The cell nucleus consists of three major components:

1. The nuclear envelope.

2. Chromatin.

3. The nucleolus.

The nuclear envelope consists of two concentric membranes separated by a perinuclear space. The inner nuclear membrane is associated with the nuclear lamina, chromatin, and ribonucleoproteins. The outer nuclear membrane is continuous with the membranes of the endoplasmic reticulum and can be associated with ribosomes.

The nuclear pore complex has a tripartite structure, composed of a central cylindrical body placed between inner and outer octagonal rings, each consisting of eight protein particles. The central cylinder consists of a central plug and eight radiating spokes. The exact role of individual nuclear pore complex proteins in nucleocytoplasmic trafficking is unclear.

Nuclear pore complexes embedded in the nuclear envelope establish bidirectional communication gates for the trafficking of macromolecules between the cytoplasm and the nucleus. Small molecules (less than 40 to 60 kd) can diffuse passively through the nuclear pore complex. Proteins of any size, containing a nuclear localization amino acid sequence (NLS, Pro-Lys-Lys-Lys-Arg-Lys-Val), can be imported into the nucleus, however, by an energy-dependent mechanism (requiring ATP and GTP).

Nucleocytoplasmic Transport: Ran-GTPase

Protein nuclear import/export is controlled by Ran (for Ras-like nuclear GTPase), a small GTPase of the Ras superfamily that dictates the directionality of nucleocytoplasmic transport.

Ran shuttles across the nuclear pores and accumulates inside the nucleus by an active transport mechanism.

1. In the nucleus, a high concentration of Ran-GTP is achieved by RCC1, a GDP-GTP exchanger protein bound to chromatin. Ran-GTP determines the dissociation of imported proteins containing NLS by binding to importin beta, the transporter receptor protein.

2. In the opposite direction, from the nucleus to the cytoplasm, binding of Ran-GTP to the carrier protein exportin/Crm1 facilitates the assembly of complexes containing proteins with nuclear export sequence (NES).

3. In the cytoplasm, Ran-GTP is converted to Ran-GDP by Ran-GTPase, which is activated by two cooperating proteins: Ran-GAP (Ran-GTPase-activating protein) and RanBP (Ran-GTP binding protein). Consequently, the exported protein is dissociated from its transporter receptor protein exportin/ Crm1 and Ran-GTP. Importin and exportins are recycled by transport back across the nuclear pore complex.

Chromatin

Chromatin is defined as particles or "beads" (called nucleosomes) on a double-stranded DNA string. Each nucleosome consists of a histone octamer core and about two turns of DNA wound around the histone core. The histone octamer contains two molecules each of H2A, H2B, H3, and H4 histones. H1 histone cross-links the DNA molecule wrapped around the octamer.

Chromatin is packed in separate chromosomes that can be visualized during mitosis (or meiosis). During interphase (phases G₁, S, and G₂ of the cell cycle), individual chromosomes cannot be identified as such, but are present in a diffuse or noncondensed state.

Diffuse chromatin, called euchromatin ("good chromatin"), is transcriptionally (RNA synthesis) active and represents about 10% of total chromatin. Euchromatin is the site of synthesis on nonribosomal RNAs, including mRNA and transfer RNA (tRNA) precursors.

Condensed chromatin, called heterochromatin ("different chromatin"), is transcriptionally inactive and represents about 90% of total chromatin.

Dosage Compensation: X Chromosome Inactivation

X chromosome inactivation, known as dosage compensation, starts early in embryonic stem cell differentiation and is characterized by four features:

1. All but one of the X chromosomes undergoes inactivation.

2. The choice of the inactivated X chromosome is random. Either the paternal or the maternal X chromosome is inactivated.

3. The inactivation processes is heritable through subsequent rounds of cell division. The choice remains nonrandom for all subsequent cell descendants.

4. Both X chromosomes in oocytes remain active.

The transcriptional inactivation of one of the two X chromosomes is observed in the trophoblast on day 12 after fertilization and on day 16 in the embryo.

In humans, the inactivated X chromosome is recognized by the presence of the Barr body, a heterochromatin mass observed adjacent to the nuclear envelope or in the form of a drumstick in polymorphonuclear leukocytes. If a cell has more than two X chromosomes, the extra X chromosomes are inactivated, and more than one Barr body is visualized.

The concept of dosage compensation is relevant to the understanding of tumor-suppressor inactivation and oncogene inactivation when a single active copy of an X-linked genes is affected. Some genes located on the inactivated X chromosome escape inactivation in normal cells and several of these genes, most of which encode growth factors, are implicated in human cancer. For example, the gene encoding gastrin-releasing peptide receptor is associated with an increased risk in lung cancer in women.

Nucleolus

The nucleolus is the site of synthesis and processing of ribosomal RNA (rRNA) and assembly of ribosomal subunits. The rRNA genes are arranged in an array of multiple copies transcribed by RNA polymerase I.

The nucleolus houses several proteins, including fibrillarin and nucleolin, required for pre-rRNA processing. In addition, the nucleolus contains nucleostemin, a protein unrelated to ribosomal biogenesis. Nucleolin and nucleostemin are shuttling proteins; they relocalize from the nucleolus to the nucleoplasm where they interact with protein p53, a protector of DNA damage by preventing DNA replication in response to genomic stress.

Essentially, the nucleolus is a multifunctional nuclear structure consisting of stable proteins involved in ribosomal synthesis and molecules shuttling between the nucleolus and nucleoplasm to fulfill non-nucleolar functions.

Structurally, the nucleolus consists of three major components:

1. A fibrillar center (corresponding to chromatin containing repeated rRNA genes and the presence of RNA polymerase I and signal recognition particle [SRP] RNA).

2. A dense fibrillar component (where nascent rRNA is present and undergoing some of its processing). Fibrillarin and nucleolin are found in the fibrillar dense component.

3. A granular component (where the assembly of ribosomal subunits, containing 18S rRNA [small subunit] and 28S rRNA [large subunit], is completed). Nucleostemin, a protein unrelated to ribosomal biogenesis, coexists with the granular components.

Nucleoli are typically surrounded by a shell of heterochromatin, mostly from centromeric and pericentromeric chromosomal regions.

The nucleolus dissociates during mitosis, then reappears at the beginning of the G₁ phase. More than one nucleolar mass, each representing the product of a chromosome with a nucleolar organizing region (NOR), can be observed in the nucleus. In some cells with an extended interphase, such as neurons, a single large nucleolus is organized by the fusion of several nucleolar masses.

The active process of rRNA synthesis can be visualized at the electron microscopic level by spreading the contents of nuclei of cells with hundreds of nucleoli (e.g., amphibian oocytes). rRNA genes can be seen as repeating gene units along the chromatin axis, like "Christmas trees," pointing in the same direction and separated by nontranscribed spacers. The entire rRNA gene region is covered by more than 100 RNA polymerase I molecules synthesizing an equivalent number of fibrils, each with a terminal granule.

Each fibril represents an rRNA precursor (45S) ribonucleoprotein molecule oriented perpendicularly to the chromatin axis similar to the branches of a tree. The 45S rRNA precursor is detached from the chromatin axis and cleaved into 28S, 18S, and 5.8S rRNAs.

The 18S rRNA and associated proteins form the small ribosomal subunit. The 28S and 5.8S, together with 5S rRNA made outside the nucleolus, and associated proteins form the large ribosomal subunit.

The mRNA precursor is transcribed by RNA polymerase II, and the tRNA precursor is transcribed by RNA polymerase III.

Localization of Nucleic Acids

Cytochemistry and autoradiography provide information about the cellular distribution and synthesis of nucleic acids. The Feulgen reaction is specific for the localization of DNA. Basic dyes, such as toluidine blue, stain DNA and RNA. Pretreatment with deoxyribonuclease (DNAse) and ribonuclease (RNAse) defines the distribution sites of DNA and RNA by selective removal of one of the nucleic acids.

Autoradiography and radiolabeled precursors for one of the nucleic acids can determine the timing of their synthesis. In this technique, a radioactive precursor of DNA ([³H]thymidine) or RNA ([³H]uridine) is exposed to living cells. As a result of exposure to the radiolabel, any synthesized DNA or RNA contains the precursor. The radioactivity is detected by coating the cells with a thin layer of a photographic emulsion. Silver-containing crystals of the emulsion are exposed to structures of the cell containing radioactive DNA or RNA. After development of the emulsion, silver grains indicate the location of the labeled structures. This approach has been used extensively for determining the duration of several phases of the cell cycle.