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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 9.5Organizing Cellular DNA into Chromosomes

We turn now to the question of how DNA molecules are organized within cells into the structures we observe as chromosomes. Because the total length of cellular DNA in cells is up to a hundred thousand times the cell’s length, the packing of DNA into chromosomes is crucial to cell architecture.

Most Bacterial Chromosomes Are Circular with One Replication Origin

In most bacterial cells, genes are encoded on large circular chromosomes. The circular nature of bacterial chromosomes was first discovered by analyzing the frequency of genetic recombination between mutant genes that produced easily assayed phenotypes, such as the inability to grow in the absence of a specific amino acid or the inability to grow on a particular sugar. As multiple genes were mapped on the E. coli chromosome by recombinational analysis, no ends were found in the single linkage map that developed. Rather, every gene had other identified genes that could be mapped on either side of it. The whole linkage map generated a circle (see Figure 8-14). Eventually, the circular structure of the E. coli chromosome was observed directly in autoradiographs of DNA molecules from cells grown in the presence of 3Hlabeled thymine, which is incorporated only into DNA. Such studies showed that the E. coli chromosome has a total length of 1 mm. The structure of the partially replicated E. coli chromosomal DNA molecule shown in Figure 9-27 demonstrates that the chromosome replicates from a single replication origin, similar to the small circular plasmid DNA molecule diagrammed in Figure 7-2.

Figure 9-27. Autoradiograph of the E. coli chromosome labeled with [3H]-thymine.

Figure 9-27

Autoradiograph of the E. coli chromosome labeled with [3H]-thymine. Points X and Y indicate the positions of DNA replication forks. Region B is the unreplicated parental chromosome. Regions A and C are the newly replicated nascent daughter chromosomes (more...)

The 1-mm-long DNA molecule of the E. coli chromosome is contained within cells that are only about 2 μm long and about 0.5 – 1 μm wide. A free DNA molecule of this size would form a random coil about 1000 times the volume of an E. coli cell. However, several mechanisms operate to compact E. coli chromosomal DNA sufficiently to fit inside the bacterial cell. For example, the large volume filled by free DNA is due largely to charge repulsion between the negatively charged phosphate groups. In the cell, this effect is counteracted by association of the DNA with positively charged polyamines, such as spermine and spermidine, which shield the negative charges of the DNA phosphate groups:

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In addition, numerous small protein molecules associate with the chromosomal DNA, causing it to fold into a more compact structure. The most abundant of these proteins, H-NS, is a dimer of a 15.6-kDa polypeptide. H-NS binds DNA tightly and compacts it considerably, as indicated by the increased sedimentation rate and decreased viscosity of DNA associated with H-NS compared with free DNA. There are about 20,000 H-NS molecules per E. coli cell, enough for one H-NS dimer per ≈400 base pairs of DNA.

Finally, E. coli chromosomal DNA is tightly supercoiled — that is, twisted upon itself like the circular SV40 DNA shown in Figure 4-11. As discussed in Chapter 12, an E. coli enzyme called DNA gyrase uses energy from ATP hydrolysis to wind supercoils into DNA. Supercoiling contributes to the compaction necessary to fit chromosomal DNA into the bacterial cell. Figure 9-28 shows an isolated, highly supercoiled E. coli chromosome attached to a fragment of cell membrane. If all the supercoils were relaxed and the DNA spread out, it would appear as a single, replicating, circular DNA molecule similar to that in Figure 9-27.

Figure 9-28. Electron micrograph of an isolated folded E. coli chromosome.

Figure 9-28

Electron micrograph of an isolated folded E. coli chromosome. The highly supercoiled DNA is attached to a fragment of the cell membrane, which appears as the most darkly staining material in the micrograph. Although this chromosome exhibits extensive supercoiling, (more...)

Eukaryotic Nuclear DNA Associates with Histone Proteins to Form Chromatin

The problem of compacting genomic DNA to fit into the nucleus of an eukaryotic cell is solved in a different way. When the DNA from eukaryotic nuclei is isolated in isotonic buffers (i.e., buffers with the same salt concentration found in cells, ≈0.15 M KCl), it is associated with an equal mass of protein in a highly compacted complex called chromatin. The general structure of chromatin has been found to be remarkably similar in the cells of all eukaryotes including fungi, plants, and animals.

The most abundant proteins associated with eukaryotic DNA are histones, a family of basic proteins present in all eukaryotic nuclei. The five major types of histone proteins — termed H1, H2A, H2B, H3, and H4 — are rich in positively charged basic amino acids, which interact with the negatively charged phosphate groups in DNA. In a fraction of the histone proteins of most cells, some of the basic amino acid side chains are modified by post-translational addition of acetyl (CH3COO−), phosphate, or methyl groups, neutralizing the positive charge of the side chain or converting it to a negative charge.

The amino acid sequences of four histones (H2A, H2B, H3, and H4) are remarkably similar among distantly related species. For example, the sequences of histone H3 from sea urchin tissue and of H3 from calf thymus are identical except for a single amino acid, and only four amino acids are different in H3 from the garden pea and that from calf thymus. Minor histone variants encoded by genes that differ from the highly conserved major types also exist, particularly in vertebrates.

The amino acid sequence of H1 varies more from organism to organism than do the sequences of the other major histones. In certain tissues, H1 is replaced by special histones. For example, in the nucleated red blood cells of birds, a histone termed H5 is present in place of H1. The similarity in sequence among histones from all eukaryotes indicates that they fold into very similar three-dimensional conformations, which were optimized for histone function early in evolution in a common ancestor of all modern eukaryotes.

Chromatin Exists in Extended and Condensed Forms

When chromatin is extracted from nuclei and examined in the electron microscope, its appearance depends on the salt concentration to which it is exposed. At low salt concentration, isolated chromatin resembles “beads on a string” (Figure 9-29a). In this extended form, the string is a thin filament of “linker” DNA connecting the beadlike structures termed nucleosomes. Composed of DNA and histones, nucleosomes are about 10 nm in diameter and are the primary structural units of chromatin. If chromatin is isolated at physiological salt concentration (≈0.15 M KCl), it assumes a more condensed fiberlike form that is 30 nm in diameter (Figure 9-29b).

Figure 9-29. Electron micrographs of extracted chromatin in extended and condensed forms.

Figure 9-29

Electron micrographs of extracted chromatin in extended and condensed forms. (a) Chromatin isolated in low ionic strength buffer has an extended “beads-on-a-string” appearance. The “beads” are nucleosomes (10-nm diameter) (more...)

Structure of Nucleosomes

The DNA component of nucleosomes is much less susceptible to digestion than is the linker DNA between them. If the nuclease treatment is carefully controlled, all the linker DNA can be digested releasing individual nucleosomes with their DNA component. A nucleosome comprises a protein core with DNA wound around its surface like thread around a spool. The core is an octamer containing two copies each of histones H2A, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shaped molecule made of interlocking histone subunits (Figure 9-30). Nucleosomes from all eukaryotes contain about 146 base pairs of DNA wrapped slightly less than two turns around the protein core. The length of the linker DNA is more variable among species, ranging from about 15 to 55 base pairs.

Figure 9-30. Structure of the nucleosome.

Figure 9-30

Structure of the nucleosome. (a) Ribbon diagram of the nucleosome shown face-on (left) and from the side (right). One DNA strand is shown in green and the other in brown. H2A is yellow; H2B, red; H3, blue; H4, green. (b) Space-filling model shown (more...)

In cells, newly replicated DNA is assembled into nucleosomes shortly after the replication fork passes, but when isolated histones are added to DNA in vitro at physiological salt concentration, nucleosomes do not spontaneously form. However, nuclear proteins that bind histones and assemble them with DNA into nucleosomes in vitro have been characterized. Proteins of this type are thought to assemble histones and newly replicated DNA into nucleosomes in vivo as well.

Structure of Condensed Chromatin

When extracted from cells in isotonic buffers, most chromatin appears as fibers ≈30 nm in diameter (see Figure 9-29b). In these condensed fibers, nucleosomes are thought to be packed into a spiral or solenoid arrangement, with six nucleosomes per turn (Figure 9-31). A fifth histone, H1, is bound to the DNA on the inside of the solenoid, with one H1 molecule associated with each nucleosome. Recent electron microscopic studies suggest that the 30-nm fiber is less uniform than the solenoid model predicts. Condensed chromatin may in fact be quite dynamic with regions partially unfolding and then refolding into a solenoid structure occasionally.

Figure 9-31. Solenoid model of the 30-nm condensed chromatin fiber in a side view.

Figure 9-31

Solenoid model of the 30-nm condensed chromatin fiber in a side view. The octameric histone core (see Figure 9-30) is shown as an orange disk. Each nucleosome associates with one H1 molecule, and the fiber coils into a solenoid structure with a diameter (more...)

The chromatin in chromosomal regions that are not being transcribed exists predominantly in the condensed, 30-nm fiber form. The regions of chromatin actively being transcribed are thought to assume the extended beads-on-a-string form.

Acetylation of Histone N-Termini Reduces Chromatin Condensation

Each of the histone proteins making up the nucleosome core contain flexible amino termini of 20 to 40 residues extending from their globular domains (see Figure 9-30b). The N-termini contain several positively charged lysine groups. Some of these interact with phosphates in DNA of the same nucleosome, and some may interact with linker DNA or with neighboring nucleosomes. These lysines undergo reversible acetylation and deacetylation by enzymes that act on specific lysines in the N-termini of the different histones. In the acetylated form, the positive charge of the lysine ϵ-amino group is neutralized and its interaction with a DNA phosphate group is eliminated. Thus the greater the extent of acetylation of his-tone N-termini, the less likely chromatin is to form condensed 30-nm fibers and possibly higher-order folded structures.

The extent of histone acetylation also is correlated with the relative resistance of chromatin DNA to digestion by nucleases. This phenomenon can be demonstrated by digesting isolated nuclei with DNase I. Following digestion, the DNA is completely separated from chromatin protein, digested to completion with a restriction enzyme, and analyzed by Southern blotting (see Figure 7-32). When a gene is cleaved at random sites by DNase I, the Southern-blot band corresponding to that gene is lost. This method has been used to show that the inactive β-globin gene in nonerythroid cells, where it is associated with relatively unacetylated histones, is much more resistant to DNase I than is the active β-globin gene in erythroid precursor cells, where it is associated with acetylated histones (Figure 9-32). This relative resistance to nuclease indicates that the chromatin structure of nonexpressed DNA is more condensed than that of transcribed DNA. In condensed chromatin, the DNA is largely inaccessible to DNase I because of its close association with histones and possibly other less-abundant chromatin proteins. In contrast, actively transcribed DNA is much more accessible to DNase I digestion because it is present in the extended, beads-on-a-string form of chromatin.

Figure 9-32. Demonstration that transcriptionally active genes are more susceptible than inactive genes to DNase I digestion.

Figure 9-32

Demonstration that transcriptionally active genes are more susceptible than inactive genes to DNase I digestion. Chick embryo erythroblasts at 14 days actively synthesize globin, whereas cultured undifferentiated MSB cells do not. Nuclei from each type (more...)

Recent genetic studies in yeast indicate that specific histone acetylases are required for the full activation of transcription of a number of genes. Consequently, as discussed in Chapter 10, the control of acetylation of histone N-termini in specific chromosomal regions is thought to contribute to gene control by regulating the strength of the interaction of histones with DNA and the folding of chromatin into condensed structures. Genes in condensed, folded regions of DNA are inaccessible to RNA polymerase and other proteins required for transcription.

Eukaryotic Chromosomes Contain One Linear DNA Molecule

As discussed in the next section, eukaryotic chromosomes can be visualized when they condense during mitosis. The general belief is that each of the several chromosomes in eukaryotic cells contains a single long DNA molecule. Because the longest DNA molecules in human chromosomes are almost 10 cm long (2 – 3 × 108 base pairs), they are difficult to handle experimentally without breaking. However, in lower eukaryotes, the sizes of the largest DNA molecules that can be extracted are consistent with the hypothesis that each chromosome contains a single DNA molecule. For example, physical analysis of the largest DNA molecules extracted from several genetically different Drosophila species and strains shows that they are from 6 × 107 to 1 × 108 base pairs long. These sizes match the DNA content of single stained metaphase chromosomes of Drosophila melanogaster, as measured by the amount of DNA-specific stain absorbed. Therefore, each chromosome probably contains a single DNA molecule.

The correspondence between the number of DNA molecules per cell and the number of chromosomes has been conclusively demonstrated in yeast cells. The DNA from each S. cerevisiae chromosome can be separated and individually identified by pulsed-field gel electrophoresis (see Figure 7-26). The number of separated DNA molecules equals the number of genetic linkage groups (i.e., chromosomes) in yeast, and the entire sequence of each chromosome has been determined and directly compared with the genetic map. The length of yeast chromosomal DNA ranges from about 1.5 × 105 to 106 base pairs.


  •  Genomic DNA in both bacteria and eukaryotes must be highly compacted in order to fit within cells.
  •  Bacterial chromosomes usually are circular DNA molecules that replicate from a single origin. Bacterial DNA is highly supercoiled and associated with polyamines and low-molecular-weight basic proteins, which permit the DNA to fold tightly in the central portion of a bacterial cell.
  •  In eukaryotic cells, DNA is associated with about an equal mass of histone proteins in a highly condensed structure called chromatin. The building block of chromatin is the nucleosome, consisting of a histone octamer around which is wrapped about 146 bp of DNA (see Figure 9-30).
  •  When extracted under physiological conditions, chromatin is visualized in the electron microscope as a 30-nm fiber made up of nucleosomes, the linker DNA between them, and histone H1 (see Figure 9-31). Transcriptionally inactive regions of DNA within cells is thought to exist in this condensed form and possibly higher-order structures built from it.
  •  When extracted at low salt concentrations, chromatin is visualized as an extended beads-on-a-string structure, which lacks histone H1. Transcriptionally active regions of DNA within cells are thought to resemble this extended form of chromatin.
  •  The reversible acetylation and deacetylation of lysine residues in the N-termini of histones H2A, H2B, H3, and H4 controls how tightly DNA is bound by the histone octamer and affects the assembly of nucleosomes into the condensed forms of chromatin. Hypoacetylated chromatin assumes a more condensed structure than hyperacetylated chromatin.
  •  The more open chromatin structure of active genes makes them more sensitive to nuclease digestion than inactive genes.
  •  Each eukaryotic chromosome contains a single linear DNA molecule.
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21500


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