Genetic information in the form of DNA is stored within the interphase nucleus in thread-like, tangled structures called chromatin. During the process of cell division, the DNA condenses and divides into several distinct pairs of linear segments or chromosomes. Each time the cell divides, the hereditary information carried in the chromosomes is passed on to the two newly formed cells. The DNA in the nucleus contains the instructions for regulating the amount and types of proteins made by the cell. These instructions are copied, or transcribed, into messenger RNA (mRNA), which is transported from the nucleus to the ribosomes located in the cytoplasm, where proteins are assembled (see Chap. 3).

Most somatic cells spend the greater part of their lives in G₀, or the resting phase of the cell cycle, because such cell populations are not actively dividing.

Before a cell can divide, it must double its mass and duplicate all of its contents. This ensures the ability of the daughter cells to begin their own cycle of growth followed by division. Most of the work involved in preparing for division goes on invisibly during the growth phase of the cell cycle, known as the interphase, which comprises the G₁, S, and G₂ phases of the cell cycle (Fig. 4-1). The interphase nucleus is the seat of crucial biochemical activities including the synthesis of proteins and the duplication of its chromosomal DNA in preparation for subsequent cell division.

The process of cell division (see Fig. 4-1) can be readily visualized in the microscope and consists of two sequential
events: nuclear division (mitosis) followed by cytoplasmic division (cytokinesis). The cell-division phase is designated as the M phase (M = mitosis). The period between the end of the M phase and the start of DNA synthesis is the G₁ phase (G = gap). In G₁, RNAs and proteins, including the essential components needed for DNA replication, are synthesized without replication of DNA. Once all the ingredients are synthesized in G₁, DNA replication takes place in the ensuing synthesis phase (S-phase) of the cell cycle.

The period between the completion of DNA synthesis and the M phase is known as the G₂ phase, in which additional cellular components are synthesized in preparation for the cell’s entry into mitosis. The interphase thus consists of successive G₁, S, and G₂ phases that normally constitute 90% or more of the total cell cycle time (see Fig. 4-1).

However, following the completion of mitotic division, most normal somatic cells leave the division cycle and enter a postmitotic resting phase (G₀), rather than the new G₁ phase. The unknown trigger mechanism for cell division is activated during the G₀ phase; as a result, the cell enters G₁ phase and is committed to divide (Brachet, 1985; Levitan, 1987; Therman, 1993; Nicklas, 1997; Hixon and Gualberto, 2000). In fact, experiments have shown that the point of no return, known as the restriction point (R point), occurs late in G₁. After cells have passed this point, they will complete the rest of the cycle at their normal rate, regardless of external conditions. The time spent by cells in G₂ and S phases is relatively constant (Brachet, 1985; Gardner, 2000). One interesting exception is the epidermis of the skin, in which some cells remain in the G₂ phase and thus are able to undergo rapid division in wound healing.

Studies of the cell cycle in yeast have shown that the cell proceeds from one phase of the cell cycle to the next by passing through a series of molecular checkpoints (Li and Murray, 1983). These checkpoints determine whether the cell is ready to enter into the next phase of the cell cycle. These biochemical checkpoints involve the synthesis of new proteins and degradation of already existing proteins. Both the S phase and the M phase are activated by related protein kinases, which function at specific stages of the cell cycle. Each kinase consists of at least two subunits, one of which is cyclin, so named because of its role in the cell cycle. There are several cyclins involved in regulating entry into different parts of the cell cycle, and they are degraded after serving their purpose or as the cell progresses in the cycle and through mitosis (Rudner and Murray, 1996; Amon, 1999; Cerrutti et al, 2000; Gardner, 2000).

The cells of the human body divide at very different rates. Some cells, such as mature neurons, heart and skeletal muscle, and mature red blood cells, do not divide at all or perhaps only under most exceptional circumstances. Other cells, such as the epithelial cells that line the inside and outside surfaces of the body (e.g., the intestine, lung, and skin), divide continuously and relatively rapidly throughout the life of the individual. The behavior of most cells falls somewhere between these two extremes. Most somatic cells rarely divide, and the duration of their cell cycle may be 100 days or more.

The average time for the mitotic cycle in most cell types is about 16 hours in human and other mammalian cells, distributed as follows: S phase, approximately 6 to 8 hours; G₁ phase, 6 to 12 hours; G₂ phase, 4 hours; and M phase, 1 to 2 hours (see Fig. 4-1). The M, and especially G₁, phases may show considerable variation in duration. Most of the available evidence suggests that these periods are longer in cancer cells than in benign cells, or at least in benign cell populations that normally have a rapid turnover. Many tissues require more than 16 hours to complete the mitosis (Miles, 1979).

Even though it takes a minimum of 7 to 8 hours for a cell to duplicate its entire chromosomal DNA, individual chromosomes or segments of chromosomes are replicated asynchronously, some of them sooner and faster than others. Thus, some chromosomes, or their segments, will have completed DNA synthesis before others begin. This asynchrony does not follow a simple pattern. The synthesis does not necessarily begin at one point and spread uniformly along the chromosome, but may start at several places on a single chromosome, while others wait their turn for DNA replication. A reproducible phenomenon is the late replication of one of the two X chromosomes in normal female cells or in cells with more than one X chromosome. Apparently, this X chromosome finishes its DNA replication later than any other chromosome in the cell. The number of late-replicating X chromosomes is usually one less than the total number of X chromosomes in the cell (Moore, 1966; Sandberg, 1983a, 1983b).

The chromosomes are not visible under the light microscope except during the M phase of the cell cycle. The physical condition of the chromosomes during interphase (e.g., G₁, S, and G₂) is not known, but their invisibility is probably caused, at least in part, to their enormous elongation. The older notion that the chromosomes lose their linear structure and become dissolved in the nucleoplasm is unlikely, and it introduces unnecessary complexities into the analysis of nuclear and chromosomal dynamics (van Holde, 1989; Miles, 1964, 1979).

Recent studies of chromosomes, utilizing fluorescent probes for chromosomal “painting,” suggest that the chromosomes retain their distinct identity during the interphase and that their position in the nucleus may be relatively constant throughout the life of the cell (Nagele et al, 1995; Koss, 1998).

The transition from the G₂ phase to the M phase of the cell cycle is not a sharply defined event. The chromatin, which is diffuse in interphase, slowly condenses into welldefined chromosomes, the exact number of which is a characteristic of the particular species; each chromosome has duplicated during the preceding S phase and consists of two sister chromatids joined at a specific point along their length by the centromere. While the chromosomes are condensing, the nucleolus begins to disassemble and gradually disappears.

Within the nucleus itself, the first sign of prophase is an accentuation of the chromocenters and a net-like pattern (Fig. 4-7B; see Fig. 4-6A). Several condensations of chromatin appear at the periphery of the nucleus, whence thin strands of chromatin extend into the center of the nucleus (see Fig. 4-7B). In females, the inactive X chromosome (Barr body) is larger than other chromocenters and is readily visible as a triangular condensation of chromatin (see Fig. 4-7). These strands and chromocenters are the condensing chromosomes. By this time, the chromosomes are probably doubled into the two chromatids or daughter chromosomes-to-be, but the double structure is sometimes difficult to visualize (Fig. 4-8A; see Fig. 4-6B). It is more evident in chromosomes that have been exposed to colchicine (a drug that inhibits mitosis) and which have been treated with hypotonic salt solutions. With the breakdown of the nuclear membrane, the chromosomes are quite distinct and are arranged into a circular position, known as a hollow spindle or prometaphase rosette (see below) (see Figs. 4-6C and 4-8B,C).

At the beginning of prophase, the cytoplasmic microtubules, which are part of the cytoskeleton (see Chap. 2), disassemble, forming a large pool of tubulin molecules. These molecules are then reused in the construction of the main component of the mitotic apparatus, the mitotic spindle. This is a bipolar fibrous structure, largely composed of microtubules, that assembles initially outside the nucleus. The focus for the spindle formation is marked in most animal cells by the centrioles (see Chap. 2). The cell’s original pair of centrioles replicates by a process that begins immediately before the S phase to give rise to two pairs of centrioles, which separate and travel to the opposite poles of the cell (see Fig. 4-6D). Each centriole pair now becomes part of a mitotic center that forms the focus for a radial array of microtubules, the aster (from Latin, aster = star). Initially, the two asters lie side by side, close to the nuclear envelope. By late prophase, the bundles of polar microtubules that interact between the two asters (seen as polar fibers in the light microscope) preferentially elongate and appear to push the two asters apart along the outer part of the nucleus. In this way, a bipolar mitotic spindle is formed.

Prometaphase starts abruptly with the disruption of the nuclear envelope, which breaks up into membrane fragments that are indistinguishable from bits of endoplasmic reticulum (see Fig. 4-6C). These fragments remain visible around the spindle during mitosis. Specialized structures called kinetochores develop on either face of the centromeres and become attached to a special set of microtubules, called kinetochore fibers or kinetochore microtubules. These fibers radiate in opposite directions from the sides of each chromosome and interact with the fibers of the bipolar spindle. The chromosomes are thrown into agitated motion by the interactions of their kinetochore fibers with other components of the spindle.

In phase cinematography of living cells, the chromosomes may be seen to undergo slow to and fro writhing movements until they finally become aligned on an equatorial plane. This plane bisects the mitotic spindle. As a result of their prometaphase oscillations, arrangement of all the chromosomes is such that their centromeres lie in one plane. The kinetochore fibers seem to be responsible for aligning the chromosomes halfway between the spindle poles and for orienting them with their long axes at right angles to the spindle axis. Each chromosome is held in tension at the metaphase plate by the paired kinetochores, with their associated fibers pointing to opposite poles of the spindle (see Figs. 4-6D and 4-8D).

The metaphase may last for several hours. As if triggered by a special signal, anaphase begins abruptly as the paired kinetochores on each chromosome separate, allowing each chromatid to be pulled slowly toward a spindle pole (see Fig. 4-6E). All chromatids are moved toward the pole they face at the same speed. During these movements, kinetochore fibers shorten as the chromosomes approach the poles. At about the same time, the spindle fibers elongate and the two poles of the polar spindle move farther apart. Soon after separation, the chromosomes appear at both poles as dark-staining masses (see Fig. 4-8E). The anaphase stage typically lasts only a few minutes.

In the meantime, the cell has become elongated, and a constriction furrow begins to appear at the level of the metaphase equator (see Figs. 4-9A and Fig. 4-6F). This process of cytoplasmic division is called cytokinesis. Although cytokinesis usually follows chromosomal division, the two processes are not necessarily dependent on one another. Chromosomal division may occur without cytokinesis (thereby producing a cell with double the normal complement of chromosomes). Less commonly, in some lower species, anucleated cytoplasm may undergo successive divisions.

The constriction furrow extends between the two daughter cells until only a narrow strand of cytoplasm is left. At this point, a distinct granule, the midbody, may sometimes be seen at the narrowest part of the cytoplasmic strand (see Fig. 4-9B). The midbody is formed, at least in part, by the spindle fibers compressed into a tight bundle. The precise significance and fate of the midbody are not known.

Some details of telophase are worthy of attention. In late anaphase, after or during cytokinesis, the compact mass of chromosomes begins to swell. In optimal material, each chromosome appears to form a distinct small vesicle, possibly by inducing the formation of a proprietary segment of the nuclear membrane, as suggested by Koss (1998) (Fig. 4-10; see Fig. 4-6F). In abnormal divisions, the process may sometimes end at this stage, with the cell thus containing numerous micronuclei (Fig. 4-11). Normally, the vesicles seem to fuse rapidly together to form a convoluted tubule (see Figs. 4-12 and 4-6G). Probably the vesicle and tubule membranes break down at points of contact so that, ultimately, a continuous nuclear membrane is formed around both groups of daughter chromatids.

The elongating chromosomes now appear at right angles to the tubule walls, thus to some extent, mimicking prophase appearances (see Figs. 4-13, 4-6H, and 4-12B). The outline of the nucleus gradually becomes less convoluted, and nucleolar material appears at the inner edges of the nucleus. The reticular appearance of the telophase nucleus (Fig. 4-14) gradually fades into the less-distinct interphase pattern.

As the separated daughter chromatids arrive at the poles, the kinetochore fibers disappear. The polar fibers elongate still farther, the condensed chromatin expands once more, the nucleoli begin to reappear, and the mitosis comes to an end.

As described above, the cytoplasm divides by a process known as cleavage, which usually starts sometime during late anaphase or telophase. The membrane around the middle of
the cell, perpendicular to the spindle axis and between the daughter nuclei, is drawn inward to form a cleavage furrow, which gradually deepens until it encounters the remains of the mitotic spindle between the two nuclei (see Figs. 4-6F and 4-9). This narrow bridge, which contains a dark granule, the midbody, may persist for some time before it finally breaks at each end, leaving two completed, separated daughter cells (Miles, 1979; Alberts, 1983; Brachet, 1985; Levitan, 1988; Edlin, 1990; Therman, 1993).

Another feature of chromosomes that shows familial differences, probably unrelated to disease, is the secondary constriction. (The primary constriction is at the centromere where the spindle fibers attach during mitosis; see earlier.) Readily visible in the microscope are secondary constrictions in the long arms near the centromere on chromosomes 1, 9, and 16. In normal cells, these constrictions are seen only occasionally and seldom in more than one homologue in a given cell. These constrictions are usually observed near centromeric sites on most chromosomes. At these sites, most chromosomes have small blocks of chromatin that replicate their DNA after the other chromosomal segments have completed DNA synthesis (e.g., late-labeling DNA). Such sites can also be selectively stained with the C-banding technique, centromeric heterochromatic stain (Fig. 4-18). In many species, such dark-staining, late-labeling segments are referred to as heterochromatin. In some species, these segments do not decondense in the interphase nucleus but rather remain as dark-staining masses of chromatin called chromocenters. In general, such heterochromatin segments are genetically inert (do not contain functioning genes and do not synthesize RNA). They are believed to have something to do with maintaining the structure of the chromosome; the material, therefore, is called constitutive heterochromatin. The latter is differentiated from facultative heterochromatin, which is condensed in some cells and not in others and, in contrast to constitutive heterochromatin, reflects some of the stable differences in genetic activity adopted by different cell types (e.g., embryonic cells seemingly contain very little, and some highly specialized cells contain a great deal of heterochromatin). Facultative heterochromatin is not known to contain the large number of highly repeated DNA sequences (satellite DNAs), which is characteristic of constitutive heterochromatin (Bahr, 1977; Lima-de-Faria, 1983; Therman, 1993). Although chromocenters (except for the sex chromatin body; see below) may vary in their appearance in the nuclei of human cells and, in some, they are difficult to visualize, only polymorphonuclear leukocytes are an exception. In the nuclear lobes of these cells, the constitutive heterochromatin of chromosome 1 and, perhaps other chromosomes, is observed as a peripheral chromocenter. With a somewhat similar technique, the C-band heterochromatin of chromosome 9 can be identified in interphase nuclei of lymphocytes.

As has been stated, most human cells contain 46 chromosomes. The germ cells, sperm and ovum, constitute an important exception. Since the individual develops from the union of sperm and ovum, to preserve the proper somatic number of 46, these cells can have only 23 chromosomes each. Thus, the developing germ cell must lose half its chromosomes. The product of the union of the spermatozoon and the ovum, or the zygote, will then receive 23 chromosomes from the mother and 23 from the father. A type of cell division known as meiosis fulfills these requirements (Fig. 4-19).

It is clear that normal development will require that the zygote receive a set of similar chromosomes (e.g., one No. 1, one No. 2, and so on) from each parent. A set of 23 maternal or paternal chromosomes is a haploid set, and the final two sets of homologues form a diploid set.

The fundamental mechanism of meiosis serves to ensure that each germ cell acquires a precise set of 23 homologues, including either an X or a Y chromosome. Meiosis essentially consists of two separate divisions, referred to as the first and the second meiotic divisions (see Fig. 4-19).

The sex chromosomes, the male Y and the female X, differ from the nonsex chromosomes or autosomes. Whereas the female has two X chromosomes, the male has only one X and one Y. During the process of meiosis, each germ cell ends up with a precise haploid set of autosomes. In sperm, each haploid set will also include either an X or a Y chromosome. Thus, by chance, roughly 50% of sperm will bear an X and 50% a Y. Since the mother has only one kind of sex chromosome, all of the ova contain a single X. If an ovum is fertilized by an X-bearing sperm, a female zygote will result (46,XX); if by a Y-bearing sperm, the offspring will be male (46,XY). Thus, it is the paternal chromosome that determines the sex of the child (Ohno et al, 1962

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