Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intracellular macromolecular sorting and maintenance of cellular compartments
Chromosome structure

Having discussed The Structure of DNA and the Proteins that make up Chromosomes, let us now examine chromosome Organization as a whole. It turns out that DNA within a chromosome is not only packaged by Histones into regularly repeating nucleosomes, but is also intricately arranged with other proteins into a series of subdomains with distinct properties. These higher-order structures are a striking feature of eukaryotic chromatin; exactly how such domains function remains an enigma.

9.2.1. Chromosomes appear to consist of a series of loops [21]

The nuclear diameter typically does not exceed 5 µm (5 x 10⁴ cm). Because DNA packaging within the chromatin fibril reduces its linear dimensions to 1 mm, there must be higher levels of compaction. One of the principles of further chromatin Condensation was suggested by the appearance of certain specialized chromosomes—the so-called lampbrush chromosomes from the oocytes of many animals and the polytene chromosomes of specific insect Cells. Both of these chromosome types exhibit a well-defined loop structure, meaning they possess a series of loop domains that project radially from the main chromosome axis. It has been established that the chromosome of the bacterium E. coli (a circular DNA molecule about 0.1 cm long, lacking histones) is also organized into loops. Current evidence suggests that loop arrangement is a universal principle of Chromatin Structural Organization.

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Fig. 9-34. Schematic representation of a chromosome region with loop organization. Each loop contains approximately 20,000 to 100,000 Base Pairs of double-stranded DNA incorporated into the 30 nm chromatin fibril. A. A packaging model in which a site-specific DNA-binding protein is located at each Base of the loop. B. A packaging model involving a chromosome scaffold. The actual mechanism of packaging remains unknown, although cytological evidence indicates that the axial region of isolated mitotic chromosomes (the localization site of loop bases) is heavily enriched with DNA topoisomerase II, an enzyme abundant in The Cell.

Fig. 9-35. Schematic representation of a typical metaphase chromosome. Each chromatid contains one of two identical daughter DNA molecules (one is highlighted in color) generated by Replication earlier in the Cell Cycle.

It has been hypothesized that chromatin loops are formed and maintained by DNA-binding proteins that recognize specific nucleotide sequences at two separate sites on the chromatin fibril and bring them together (Fig. 9-34A). As a result, these sites form the base of the loop. Loops can also form alternatively through the binding of DNA to the chromosome axis (Fig. 9-34B). Indirect estimates indicate that in taxonomically distant organisms such as Drosophila and humans, the average loop length is remarkably similar; a typical loop contains roughly 20,000 to 100,000 base pairs, which corresponds to about 0.5 µm of a 30 nm-diameter fibril. If a typical human chromosome consists primarily of loops, it could comprise over 2,000 such domains.

9.2.2. Mitotic chromosomes consist of maximally condensed chromatin [22]

Most chromosomes are extremely extended and intertwined, making them invisible during all Phases of the cell cycle except mitosis. During this period, chromosomes coil, condense, and acquire distinct shapes. This supercoiling, which reduces the linear dimensions of DNA from 5 cm to 5 µm, is accompanied by the phosphorylation of all cellular histone H1 molecules at five Serine residues. Because histone H1 links adjacent nucleosome particles (see Fig. 9-28), its phosphorylation may play a pivotal role in chromosome condensation during mitosis. Fig. 9-35 illustrates a typical mitotic chromosome at metaphase. The two daughter DNA molecules are packaged separately to form sister chromatids, which are held together by the centromere. Mitotic chromosomes typically bear numerous other molecules on their surface, including a large quantity of ribonucleoproteins. When this coat is removed, electron micrographs clearly reveal that each chromatid is built from chromatin loops radiating from a central axis (Figs. 9-36 and 9-37). Experiments have proven that the transverse banding pattern characteristic of mitotic chromosomes reflects, to some extent, the linear order of genes along the DNA molecule. Various modes of packing the long DNA helix are shown in Fig. 9-38.

Fig. 9-36. Scanning electron micrograph of a region of a highly condensed mitotic chromosome. Each nodule-like protrusion is thought to represent the base of an individual loop domain. Note the clear distinction between the two identical paired chromatids diagrammed in Fig. 9-35. (From Marsden M.P., Laemmli U.K. Cell 17: 849-858, 1979.)

Fig. 9-37. Transmission electron micrograph of a single chromatid from an insect mitotic chromosome (Oncopeltus). Special preparation allows visualization of the chromatin loops extending from the central axis of the chromatid. (Courtesy of Victoria Foe.)

Fig. 9-38. Diagram illustrating the various levels of chromatin packing, which presumably reflect successive stages in The formation of a highly condensed metaphase chromosome.

In the mitotic chromosome, chromatin is transcriptionally inactive: RNA Synthesis ceases upon the onset of chromosome condensation. Apparently, RNA polymerase is unable to progress along the DNA under these conditions, although other factors may also be involved.

9.2.3. Each mitotic chromosome contains a specific set of very large domains [23]

The Complement of 46 human mitotic chromosomes is referred to as the karyotype. Cytological techniques developed in the early 1970s make it possible to unambiguously identify each chromosome in the Human Karyotype. For this purpose, specially prepared mitotic chromosome spreads are treated with fluorochromes that selectively bind to specific types of DNA sequences. Although these Dyes have relatively low sequence Specificity (their main use is distinguishing AT-rich DNA regions, or G-bands, from GC-rich regions, or R-bands), staining mitotic chromosomes with them reveals a characteristic pattern of alternating dark and light bands (segments) on each chromosome (Fig. 9-39). The distribution of these bands is specific for each chromosome type, enabling their definitive identification (Fig. 9-40).

Fig. 9-39. Micrographs of three pairs of human mitotic chromosomes obtained by Fluorescence Microscopy. A. Staining with the AT-specific dye Hoechst 33258 (G-bands). B. Staining with the GC-specific dye olivomycin (R-bands). The bar indicates THE POSITION OF the centromere. Note that the banding patterns in the chromosomes of both photographs are complementary: bands that fluoresce brightly in A are dark in B, and vice versa. G-bands also appear upon staining with Giemsa (hence their name), whereas the designation R-bands reflects the fact that they are essentially the reverse of G-bands. (From K.F. Jorgenson, J.H. van de Sande and C.C. Lin, Chromosoma 68: 287-302, 1978.)

Analysis of Human chromosomes using fluorescent dyes during early mitosis—when chromosomes are significantly less compact than at metaphase—has made it possible to estimate that the entire haploid set contains at least 2,000 distinct bands corresponding to AT-rich DNA sequences.

Fig. 9-40. Standard idiogram showing the distribution of segments on each chromosome of the human karyotype at prometaphase of mitosis. Chromosomes 1 through 22 are numbered roughly in order of decreasing size. A diploid human cell contains two chromosomes of each type plus two X chromosomes (in females) or an X and a Y chromosome (in males). The 850 bands indicated in the figure are G-bands revealed by staining with Reagents specific for AT-rich DNA sequences. The highlighted "satellites" on chromosomes 13, 14, 15, 21, and 22 indicate the locations of genes encoding large Ribosomal RNAs, and the colored lines mark the position of the centromere on each chromosome. (Modified from U. Franke, Cytogenet. Cell Genet. 31: 24-32, 1981.)

During mitosis, further condensation of chromosomes occurs, resulting in the thickening of bands and a reduction in their number.

The presence of transverse banding is a common feature of mitotic chromosomes, even in species as evolutionarily distant as humans and Drosophila. Moreover, the band distribution pattern along the chromosomes has remained remarkably unchanged over long periods of evolution. For example, almost every human chromosome has a counterpart in the karyotypes of chimpanzees, gorillas, and orangutans (although due to the fusion of a single chromosome pair, humans have 46 chromosomes rather than the 48 found in apes), and their banding patterns are virtually identical. All of this further underscores the critical importance of the Spatial Organization of DNA within chromosomes for the expression of respective genes, while the very existence of these bands may reflect certain functional features of chromatin organization. Why such bands form remains a mystery to this day. Even the finest bands depicted in Fig. 9-40 must contain at least 30 loops, and the cumulative Nucleotide Composition of such long DNA sequences (over a million base pairs, which corresponds to the size of an average bacterial genome) appears to be close to the statistical average. It is known that both AT-rich and GC-rich bands contain genes.

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9.2.4. Interphase lampbrush chromosome DNA consists of a series of distinct domains [24]

Despite the high order of chromatin packing, its fibers during interphase are too thin and tangled to allow the visualization of an entire chromosome in its entirety. Nevertheless, there are specific cell types in which the overall structure of interphase chromosomes can be discerned. For example, meiotically paired chromosomes of growing oocytes (immature egg cells) actively synthesize RNA and form unusually rigid and extended chromatin loops coated with newly transcribed RNA packaged into dense RNA-Protein Complexes. Because the DNA is covered by such complexes, these chromosomes (known as lampbrush chromosomes) are clearly visible even under a Light Microscope (Fig. 9-41).

Fig. 9-41. Light micrograph of lampbrush chromosomes from an amphibian oocyte. At Cytology/cytology/16.html">Early stages of oocyte differentiation, each chromosome replicates before Meiosis begins, and the homologous pairs synapse to form the four-chromatid extended structure shown in the photograph. The lampbrush chromosome stage can last for months or even years. During this time, the oocyte accumulates a reserve of mRNA and other substances required for its subsequent development into a new Organism. Note that each chromosome axis is approximately 400 µm in length, whereas the length of most mitotic chromosomes is less than 10 µm. (Courtesy of Joseph G. Gall.)

Fig. 9-42. Structure of a lampbrush chromosome. In many amphibians, the set of lampbrush chromosomes contains a total of about 10,000 chromatin loops, although the bulk of the DNA remains in a highly condensed state located within chromomeres. Each loop corresponds to a specific DNA sequence. Every cell contains four copies of each loop because the structure shown at the top of the figure consists of two paired homologous chromosomes, with each chromosome represented by two sister chromatids. Such four-stranded structures are characteristic of this stage of oocyte development (the diplotene stage of meiosis).

The structural layout of lampbrush chromosomes is illustrated in Fig. 9-42. Large loops composed of decondensed chromatin project laterally from the chromosome axis. Nucleic acid Hybridization experiments have demonstrated that a given loop always contains the same DNA sequence, which is positioned in a strictly defined manner during oocyte growth. Consequently, these loops correspond to fixed units of chromatin packaging that have decondensed and become transcriptionally active. Because an average-sized loop contains approximately 100,000 base pairs, each loop may correspond to a single chromatin loop as described above (see Section 9.2.1). Many loops are continuously transcribed along their entire length, whereas others contain extended regions of chromatin that are not transcribed at all. The majority of chromatin is not incorporated into loops and remains highly condensed within chromomeres; as a rule, this chromatin is not transcribed. Short regions of chromatin that lack a high degree of condensation and are not actively transcribed connect adjacent chromomeres along the well-defined chromosome axis.

Lampbrush chromosomes are unusual in that their METABOLISM/31.html">Transcription rate is higher, and most of the resulting RNA transcripts are longer, than those synthesized on other chromosomes. However, evidence suggests that the DNA molecule comprising any interphase chromosome is similarly subdivided into distinct regions, each separated from its neighbors by a boundary. Here too, chromatin appears to be packaged differently in different regions (e.g., as loops, chromomeres, or interchromomeric axis-associated chromatin).

9.2.5. Ordered interphase chromatin regions can also be observed in polytene chromosomes [25]

Chromatin Structure at the level of individual loops is also clearly discernible in certain insect cells. Many fly larval cells grow to an unusually large size by undergoing several cycles of DNA replication not accompanied by Cell Division. As a result, they contain several thousand times more DNA than a normal cell. Such giant cells are called polyploid if their set of chromosome numbers exceeds the normal level. Alternatively, homologous chromosome pairs may fail to separate from one another, instead forming single massive chromosomes (polytene chromosomes). The fact that chromosomes in certain giant insect cells can transition from a polytene to a polyploid state indicates that these two chromosomal statuses are closely related and that the structure of polytene chromosomes is fundamentally analogous to that of normal chromosomes.

Fig. 9-43. Complete set of polytene chromosomes from a Drosophila salivary gland cell. These chromosomes were spread and prepared for observation by squashing the material on a microscope slide. The figure shows four pairs of chromosomes. Each chromosome is tightly paired with its homolog (such that each pair appears as a single structure), which is not observed in most interphase nuclei. The four pairs of chromosomes are linked together at their centromeric regions, forming a single large "chromocenter" (stained area). In this preparation, the chromocenter split into two parts during slide preparation. It should be noted that the side-by-side alignment of numerous chromatin threads resulted in a significant unwinding of each DNA molecule. (Modified from T. S. Painter, J. Hered. 25: 465-476, 1934.)

Fig. 9-44. Light micrograph of a region of a polytene chromosome from a Drosophila salivary gland cell. The characteristic banding pattern is visible. These bands are found in interphase chromosomes and are a hallmark of giant polytene chromosomes; they should not be confused with the much coarser bands shown in Fig. 9-40, which are revealed in normal mitotic chromosomes using special stains. (Courtesy of Joseph G. Gall.)

Fig. 9-45. Electron micrograph of a small region of a Drosophila polytene chromosome. A thin section reveals that different chromosomal bands, varying greatly in thickness (B), are separated by interband regions (I) whose chromatin is much less condensed. (Courtesy of Veikko Sorsa.)

Due to their large size, and because the tight and strictly ordered packing of individual chromatin threads within a polytene chromosome prevents them from tangling, these chromosomes are readily visible under a light microscope. Like lampbrush chromosomes, polytene chromosomes actively synthesize RNA during interphase. The phenomenon of polyteny has been most thoroughly studied in the four chromosomes of Drosophila larval salivary gland cells. In these cells, DNA replicates 10 consecutive times without the daughter chromosomes separating, resulting in giant chromosomes containing 1,024 (2¹⁰) closely appressed individual chromatin threads.

When stained polytene chromosomes are observed under a light microscope, alternating transverse bands are clearly noticeable: dark bands (chromomeres or discs) and light bands (interband regions) (Figs. 9-43 and 9-44). Each disc and interband region consists of 1,024 identical DNA sequences arranged side by side. About 85% of the DNA in polytene chromosomes is contained in the discs and 15% in the interband regions. The chromatin of each disc appears darker upon staining because it is more condensed than the chromatin of the interband regions (Fig. 9-45). It is believed that a disc consists of a repeatedly folded loop (Fig. 9-46). Depending on their size, individual bands contain from 3,000 to 300,000 nucleotide pairs. Because each disc can be identified by its thickness and position along the chromosome, all discs can be numbered, making it possible to construct a "map" of the polytene chromosome. The entire Drosophila genome contains approximately 5,000 discs and 5,000 interband regions.

9.2.6. Individual chromatin domains in polytene chromosomes can unfold and repackage as discrete units [26]

Long before the first data on chromatin structure were obtained, studies of polytene chromosomes led to the hypothesis that Gene transcription is accompanied by significant Changes in DNA packaging: an individual chromosome disc puffs out when the genes it contains are activated and re-condenses when the genes become inactive.

To identify transcribed regions on a polytene chromosome, cells can be fed a radioactive RNA precursor, [³H]-uridine, after which the growing RNA transcripts can be localized by autoradiography (Fig. 9-47). It was precisely by this method that it was established that the bulk of active chromatin is in a decondensed state and forms characteristic chromosomal puffs.

Fig. 9-46. Diagram illustrating how closely appressed homologous loop domains can give rise to discs in polytene chromosomes. Within each disc, the loops of chromatin fibrils are in very close contact with one another, forming a much more condensed structure than is depicted in this drawing.

One of the primary factors controlling gene activity in Drosophila polytene chromosomes is ecdysone, an insect hormone. The level of this hormone rises and falls periodically during larval development, inducing the transcription of various genes that encode proteins required by the larva for molting and pupariation. As the larva progresses through specific developmental stages, new puffs appear and old ones disappear, a process associated with the activation and cessation of transcription units and the synthesis of diverse mRNAs and proteins (Fig. 9-48). The Study of an individual puff, which is relatively small in size (though its corresponding band on the chromosome is still discernible), suggests that each puff is formed by the uncoiling of a single chromosome band (Fig. 9-49). Electron microscopic analysis has shown that the DNA in puffs is in a much less condensed state than the characteristic 30 nm chromatin fibril (Fig. 9-50). Apparently, an individual loop, which is believed to be packaged into a band on the chromosome (Fig. 9-46), decondenses as an independent unit during transcription.

9.2.7. Genes on polytene chromosomes are likely located in both bands and interband regions [27]

The fixed arrangement of bands and interband regions on the Drosophila polytene chromosome led cytogeneticists to suggest that each band might correspond to an individual gene. Mutation analysis not only confirmed this hypothesis but also allowed geneticists to estimate the number of vital genes in Drosophila, which turned out to be approximately 5,000—roughly equal to the number of chromosomal bands. For example, when attempting to induce Mutations mapped to a small chromosomal region (containing about 50 different bands) using Genetic Methods, researchers identified about 50 essential genes. Although this method does not pinpoint whether a specific gene is localized within a band or an interband region, these observations suggest the following Conclusion: a typical band may contain DNA sequences encoding a single essential protein.

However, subsequent experiments cast doubt on the validity of the "one band - one gene" hypothesis. For instance, a substantial region of the Drosophila genome spanning 315,000 nucleotide pairs was cloned, and its individual fragments were used as probes to identify mRNAs synthesized in this region. The number of distinct mRNAs turned out to be three times greater than the number of bands. It is very likely that a large fraction of these mRNAs correspond to genes that affect fly fitness specifically under natural conditions. In the laboratory, in the absence of selective pressure, mutations in many of these genes may go undetected. It has recently been established that mRNA is synthesized in both bands and interband regions.

Fig. 9-47. RNA synthesis on a giant polytene chromosome from the Salivary Glands of the insect Chironomus tentans. In this autoradiograph of a chromosome labeled with [³H]-uridine, regions of RNA synthesis are covered with dark silver grains corresponding to The activity of each region. (After S. Felling, Chromosoma 15: 71-122, 1964.)

Fig. 9-48. A series of photographs illustrating successive stages in the formation and regression of puffs in polytene chromosomes of Drosophila melanogaster. A fragment of the left arm of chromosome 3 containing five major puffs is shown. Each of these puffs is active only for a short period: The sequence of events captured in the photographs takes 22 hours and is repeated in every generation of flies. (Courtesy of Michael Ashburner.)

Fig. 9-49. A. Simplified diagram of puff formation on a polytene chromosome. B. Autoradiograph of an individual puff in which RNA was synthesized in the indicated region, resulting in labeling with [³H]-uridine. (Courtesy of Jose Bonner.)

Fig. 9-50. ELECTRON MICROGRAPHS OF a series of thin sections through a large chromosomal puff, showing the conformation of an unusually long transcription unit. Diagram D illustrates a spatial model of the investigated region of this transcription unit. Knob-like ends of the RNA transcripts are attached to a single chromatin fibril. The "knobs" at the 5'-end of most transcripts are formed as a result of the packaging of the RNA molecule into ribonucleoprotein particles. (After K. Andersson, B. Bjorkroth, and B. Daneholt, Exp. Cell Res. 130: 313-326, 1980.)

Although the Structural organization of chromosomes is most clearly revealed by studying unusual interphase chromosomes, such as polytene or lampbrush chromosomes, all chromosomes of higher eukaryotes likely share a similar organization. Studies of Drosophila polytene chromosomes have shown that genes can be located both in the condensed chromatin of bands and in the less condensed interband regions; furthermore, it has been established that each band may contain multiple genes. Why, then, is each long chromatin fiber in a chromosome subdivided into A large number of distinct regions? Although the answer is unknown, it is very likely that this architecture serves to: 1) preserve DNA organization; 2) separate genes from one another, thereby avoiding biological "Interference"; and 3) regulate gene transcription (for example, constitutively expressed housekeeping genes may be located in interband regions, whereas cell-type-specific genes may reside within bands).

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9.2.8. Chromatin is less condensed in transcriptionally active regions [28]

Treatment of vertebrate cell nuclei with DNase I (the same enzyme that, at lower concentrations, reveals nuclease-hypersensitive regions devoid of nucleosomes) has shown that a specific fraction of chromatin containing active genes exists in a decondensed state. This finding Supports results obtained from insect polytene chromosomes. At a specific concentration of DNase, about 10% of the vertebrate genome is preferentially degraded. In different Cells of the same organism, the nuclease cleaves different DNA sequences that correspond to the various types of RNA produced by those cells. Using hybridization with specific DNA probes, it was found that the degraded sequences belong primarily to those Regions of the genome that are actively transcribed in the given cell type. Interestingly, even genes that are transcribed only a few times in each cell generation exhibit nuclease sensitivity. This fact suggests that a region becomes nuclease-hypersensitive due to a specific chromatin state rather than transcription per se (Fig. 9-51). Chromatin in such a nuclease-sensitive state is often referred to as active chromatin. It is believed that the nucleosomes comprising it are packed less tightly. Even when mitotic chromosomes are subjected to nuclease treatment, it is predominantly the active chromatin that gets cleaved. Evidently, even in mitosis, despite the extremely tight packaging of The Genome, certain differences between active and inactive chromatin are preserved.

Fig. 9-51. Treatment of chromatin with pancreatic DNase I. The enzyme first cuts sites with heightened nuclease sensitivity (not shown), after which the DNA sequence encompassing actively transcribed and potentially active genes undergoes degradation.

Fig. 9-52. Electron micrograph of spread chromatin from insect embryo cells (Oncopeltus). Significant Changes in the chromatin structure of two tandemly arranged rRNA genes are visible. Apparently, in these embryos, rRNA synthesis is preceded by the transition of chromatin from a "beads-on-a-string" state to a smooth conformation. It is possible that the smooth chromatin represents a region where DNA has "slipped off" the nucleosomes in preparation for transcription. It should be noted that the chromatin STRUCTURE OF THE gene located to the left of the transcribed region has undergone changes, even though it is not being transcribed at the moment. (After V. E. Foe, Cold Spring Harbor Symp. Quant. Biol. 42: 723-740, 1978.)

Electron microscopic analysis generally reveals no changes in the structure of nucleosomes that make up active chromatin. However, There is a striking exception: in some insect embryos, extended stretches of chromatin apparently devoid of nucleosomes can be observed in the regions of active rRNA genes (Fig. 9-52). According to one hypothesis, each nucleosome in such regions opens up to form an unfolded structure. This likely involves the unwinding of the two turns of the DNA helix that are normally wrapped around the nucleosome. These structural changes presumably facilitate transcription efficiency.

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9.2.9. Active chromatin possesses distinct biochemical properties [29]

To understand the structure of chromatin condensed to varying degrees, it is necessary to isolate and characterize the chromosomal proteins associated with each of these states. Certain progress has been achieved along this path regarding active chromatin. It has been established that: 1) histone H1 is not very tightly bound to at least some fraction of active chromatin; 2) the four core histones that make up the nucleosome are present in normal amounts but are characterized by an unusually high level of Acetylation of Lysine residues located near the amino terminus of these proteins. Acetyl groups are attached to them by the enzyme histone acetylase and removed by histone deacetylase, with each acetyl group persisting for an average of about 10 minutes; 3) nucleosomes in active chromatin selectively bind two closely related small chromosomal proteins, HMG14 and HMG17. Because these proteins are present only in active chromatin, their Abundance strictly corresponds to that required to bind approximately one in every 10 nucleosomes. The Amino acid sequences of both proteins are highly conserved, underscoring The Importance of their function; 4) in some organisms, such as Tetrahymena, active chromatin is significantly enriched in a minor variant of histone H2A. A similar histone variant has also been found in Drosophila, chickens, and humans. Any of these described properties may play an important role in chromatin decondensation and thereby facilitate RNA synthesis, although direct experiments are required to confirm this hypothesis. Further biochemical studies can be pursued using novel purification methods for active chromatin based on selective binding to an affinity matrix containing Antibodies specific either to the HMG protein or to acetylated lysine. Mechanisms controlling the formation of active chromatin are discussed in Chapter 10.

9.2.10. Heterochromatin is heavily condensed and transcriptionally inactive [30]

Evidence for the existence of a second unusual form of chromatin emerged in 1930, when light microscopy of interphase nuclei from higher eukaryotes revealed a distinct, highly condensed form of chromatin termed heterochromatin (with the less condensed portion designated as euchromatin). During interphase, heterochromatin remains remarkably compact, maintaining a structure that most chromatin assumes only during mitosis. It was later discovered that, much like mitotic chromatin, heterochromatin is transcriptionally inactive (Fig. 9-53). It is estimated that in most cells, approximately 90% of the chromatin is transcriptionally inactive. Although the relative resistance of this inactive chromatin to nuclease Digestion suggests that it is more condensed than the 10% found in transcriptionally active regions, only a fraction of it (perhaps 10% to 20%) adopts the heavily condensed conformation known as heterochromatin. Thus, heterochromatin likely represents a specialized class of transcriptionally inert chromatin that serves specific Functions. In mammals and many other higher eukaryotes, the DNA surrounding each centromere consists of relatively simple, repetitive nucleotide sequences; it is precisely these "satellite DNAs" that make up the bulk of the heterochromatin in these organisms.

Fig. 9-53. Autoradiograph of a thin section of a Cell Nucleus pulse-labeled with [³H]-uridine to reveal regions of active RNA synthesis (silver grains). The light areas represent regions of heterochromatin, which typically concentrates along the inner surface of the nuclear envelope. The light staining of these heterochromatic regions is an artifact of the Sample preparation method. RNA synthesis is observed predominantly in the euchromatic regions surrounding the heterochromatin. (Courtesy of Stan Fakan.)

Fig. 9-54. A region of a Drosophila polytene chromosome viewed under phase-contrast (top) and fluorescence (bottom) microscopy following immunofluorescent labeling of heterochromatin (left) and active chromatin (right). A. Staining with Monoclonal Antibodies specific for proteins localized in heterochromatic regions. The chromocenter (indicated by an arrow) and several individual bands are stained. B. Staining with monoclonal antibodies specific for proteins found in active chromatin. A subset of bands is stained. (Left: after T.S. James and S.C.R. Elgin, Mol. Cell. Biol. 6: 3862–3872, 1986; Right: after G.C. Howard, S.M. Abmayr, Z.A. Shinefeld, V.Z. Sato, and S.C.R. Elgin, J. Cell Biol. 88: 219–225, 1981. © 1981 Rockefeller University Press.)

In Drosophila, heterochromatin is organized at the centromeric regions as well as in randomly scattered short segments. These heterochromatic regions are under-replicated during early Stages of DNA synthesis—specifically, during the period when polytene chromosomes are formed. Consequently, these sequences are present in relatively lower amounts in polytene chromosomes. The Biochemical characteristics of this type of heterochromatin can be studied at THE MOLECULAR LEVEL by binding antibodies that target chromosomal proteins found exclusively in heterochromatin (Fig. 9-54). Cloning the genes that encode heterochromatin-specific proteins also holds great promise for its biochemical analysis.

Conclusion

All chromosomes undergo marked condensation during mitosis. Special staining techniques reveal distinct banding patterns on mitotic chromosomes, allowing for the precise identification of each individual chromosome. Comprising millions of DNA nucleotide pairs, these bands reflect a significant degree of heterogeneity within the chromosome, the exact nature of which remains poorly understood.

During interphase, chromosomes typically undergo decondensation to such an extent that their structure becomes difficult to resolve. Striking exceptions include specialized lampbrush chromosomes in vertebrate oocytes and polytene chromosomes in giant secretory cells of insects. Studies of these Two Types of interphase chromosomes have demonstrated that each long DNA molecule comprising a chromosome consists of numerous discrete, differentially packaged domains. A key feature of these chromosomes is the presence of a large number of chromatin loops, most of which contain between 20,000 and 100,000 nucleotide pairs. In both lampbrush and polytene chromosomes, the least condensed regions are those most actively engaged in RNA synthesis. Judging by DNase sensitivity, only about 10% of the DNA exists in a relatively decondensed, actively transcribed state. Such "active" chromatin exhibits biochemical properties distinct from those of more heavily condensed regions.