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Modern Genetic Analysis Chapter 2. The Structure of Genes and Genomes
The Nature of Eukaryotic Nuclear Chromosomes

The set of chromosomes as viewed under the microscope is called a karyotype ("nuclear type"). The karyotype is defined by chromosome number and by other visible landmarks.

Visible Chromosomal Landmarks

The microscopic study of chromosomes and analysis of their genetic properties is called cytogenetics, a discipline that combines cytology with genetics. Since a great deal of space in this book will be devoted to the properties of chromosomes, we will begin with a look at the visible morphology of chromosomes, the features that are commonly used in cytogenetic analysis for telling chromosomes apart.

Chromosomes can be viewed relatively easily under the microscope, but only just before, during, and immediately after cell division. When a cell divides, the nucleus and its chromosomes also divide. The topics of cell and nuclear division will be covered fully in Chapter 4. At present we need note only that chromosomes must become very compact during cell division, since they are moved around a great deal during their apportionment into daughter cells. Thus, the components of a chromosome (including its DNA) become condensed from the extended form found in nondividing cells into a shorter, fatter form that can be easily handled by the division apparatus of the cell. It is the landmarks of these condensed chromosomes that are seen under the microscope, but it can be presumed that these landmarks are in the same relative positions on the extended chromosomes of nondividing cells.

Chromosome size.

The chromosomes of a single genome may differ considerably in size. In the human genome, for example, there is about a fourfold range in size from chromosome 1 (the biggest) to chromosome 21 (the smallest), as shown in Table 2-4. In studying the chromosomes of a species, a cytogeneticist may have difficulty identifying individual chromosomes by size alone but may be able to group chromosomes of similar size. For example, in humans the chromosomes are placed into seven groups, labeled A (the largest chromosomes) through G (the smallest), as shown in the table.

Centromere position.

The molecular strings (spindle fibers) that move chromosomes around during cell division attach to a specialized region of the chromosome called the centromere. The centromeric region usually appears as a constriction or neck at a specific position along the chromosome. This constriction divides the chromosome into two "arms." The shorter arm is called p and the longer arm q. The position of the constriction defines the ratio between the lengths of the two chromosome arms. This arm ratio is a useful characteristic for identifying individual chromosomes (Table 2-4). Centromere positions also can be categorized as telocentric (at one end), acrocentric (close to one end), or metacentric (in the middle).

The tips of the chromosomes are called telomeres. The telomeres generally are not visibly distinct from the other parts of the chromosome. The telomeres and the centromeres have unique molecular structures that are crucial to normal chromosome behavior.

Position of nucleolar organizers.

Nucleoli are spherical structures found associated with constrictions of the chromosomes called nucleolar organizers. Different organisms are differently endowed with nucleoli, which range in number from one to many per chromosome set. The diploid cells of many species have just a pair of nucleoli. Nucleolar organizers contain numerous tandem copies of the genes that code for ribosomal RNA, an untranslated RNA that is a component of the ribosomes. Ribosomal RNA is synthesized at the nucleolar organizers, deposited into the nucleoli, and later exported to the cytoplasm to become incorporated into ribosomes. The positions of nucleoli, like the positions of centromeres, are quite useful landmarks for cytogenetic analysis.

Chromomere patterns.

The chromomeres are beadlike, localized thickenings found along the chromosome during the early stages of nuclear division. The positions of chromomeres are the same in all homologous chromosomes. Although chromomeres can be useful as markers, their molecular nature is not known.

Heterochromatin patterns.

When chromosomes are treated with chemicals that react with DNA, such as Feulgen reagent, distinct regions with different staining characteristics are revealed. Densely staining regions are called heterochromatin and reflect a high degree of compactness; poorly staining regions are called euchromatin and indicate less tightly packed regions. Most of the active genes are in euchromatin. Figure 5-15 shows examples of heterochromatin in the tomato.

Banding patterns.

Special chromosome staining procedures have revealed intricate sets of bands (transverse stripes) in many different organisms. The positions and sizes of the bands are highly chromosome-specific; therefore, they represent useful landmarks. There are Q bands (produced by quinacrine hydrochloride), G bands (produced by Giemsa stain), and R bands (produced by reversed Giemsa). The G banding patterns in two human chromosomes and the locations of some of the genes on those chromosomes are shown in Figure 2-14.

A rather specialized kind of banding occurs in a few organisms whose chromosomes can replicate their DNA many times without separating. This produces giant chromosomes, which are in essence magnified versions of the unreplicated forms. Consequently, the natural banding patterns of the chromosomes become readily visible and can serve as landmarks. These polytene chromosomes (polytene means "many-stranded") are found in highly specialized cells of Malpighian tubules, rectum, gut, footpads, and salivary glands of the dipteran insects such as houseflies, mosquitoes, and fruit flies.

The fruit fly Drosophila melanogaster is a much-studied example. This insect (a diploid) has a 2n number of 8, and these eight chromosomes (see Figure 2-15a) are present in most cells. However, in the cells of the special organs that contain the polytene chromosomes we see some interesting peculiarities (Figure 2-15b). First, there are only four polytene chromosomes per cell (not eight) because during the specialized replication process the members of each homologous pair unexpectedly unite with each other. Second, all four polytene chromosomes become joined at a structure called the chromocenter, which is a coalescence of the heterochromatic areas around the centromeres of all four chromosomes. The chromocenter of Drosophila salivary gland chromosomes is shown in Figure 2-15b, where L and R stand for arbitrarily assigned left and right arms.

Along the length of a polytene chromosome, there are transverse bands (Figure 2-15c). Polytene bands are much more numerous than Q, G, or R bands, numbering in the hundreds on each chromosome. The bands differ in width and appearance, so that the banding pattern of each chromosome is unique and characteristic of that chromosome.

Molecular studies have shown that in any chromosomal region of Drosophila there are more genes than there are polytene bands, so the bands do not represent genes. Similarly, the significance of the Q, G, and R patterns in other eukaryotic cells is not clear. They probably reflect the degree of compactness of the DNA, but it is not known how this pattern is maintained.

Using all the available chromosomal landmarks together, cytogeneticists can distinguish each of the chromosomes of many species. Figure 2-16 shows a map of the chromosomal landmarks of the genome of corn.

MESSAGE
Such features as size, arm ratio, heterochromatin, number and position of chromomeres, number and location of nucleolar organizers, and banding pattern identify the individual chromosomes within the set that characterizes a species.

Three-Dimensional Structure of Nuclear Chromosomes

A human cell contains about 2 meters of DNA (1 m per chromosome set). The human body consists of approximately 1013 cells, and each cell is diploid; therefore, the body contains a total of about 2 × 1013 m of DNA. Some idea of the extreme length of this DNA can be obtained by comparing it with the distance from Earth to the sun, which is 1.5 × 1011 m. You can see that the DNA in your body could stretch to the sun and back almost 100 times. This peculiar fact makes the point that the DNA of eukaryotes is obviously efficiently packed. In fact, the packing occurs at the level of the nucleus, where the 2 m of DNA in a human cell are packed into 46 chromosomes, all inside a nucleus only 0.006 mm in diameter. How can such long molecules be packaged into to the wormlike structures, visible under the light microscope, that we call chromosomes? To answer this question we need to understand the three-dimensional structure of eukaryotic chromosomes.

If eukaryotic cells are broken, and the contents of their nuclei are examined under the electron microscope, chromosomes appear as masses of spaghetti-like fibers with diameters of about 30 nm. Some examples are shown in the electron micrograph in Figure 2-17. If such chromosomes are studied carefully, it becomes clear that there are no ends protruding from the fibrillar mass. This suggests that each chromosome is one long, fine fiber folded up in some way. If the fiber somehow corresponds to a DNA molecule, then we arrive at the idea that each chromosome is one densely folded DNA molecule. Measurements of the elasticity of Drosophila DNA in solution further support this concept, as does electron microscopy (Figure 2-18).

Perhaps the best evidence that a chromosome consists of a single long DNA molecule comes from a technique called pulsed field gel electrophoresis. If DNA from a large number of cells is extracted, purified with great care to avoid breaking the molecules, and placed on a gelatinous matrix under the influence of powerful, alternating, crossed electric fields, the DNA molecules of each chromosome move through the gel at speeds proportional to their size. All the DNA of one chromosomal type ends up in one position (called a band), so if the gel is stained with a dye that binds to DNA, the number of bands of DNA is always equal to the number of chromosomes, whether the cell is diploid or haploid.

MESSAGE
Each eukaryotic chromosome contains a single, long, folded DNA molecule.

Histone proteins.

What are the precise mechanisms that pack DNA into chromosomes? How is the very long DNA thread converted into the relatively thick, dense, wormlike structure visible to us during cell division? The overall mixture of material that comprises chromosomes is given the general name chromatin. It is DNA and protein. If chromatin is extracted and treated with differing concentrations of salt, different degrees of compaction, or condensation, are observed under the electron microscope. With low salt concentrations, a structure about 10 nm in diameter is seen that resembles a beaded necklace. The string between the beads of the necklace can be digested away with the enzyme DNase, so the string can be inferred to be DNA. The beads on the necklace are called nucleosomes; these can be shown to be complexes of DNA and special chromosomal proteins called histones. Histone structure is remarkably conserved across the range of eukaryotic organisms, and nucleosomes are always found to contain an octamer of two units each of histones H2A, H2B, H3, and H4. The DNA is wrapped twice around the octamer as shown in Figure 2-19a. When salt concentrations are higher, the nucleosome beaded necklace gradually assumes a coiled form called a solenoid (Figure 2-19b). This solenoid, produced in vitro, is 30 nm in diameter and probably corresponds to the in vivo spaghetti-like structures we saw in Figure 2-17. The solenoid is stabilized by another histone, H1, that runs down the center of the structure, as Figure 2-19b shows.

We see then that to achieve its first level of packaging, DNA winds onto histones, which act somewhat like spools. Further coiling results in the solenoid conformation. However, it takes one more level of packaging to convert the solenoids into the three-dimensional structure we call the chromosome.

Higher-order coiling.

Many cytogenetic studies show that chromosomes are visibly coiled; Figure 2-20 shows a good example from the nucleus of a protozoan. Whereas the diameter of the solenoids is 30 nm, the diameter of these "supercoils" is the same as the diameter of the chromosome during cell division, often about 700 nm. What produces the supercoils? One clue comes from observing chromosomes from which the histone proteins have been removed chemically. After such treatment, the chromosomes have a densely staining central core of nonhistone protein called the scaffold, as shown in Figure 2-21. Projecting laterally from this protein scaffold are loops of DNA. The fibers forming these loops are believed to be the solenoids. At high magnifications, it is clear from electron micrographs that each DNA loop begins and ends at the scaffold. It has been discovered that the central scaffold is largely composed of the enzyme topoisomerase II, which has the ability to pass a strand of DNA through another strand, rather like a magician's rings. Evidently, the central scaffold organizes the vast skein of DNA during replication, a time when the two strands of DNA must unwind to allow new synthesis. Anyone who has tried to unwind the two strands of a piece of rope has experienced the difficulties inherent in this process.

Now, to return to the question of how the supercoiling of the chromosome is produced. The best evidence suggests that the solenoids arrange in loops emanating from the central scaffold matrix, which itself is in the form of a spiral. We see the general idea in Figure 2-22, which shows a representation of a loosely coiled chromosome from a nondividing cell and a more tightly coiled chromosome during cell division. The loops attach to the scaffold by special regions along the DNA called scaffold attachment regions, or SARs. Some SARs that have been mapped in Drosophila are shown in Figure 2-23.

As we saw in Figure 2-22, chromosomes are much less supercoiled (but still not naked DNA) in the nondividing nucleus than during cell division. Hence they are essentially invisible at the light microscopic level. This more relaxed state is probably necessary for most genes to be expressed.

MESSAGE

In the progressive levels of chromosome packing

1. DNA winds onto nucleosome spools.
2. The nucleosome chain coils into a solenoid.
3. The solenoid loops, and the loops attach to a central scaffold.
4. The scaffold plus loops arrange into a giant supercoil.

Representations of chromosomes.

We have seen that eukaryotic chromosomes are complex structures, and, not surprisingly, they are difficult to represent diagrammatically. Figure 2-24 shows three shorthand representations of chromosomes in diploid and haploid nondividing cells, focusing on coiled structure, linear DNA content, and chromosome length and arm ratio. Shorthand representations such as these are used for illustrating specific chromosomal properties under discussion, but it should be emphasized that none is a perfect representation. Indeed the diagrams shown in Figure 2-24 on the following page are representations of the chromosomes in a nondividing cell, and as noted before, at this stage the chromosomes are not even visible under the microscope. It is important to note that in the diploid condition shown homologous chromosomes constitute a pair, but they are generally not in a paired configuration. In fact, homologous chro-mosomes pair physically only during the specialized type of nuclear division that occurs during the production of sex cells (meiosis), as we shall see in Chapter 4.

© 1999 by W. H. Freeman and Company. All rights reserved.