a seeding event. Moreover, once formed, the structure seems to be directly inherited on the DNA as part of each round of chromosome replication. Thus, for example, some regions of our chromosomes contain nonfunctional alpha satellite DNA sequences that seem to be identical to those at the centromere; these sequences are presumed to have arisen from a chromosome-joining event that initially created one chromosome with two centromeres (an unstable, dicentric chromosome; Figure 4-51A). Moreover, in some unusual cases, new human centromeres (called neocentromeres) have been observed to form spontaneously on fragmented chromosomes. Some of these new positions were originally euchromatic and lack alpha satellite DNA altogether (Figure 4-51B).

Figure 4-51. The plasticity of human centromere formation.

Figure 4-51

The plasticity of human centromere formation. (A) Owing to an ancient chromosome breakage and rejoining event, some human chromosomes contain two blocks of alpha satellite DNA (red), each of which presumably functioned as a centromere in its original (more...)

To explain these observations it has been proposed that de novo centromere formation requires an initial marking (or seeding) event, perhaps the formation of a specialized DNA-protein structure, which, in humans, happens more readily on arrays of alpha satellite DNA than on other DNA sequences. This mark would be duplicated when the chromosome divides, and the same centromere would then function in the next cell division. Very rarely, the mark would be lost after chromosome replication, in which case it would be very difficult to establish again (Figure 4-51C). Although the self-renewing nature of centromeres is not understood in detail, the type of models described for heterochromatin inheritance in Figure 4-48 could also be critical here.

The plasticity of centromeres may provide an important evolutionary advantage. We have seen that chromosomes evolve in part by breakage and rejoining events (see Figure 4-19). Many of these events produce chromosomes with two centromeres, or chromosome fragments with no centromeres at all. Although rare, the inactivation of centromeres and their ability to be activated de novo may occasionally allow newly formed chromosomes to be maintained stably and thereby facilitate the process of chromosome evolution.

Heterochromatin May Provide a Defense Mechanism Against Mobile DNA Elements

DNA packaged in heterochromatin often consists of large tandem arrays of short, repeated sequences that do not code for protein, as we saw above for the heterochromatin of mammalian centromeres. In contrast, euchromatic DNA is rich in genes and other single-copy DNA sequences. Although this correlation is not absolute (some arrays of repeated sequences exist in euchromatin and some genes are present in heterochromatin), this trend suggests that some types of repeated DNA may be a signal for heterochromatin formation. This idea is supported by experiments in which several hundred tandem copies of genes have been artificially introduced into the germ lines of flies and mice. In both organisms these gene arrays are often silenced, and in some cases, they can be observed under the microscope to have formed regions of heterochromatin. In contrast, when single copies of the same genes are introduced into the same position in the chromosome, they are actively expressed.

This feature, called repeat-induced gene silencing, may be a mechanism that cells have for protecting their genomes from being overtaken by mobile genetic elements. These elements, which are discussed in Chapter 5, can multiply and insert themselves throughout the genome. According to this idea, once a cluster of such mobile elements has formed, the DNA that contains them would be packaged into heterochromatin to prevent their further proliferation. The same mechanism could be responsible for forming the large regions of heterochromatin that contain large numbers of tandem repeats of a simple sequence, as occurs around centromeres.

Mitotic Chromosomes Are Formed from Chromatin in Its Most Condensed State

Having discussed the dynamic structure of interphase chromosomes, we now turn to the final level of DNA packaging, that observed for mitotic chromosomes. With the exception of a few specialized cases, such as the lampbrush and polytene chromosomes discussed above, most interphase chromosomes are too extended and entangled for their structures to be clearly seen. In contrast, the chromosomes from nearly all eucaryotic cells are readily visible during mitosis, when they coil up to form highly condensed structures. It is remarkable that this further condensation, which reduces the length of a typical interphase chromosome only about tenfold, produces such a dramatic change in the appearance of chromosomes.

Figure 4-52 depicts a typical mitotic chromosome at the metaphase stage of mitosis. The two daughter DNA molecules produced by DNA replication during interphase of the cell-division cycle are separately folded to produce two sister chromosomes, or sister chromatids, held together at their centromeres (see also Figure 4-21). These chromosomes are normally covered with a variety of molecules, including large amounts of RNA-protein complexes. Once this covering has been stripped away, each chromatid can be seen in electron micrographs to be organized into loops of chromatin emanating from a central scaffolding (Figures 4-53 and 4-54). Several types of experiment demonstrate that the order of visible features along a mitotic chromosome at least roughly reflects the order of the genes along the DNA molecule. Mitotic chromosome condensation can thus be thought of as the final level in the hierarchy of chromosome packaging (Figure 4-55).

Figure 4-52. A typical mitotic chromosome at metaphase.

Figure 4-52

A typical mitotic chromosome at metaphase. Each sister chromatid contains one of two identical daughter DNA molecules generated earlier in the cell cycle by DNA replication.

Figure 4-53. A scanning electron micrograph of a region near one end of a typical mitotic chromosome.

Figure 4-53

A scanning electron micrograph of a region near one end of a typical mitotic chromosome. Each knoblike projection is believed to represent the tip of a separate looped domain. Note that the two identical paired chromatids drawn in Figure 4-52 can be clearly (more...)

Figure 4-54. An electron micrograph of a mitotic chromosome.

Figure 4-54

An electron micrograph of a mitotic chromosome. This chromosome (from an insect) was treated to reveal loops of chromatin fibers that emanate from a central scaffold of the chromatid. Such micrographs support the idea that the chromatin in all chromosomes (more...)

Figure 4-55. Chromatin packing.

Figure 4-55

Chromatin packing. This model shows some of the many levels of chromatin packing postulated to give rise to the highly condensed mitotic chromosome.

The compaction of chromosomes during mitosis is a highly organized and dynamic 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 easily separate when the mitotic apparatus begins pulling them apart. Second, the compaction of chromosomes protects the relatively fragile DNA molecules from being broken as they are pulled to separate daughter cells.

The condensation of interphase chromosomes into mitotic chromosomes occurs in M phase, and it is intimately connected with the progression of the cell cycle, as discussed detail in Chapters 17 and 18. It requires a class of proteins called condensins which using the energy of ATP hydrolysis, drive the coiling of each interphase chromosome that produces a mitotic chromosome. Condensins are large protein complexes that contain SMC proteins: long, dimeric protein molecules hinged in the center, with globular domains at each end that bind DNA and hydrolyze ATP (Figure 4-56). When added to purified DNA, condensins use the energy of ATP hydrolysis to make large right-handed loops in the DNA. Although it is not yet known how they act on chromatin, the coiling model shown in Figure 4-56C is based on the fact that condensins are a major structural component of mitotic chromosomes, with one molecule of condensin being present for every 10,000 nucleotides of mitotic DNA.

Figure 4-56. The SMC proteins in condensins.

Figure 4-56

The SMC proteins in condensins. (A) Electron micrographs of a purified SMC dimer. (B) The structure of an SMC dimer. The long central region of this protein is an antiparallel coiled coil (see Figure 3-11) with a flexible hinge in its middle, as demonstrated (more...)

Each Mitotic Chromosome Contains a Characteristic Pattern of Very Large Domains

As mentioned earlier, the display of the 46 human chromosomes at mitosis is called the human karyotype. When stained with dyes such as Giemsa, mitotic chromosomes show a striking and reproducible banding pattern along each chromosome, as shown in Figure 4-11. These bands are unrelated to those described earlier for the insect polytene chromosomes, which correspond to relatively small regions of interphase chromatin. In a human mitotic chromosome, all the chromatin is condensed and the bands represent a selective binding of the dyes.

By examining human chromosomes very early in mitosis, when they are less condensed than at metaphase, it has been possible to estimate that the total haploid genome contains about 2000 distinguishable bands. These coalesce progressively as condensation proceeds during mitosis, producing fewer and thicker bands. As we saw earlier, cytogeneticists routinely use the pattern of these chromosome bands to discover in patients genetic alterations such as chromosome inversions, translocations, and other types of chromosomal rearrangements (see Figure 4-12).

Mitotic chromosome bands are detected in chromosomes from species as diverse as humans and flies. Moreover, the pattern of bands in a chromosome has remained unchanged over long periods of evolutionary time. Each human chromosome, for example, has a clearly recognizable counterpart with a nearly identical banding pattern in the chromosomes of the chimpanzee, gorilla, and orangutan—although there are also clear differences, such as chromosome fusion, that give the human 46 chromosomes instead of the ape's 48 (Figure 4-57). This conservation suggests that chromosomes are organized into large domains that may be important for chromosomal function.

Figure 4-57. Comparison of the Giemsa pattern of the largest human chromosome (chromosome 1) with that of chimpanzee, gorilla, and orangutan.

Figure 4-57

Comparison of the Giemsa pattern of the largest human chromosome (chromosome 1) with that of chimpanzee, gorilla, and orangutan. Comparisons among the staining patterns of all the chromosomes indicate that human chromosomes are more closely related to (more...)

Even the thinnest of the bands in Figure 4-11 probably contains more than a million nucleotide pairs, which is nearly the size of a bacterial genome. These bands seem to reflect a rough division of chromosomes into regions of different GC content. The nucleotide sequence of the human genome has revealed large non-random blocks of sequence (some greater than 107 nucleotide pairs) that are significantly higher or lower in GC content than the genome-wide average of 41%. The blocks correlate roughly with the staining pattern of metaphase chromosomes. For example, bands that are darkly stained by Giemsa (the so-called G-bands) are correlated with DNA that is low in GC content, whereas lightly stained bands (the R-bands) correspond to DNA of higher than average GC content.

In general, GC-rich regions of the genome have a higher density of genes, especially of “house-keeping” genes, the genes that are expressed in virtually all cell types. On the basis of these observations, it has been proposed that the banding pattern may be related to gene expression. Perhaps the differentiation of chromosomes into G- and R-bands reflects subtle differences, determined by GC content, in the way in which chromatin loops are packaged in these areas. If this idea is correct, the rough division of chromosomes can be seen as a form of compartmentalization, in which the particular cellular components involved in gene expression are more concentrated in the R-bands where their activities are required. In any case, it should be obvious from this discussion that we are only beginning to glimpse the principles of large-scale chromosome organization.

Individual Chromosomes Occupy Discrete Territories in an Interphase Nucleus

We saw earlier in this chapter that chromosomes from eucaryotes are contained in the cell nucleus. However, the nucleus is not simply a bag of chromosomes; rather, the chromosomes—as well as the other components inside the nucleus which we shall encounter in subsequent chapters—are highly organized. The way in which chromosomes are organized in the nucleus during interphase, when they are active and difficult to see, has intrigued biologists since the nineteenth century. Although our understanding today is far from complete, we do know some interesting features of these chromosome arrangements.

A certain degree of chromosomal order results from the configuration that the chromosomes always have at the end of mitosis. Just before a cell divides, the condensed chromosomes are pulled to each spindle pole by microtubules attached to the centromeres; thus, as the chromosomes move, the centromeres lead the way and the distal arms (terminating in the telomeres) lag behind. The chromosomes in some nuclei tend to retain this so-called Rabl orientation throughout interphase, with their centromeres facing one pole of the nucleus and their telomeres pointing toward the opposite pole (Figures 4-58 and 4-59).

Figure 4-58. The polarized orientation of chromosomes found in certain interphase nuclei.

Figure 4-58

The polarized orientation of chromosomes found in certain interphase nuclei. (A) Fluorescent light micrograph of interphase nuclei from the rapidly growing root tip of a plant. Centromeres are stained green and teleomeres red by in situ hybridization (more...)

Figure 4-59. A polymer analogy for interphase chromosome organization.

Figure 4-59

A polymer analogy for interphase chromosome organization. (A) The behavior of a polymer in solution. Entropy drives a long polymer into a compact conformation in the absence of an externally applied force. If the polymer is subjected to shear or hydrodynamic (more...)

The chromosomes in most interphase cells are not found in the Rabl orientation; instead, the centromeres seem to be dispersed in the nucleus. Most cells have a longer interphase than the specialized cells illustrated above, and this presumably gives their chromosomes time to assume a different conformation (see Figure 4-59). Nevertheless, each interphase chromosome does tend to occupy a discrete and relatively small territory in the nucleus: that is, the different chromosomes are not extensively intertwined (Figure 4-60).

Figure 4-60. Selective “painting” of two interphase chromosomes in a human peripheral lymphocyte.

Figure 4-60

Selective “painting” of two interphase chromosomes in a human peripheral lymphocyte. The fluorescent light micrograph shows that the two copies of human chromosome 18 (red) and chromosome 19 (turquoise) occupy discrete territories of the (more...)

One device for organizing chromosomes in the nucleus may be the attachment of certain portions of each chromosome to the nuclear envelope (Figure 4-61). For example, in many cells, telomeres seem bound in this way. But the exact position of a chromosome in a nucleus is not fixed. In the same tissue, for example, two apparently identical cells can have different chromosomes as nearest neighbors.

Figure 4-61. Specific regions of interphase chromosomes in close proximity to the nuclear envelope.

Figure 4-61

Specific regions of interphase chromosomes in close proximity to the nuclear envelope. This high-resolution microscopic view of nuclei from a Drosophila embryo shows the localization of two different regions of chromosome 2 (yellow and magenta) close (more...)

Some cell biologists believe that there is an intranuclear framework, analogous to the cytoskeleton, on which chromosomes and other components of the nucleus are organized. The nuclear matrix, or scaffold, has been defined as the insoluble material left in the nucleus after a series of biochemical extraction steps. Some of the proteins that constitute it can be shown to bind specific DNA sequences called SARs or MARs (scaffold-associated or matrix-associated regions). These DNA sequences have been postulated to form the base of chromosomal loops (see Figure 4-44), or to attach chromosomes to the nuclear envelope and other structures in the nucleus. By means of such chromosomal attachment sites, the matrix might help to organize chromosomes, localize genes, and regulate gene expression and DNA replication. It still remains uncertain, however, whether the matrix isolated by cell biologists represents a structure that is present in intact cells.

Summary

Chromosomes are generally decondensed during interphase, so that their structure is difficult to visualize directly. Notable exceptions are the specialized lampbrush chromosomes of vertebrate oocytes and the polytene chromosomes in the giant secretory cells of insects. Studies of these two types of interphase chromosomes suggest that each long DNA molecule in a chromosome is divided into a large number of discrete domains organized as loops of chromatin, each loop probably consisting of a folded 30-nm chromatin fiber. When genes contained in a loop are expressed, the loop decondenses and allows the cell's machinery easy access to the DNA.

Euchromatin makes up most of interphase chromosomes and probably corresponds to looped domains of 30-nm fibers. However, euchromatin is interrupted by stretches of heterochromatin, in which 30-nm fibers are subjected to additional levels of packing that usually render it resistant to gene expression. Heterochromatin is commonly found around centromeres and near telomeres, but it is also present at other positions on chromosomes. Although considerably less condensed than mitotic chromosomes, interphase chromosomes occupy discrete territories in the cell nucleus; that is, they are not extensively intertwined.

All chromosomes adopt a highly condensed conformation during mitosis. When they are specially stained, these mitotic chromosomes have a banding structure that allows each individual chromosome to be recognized unambiguously. These bands contain millions of DNA nucleotide pairs, and they reflect a poorly-understood coarse heterogeneity of chromosome structure.

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Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
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