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NUCLEUS

The nucleus (Figs 1.1, 1.2) is generally the largest intracellular structure and is usually spherical or ellipsoid in shape, with a diameter of 3–10 μm. Histological stains used to identify nuclei in tissue sections mainly detect the acidic molecules of deoxyribonucleic acid (DNA), which are largely confined to the nucleus.

Nuclear membrane

The nucleus is surrounded by two concentric lipid bilayers which together form the nuclear membrane or envelope. The outer membrane bilayer and the lumen between the two bilayers are continuous with the rough endoplasmic reticulum. Like the rough endoplasmic reticulum, the outer membrane of the nuclear envelope is studded with ribosomes that are active in protein synthesis; the newly synthesized proteins pass into the perinuclear space between the two membrane layers.

A special class of intermediate filaments known as lamins is associated with the inner surface of the nuclear membrane. The lamins form a dense meshwork beneath the membrane, the nuclear lamina. The lamin filaments cross each other at right angles to create an irregular anastomosing network that covers the interior surface of the nuclear membrane. In so doing, they reinforce the nuclear membrane mechanically, determine the shape of the nucleus and provide a binding site for a range of proteins that anchor chromatin. Nuclear lamin A, with over 350 mutations, is the most mutated protein linked to human disease. Lamin A mutations cause a surprisingly wide range of diseases, from progeria to various dystrophies (reviewed in Mattout et al 2006 and Pollard and Earnshaw 2007).

Condensed chromatin (heterochromatin) also tends to aggregate near the nuclear membrane during interphase. At the end of mitotic and meiotic prophase (see below), the lamin filaments disassemble, causing the nuclear membranes to vesiculate and disperse into the endoplasmic reticulum. During the final stages of mitosis (telophase), proteins of the nuclear periphery, including lamins, associate with the surface of the chromosomes, providing docking sites for membrane vesicles. Fusion of these vesicles reconstitutes the nuclear compartment.

The transport of molecules between the nucleus and the cytoplasm occurs via specialized nuclear pore structures that perforate the nuclear membrane (Fig. 1.14A). They act as highly selective directional molecular filters, permitting proteins such as histones and gene regulatory proteins (which are synthesized in the cytoplasm but function in the nucleus) to enter the nucleus, and molecules that are synthesized in the nucleus but destined for the cytoplasm (e.g. ribosomal subunits, transfer RNAs and messenger RNAs), to leave the nucleus.

  

Fig. 1.14  A, Nuclear envelope with nuclear pores (arrowed) in transverse section, showing the continuity between the inner and outer phospholipid layers of the envelope on either side of the pore. The fine ‘membrane’ appearing to span the pore is formed by proteins of the pore complex. Note that the chromatin is less condensed in the region of nuclear pores. Nucleus (N); cytoplasm (C). B, Nuclear pores seen ‘en face’ as spherical structures (arrows) in a tangential section through the nuclear envelope. The appearance of the envelope varies in electron density as the plane of section passes through different regions of the curved double membrane, which is interrupted at intervals by pores through the envelope (see also Fig. 1.1). The surrounding cytoplasm with ribosomes is less electron-dense. Human tissues.

Ultrastructurally, nuclear pores appear as disc-like structures with an outer diameter of 130 nm and an inner pore with an effective diameter for free diffusion of 9 nm (Fig. 1.14B). The nuclear membrane of an active cell is bridged by up to 4000 such pores. The nuclear pore complex has an octagonal symmetry and is formed by an assembly of more than 50 proteins, the nucleoporins. The inner and outer nuclear membranes fuse around the pore complex (Fig. 1.14A). Nuclear pores are freely permeable to small molecules, ions and proteins up to about 17 kDa. Most proteins that enter the nucleus do so as complexes with specific transport receptor proteins known as importins. Importins shuttle back and forth between the nucleus and cytoplasm. Binding of the cargo to the importin requires a short sequence of amino acids known as a nuclear localization sequence (NLS), and can be either direct or via an adapter protein. Interactions of the importin with components of the nuclear pore move it together with its cargo through the pore by an energy-independent process still not understood. A complementary cycle functions in export of proteins and RNA molecules from the nucleus to the cytoplasm using transport receptors known as exportins. For further explanation, see Pollard & Earnshaw (2007).

Chromatin

DNA is organized within the nucleus in a DNA–protein complex known as chromatin. The protein constituents of chromatin are the histones and the non-histone proteins. Non-histone proteins are an extremely heterogeneous group that includes structural proteins, DNA and RNA polymerases and gene regulatory proteins. Histones are the most abundant group of proteins in chromatin, primarily responsible for the packaging of chromosomal DNA into its primary level of organization, the nucleosome. There are four core histone proteins: H2A, H2B, H3 and H4 that combine in equal ratios to form a compact octameric nucleosome core. A fifth histone, H1, is involved in further compaction of the chromatin. The DNA molecule (one per chromosome) winds twice around each nucleosome core, taking up 165 nucleotide pairs. This packaging organizes the DNA into a chromatin fibre 11 nm in diameter, and imparts to this form of chromatin the electron microscopic appearance of beads on a string, in which each bead is separated by a variable length of DNA, typically about 35 nucleotide pairs long. The nucleosome core region and one of the linker regions constitute the nucleosome proper, which is typically about 200 nucleotide pairs in length. However, chromatin rarely exists in this simple form and is usually packaged further into a 30 nm thick fibre, involving a single H1 histone per nucleosome, which interacts with both DNA and protein to impose a higher order of nucleosome packing. Usually, 30 nm fibres are further coiled or folded into larger domains. Individual domains are believed to decondense and extend during active transcription. In a typical interphase nucleus, euchromatin (nuclear regions that appear pale in appropriately stained tissue sections, or relatively electron-lucent in electron micrographs; Fig. 1.2) is likely to consist mainly of 30 nm fibres and loops, and contains the transcriptionally active genes. Transcriptionally active cells, such as most neurones, have nuclei that are predominantly euchromatic and often described as ‘open face’ nuclei.

Heterochromatin (nuclear regions that appear dark in appropriately stained tissue sections or electron-dense in electron micrographs) is characteristically located mainly around the periphery of the nucleus, except over the nuclear pores (Fig. 1.14A), and around the nucleolus (Fig. 1.2). It is a relatively compacted form of chromatin in which the histone proteins carry a specific set of post-translational modifications, including methylation at characteristic residues. This facilitates the binding of specific heterochromatin-associated proteins. Heterochromatin includes non-coding regions of DNA, such as centromeric regions, which are known as constitutive heterochromatin. DNA that is inactivated (becoming resistant to transcription) in some cells as they differentiate during development or cell maturation contributes to heterochromatin, and is known as facultative heterochromatin. The inactive X chromosome in females is an example of facultative heterochromatin and can be identified in the light microscope as the deeply staining Barr body (drumstick chromosome), often located near the nuclear periphery.

In transcriptionally inactive cells, chromatin is predominantly in the condensed, heterochromatic state, and may comprise as much as 90% of the total. Examples of such cells are mature neutrophil leukocytes (in which the condensed chromatin is present in a multilobed, densely staining nucleus), and the highly condensed nuclei of orthochromatic erythroblasts (late-stage erythrocyte precursors). In most mature cells, a mixture of the two occurs, indicating that only a proportion of the DNA is being transcribed. A particular instance of this is seen in the mature B lymphocyte (plasma cell), in which much of the chromatin is in the condensed condition and is arranged in regular masses around the perimeter of the nucleus, producing the so-called ‘clock-face’ nucleus (Figs 4.6, 4.12). Although this cell is actively transcribing, much of its protein synthesis is of a single immunoglobulin type, and consequently much of its genome is in an inactive state.

During mitosis, the chromatin is further reorganized and condensed to form the much shortened chromosomes characteristic of metaphase. This shortening is achieved through further levels of close packing of the chromatin. The mechanism of condensation is unknown, but the condensed chromosomes are stabilized by protein complexes known as condensins. Progressive folding of the chromosomal DNA by interactions with specific proteins can reduce 5 cm of chromosomal DNA by 10,000 fold, to a length of 5 μm in the mitotic chromosome.

Chromosomes and karyotypes

The nuclear DNA of eukaryotic cells is organized into linear units called chromosomes. The DNA in a normal human diploid cell contains 6 × 109 nucleotide pairs organized in the form of 46 chromosomes (44 autosomes and two sex chromosomes). The largest human chromosome (number 1) contains 2.5 × 108 nucleotide pairs, and the smallest (the Y chromosome) 5 × 107 nucleotide pairs.

Each chromosomal DNA molecule contains a number of specialized nucleotide sequences that are associated with its maintenance. One is the centromere. During mitosis, a disc-shaped structure composed of a complex array of proteins, the kinetochore, forms as a substructure of the centromeric region of DNA in order to attach it to the microtubular spindle. Another sequence, the telomere, defines the end of each chromosomal DNA molecule. Telomeres consist of hundreds of repeats of the nucleotide sequence (TTAGGG)n. The very ends of the chromosomes cannot be replicated by the same DNA polymerase as the rest of the chromosome, and are maintained by a specific enzyme called telomerase which contains an RNA subunit which acts as the template for lengthening the TTAGGG repeats. Thus telomerase is a specialized type of polymerase known as a reverse transcriptase that turns sequences in RNA back into DNA. The number of tandem repeats of the telomeric DNA sequence varies. It appears to shorten with successive cell divisions, because telomerase activity reduces or is absent in differentiated cells with a finite lifespan. It is believed that this mechanism contributes to regulation of cell senescence and may protect against proliferative disorders, including cancer (reviewed in Flores et al 2006).

Classification of human chromosomes

A number of genetic abnormalities can be directly related to the chromosomal pattern. The characterization or karyotyping of chromosome number and structure is therefore of considerable diagnostic importance. The identifying features of individual chromosomes are most easily seen during metaphase, although prophase chromosomes can be used for more detailed analyses.

Lymphocytes separated from blood samples, or cells taken from other tissues, are used as a source of chromosomes. Diagnosis of fetal chromosome patterns is generally carried out on samples of amniotic fluid containing fetal cells aspirated from the uterus by amniocentesis, or on a small piece of chorionic villus tissue removed from the placenta. Whatever their origin, the cells are cultured in vitro and stimulated to divide by treatment with agents that stimulate cell division. Mitosis is interrupted at metaphase with spindle inhibitors. The chromosomes are dispersed by first causing the cells to swell in a hypotonic solution, then the cells are gently fixed and mechanically ruptured on a slide to spread the chromosomes. They are subsequently stained in various ways to allow the identification of individual chromosomes by size, shape and distribution of stain (Fig. 1.15). General techniques show the obvious landmarks, e.g. lengths of arms and positions of constrictions. Banding techniques demonstrate differential staining patterns, characteristic for each chromosome type. Fluorescence staining with quinacrine mustard and related compounds produces Q bands, and Giemsa staining (after treatment that partially denatures the chromatin) gives G bands (Fig. 1.15A). Other less widely used methods include: reverse-Giemsa staining, in which the light and dark areas are reversed (R bands); the staining of constitutive heterochromatin with silver salts (C-banding); T-banding to stain the ends (telomeres) of chromosomes. Collectively, these methods permit the classification of chromosomes into numbered autosomal pairs in order of decreasing size, from 1 to 22, plus the sex chromosomes.

  

Fig. 1.15  Chromosomes from normal males, arranged as karyotypes. A, G-banded preparation; B, preparation stained by multiplex fluorescence in situ hybridization to identify each chromosome.
(By courtesy of Dr Denise Sheer, Cancer Research UK.)

A summary of the major classes of chromosomes is given below:

Group Features
1–3 (A) Large metacentric chromosomes
4–5 (B) Large submetacentric chromosomes
6–12 + X (C) Metacentrics of medium size
13–15 (D) Medium-sized acrocentrics with satellites
16–18 (E) Shorter metacentrics (16) or submetacentrics (17,18)
19–20 (F) Shortest metacentrics
21–22 + Y (G) Short acrocentrics; 21, 22 with satellites, Y without

Methodological advances in banding techniques improved the recognition of abnormal chromosome patterns. The use of in situ hybridization with fluorescent DNA probes specific for each chromosome (Fig. 1.15B) permits the identification of even very small abnormalities.

Nucleolus

Nucleoli are a prominent feature of an interphase nucleus (Fig. 1.2). They are the site of most of the synthesis of rRNA and assembly of ribosome subunits. Ultrastructurally, the nucleolus appears as a pale fibrillar region (non-transcribed DNA), containing dense fibrillar cores (sites of rRNA gene transcription) and granular regions (sites of ribosome subunit assembly) within a diffuse nucleolar matrix. Five pairs of chromosomes carry rRNA genes organized in clusters of tandemly repeated units on each chromosome. Each rRNA unit is transcribed individually and encodes a large precursor RNA that is processed to yield the 28S, 18S and 5.8S rRNA molecules. This processing takes place in the nucleolus, as does the processing of a number of other stable RNAs, including the RNA component of the signal recognition particle (SRP), which is essential for protein secretion. During mitosis the nucleolus breaks down. It reforms after nuclear envelope reformation in telophase, in a process associated with the onset of transcription in nucleolar organizing centres on each chromosome. The 28S, 18S and 5.8S rRNA molecules are assembled into their ribosomal subunits in the granular region of the nucleolus together with the 5S rRNA, which is not synthesized in the nucleolus. The newly formed ribosomal subunits are then translocated to the cytoplasm through the nuclear pores.

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