Chromatin

Chromatin, in nondividing nuclei, is in fact the chromosomes in a different degree of uncoiling. According to the degree of chromosome condensation, two types of chromatin can be distinguished with both the light and electron microscopes (Figures 3-2 and 3-4, in previous post). Heterochromatin (Gr. heteros, other, + chroma, color), which is electron dense, appears as coarse granules in the electron microscope and as basophilic clumps in the light microscope. Euchromatin is the less coiled portion of the chromosomes, visible as a finely dispersed granular material in the electron microscope and as lightly stained basophilic areas in the light microscope. The proportion of heterochromatin to euchromatin accounts for the light-to-dark appearance of nuclei in tissue sections as seen in light and electron microscopes. The intensity of nuclear staining of the chromatin is frequently used to distinguish and identify different tissues and cell types in the light microscope.

Chromatin is composed mainly of coiled strands of DNA bound to basic proteins (histones); its structure is schematically presented in Figure 3-5 (in previous post). The basic structural unit of chromatin is the nucleosome (Figure 3-9), which consists of a core of four types of histones: two copies each of histones H2A, H2B, H3, and H4, around which are wrapped 166 DNA base pairs. An additional 48-base pair segment forms a link between adjacent nucleosomes, and another type of histone (H1 or H5) is bound to this DNA. This organization of chromatin has been referred to as "beads-on-a-string." Nonhistone proteins are also associated with chromatin, but their arrangement is less well understood.

Figure 3-9

Schematic representation of a nucleosome. This structure consists of a core of four types of histones (two copies of each) H2A, H2B, H3, and H4 and one molecule of H1 or H5 located outside the DNA filament.

The next higher order of organization of chromatin is the 30-nm fiber (Figure 3-10). In this structure, nucleosomes become coiled around an axis, with six nucleosomes per turn, to form the 30-nm chromatin fiber. There are higher orders of coiling, especially in the condensation of chromatin during mitosis and meiosis.

Figure 3-10

The orders of chromatin packing believed to exist in the metaphase chromosome. Starting at the top, the 2-nm DNA double helix is shown; next is the association of DNA with histones to form filaments of nucleosomes of 11 nm and 30 nm. Through further condensation, filaments with diameters of 300 nm and 700 nm are formed. Finally, the bottom drawing shows a metaphase chromosome, which exhibits the maximum packing of DNA.

The chromatin pattern of a nucleus has been considered a guide to the cell's activity. In general, cells with light nuclei are more active than those with condensed, dark nuclei. In light-stained nuclei (with few heterochromatin clumps), more DNA surface is available for the transcription of genetic information. In dark-stained nuclei (rich in heterochromatin), the coiling of DNA makes less surface available.

Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin and is one of the two X chromosomes present in female cells. The X chromosome that constitutes the sex chromatin remains tightly coiled and visible, whereas the other X chromosome is uncoiled and not visible. Evidence suggests that the sex chromatin is genetically inactive. The male has one X chromosome and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is visible. In human epithelial cells, sex chromatin appears as a small granule attached to the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin. Blood smears are also often used, in which case the sex chromatin appears as a drumsticklike appendage to the nuclei of the neutrophilic leukocytes (Figure 3-11).

Figure 3-11

Morphological features of sex chromatin in human female oral (buccal) epithelium and in a polymorphonuclear leukocyte. In the epithelium, sex chromatin appears as a small, dense granule adhering to the nuclear envelope. In the leukocyte, it has a drumstick shape.

Medical Application

The study of sex chromatin discloses the genetic sex in patients whose external sex organs do not permit assignment of gender, as in hermaphroditism and pseudohermaphroditism. Sex chromatin helps the study of other anomalies involving the sex chromosomes—eg, Klinefelter syndrome, in which testicular abnormalities, azoospermia (absence of spermatozoa), and other symptoms are associated with the presence of XXY chromosomes.

The study of chromosomes progressed considerably after the development of methods that induce cells to divide, arrest mitotic cells during metaphase, and cause cell rupture. Mitosis can be induced by phytohemagglutinin (in cell cultures) and can be arrested in metaphase by colchicine. Cells are immersed in a hypotonic solution, which causes swelling, after which cells are flattened and broken between a glass slide and a coverslip.

The pattern of chromosomes obtained in a human cell after staining is illustrated in Figure 3-12. In addition to the X and Y sex chromosomes, the remaining chromosomes are customarily grouped according to their size and morphological characteristics into 22 successively numbered pairs.

Figure 3-12

Human karyotype preparation made by means of a banding technique. Each chromosome has a particular pattern of banding that facilitates its identification and also the relationship of the banding pattern to genetic anomalies. The chromosomes are grouped in numbered pairs according to their morphological characteristics.

Medical Application

The number and characteristics of chromosomes encountered in an individual are known as the karyotype (Figure 3-12). The study of karyotypes has revealed chromosomal alterations associated with tumors, leukemias, and several types of genetic diseases.

The development of techniques that reveal segmentation of chromosomes in transverse, differentially stained bands permitted a more precise identification of individual chromosomes and the study of gene deletions and translocations. These techniques are based mainly on the study of chromosomes previously treated with saline or enzyme solution and stained with fluorescent dyes or Giemsa's blood-staining technique. In situ hybridization is also a valuable technique for localizing DNA sequences (genes) in chromosomes.

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