3. The Cell Nucleus

structure of chromatin

Chromatin consists of DNA double helix complexed with histones and nonhistone proteins. It resides within the nucleus as heterochromatin and euchromatin. The euchromatin/ heterochromatin ratio is higher in malignant cells than in normal cells.

Chromatin

Chromatin, a complex of DNA and proteins, is responsible for the characteristic basophilia of the nucleus

Membrane blebbing

results from cell membrane alterations. One alteration is related to translocation of certain molecules (e.g., phosphatidylserine) from the cytoplasmic surface to the outer surface of the plasma membrane. These changes cause the plasma membrane to change its physical and chemical properties and lead to blebbing without loss of membrane integrity (see Fig. 3.18).

copy number variations (CNVs)

segments of DNA that are duplicated or deleted

Nucleostemin

-p53 binding protein found in undifferentiated cells -Decreases as cells become differentiated. -Presence in cancer cells may play a role in unchecked proliferation The presence of nucleostemin in malignant cells suggests that it could play a role in their uncontrolled proliferation (Folder 3.2). In addition, DNA, RNA, and retroviruses and their viral proteins interact with the nucleolus and cause redistribution of fibrillar and granular materials during the course of viral infection. These viruses can use components of the nucleolus as part of their own replication process. Evidence suggests that viruses may target the nucleolus and its components to favor viral transcription and translation and perhaps alter the cell cycle to promote viral replication.

telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.

Structure of a chromosome

Chromosomes consist of chromatin extensively folded into loops; this conformation is maintained by DNA-binding proteins (Figure 2.3). Each chromosome contains a long DNA molecule and associated proteins, assembled into nucleosomes, the structural unit of chromatin packaging. Each nucleosome has a core of eight histones (histone octamers) and the DNA double helix is wrapped around the histone octamers in such a fashion that it makes two spiral turns. Since the DNA double helix is extremely long, it connects a huge number of histone octamers to each other. The DNA double helix between adjacent histone octamers is not associated with histones and appears as if it were a thin string that connects neighboring histone octamers to each other; therefore, these connecting regions of the DNA double helix are known as linker DNA. Chromosomes are visible with the LM only during mitosis and meiosis, when their chromatin condenses; otherwise, the chromatin is extended and is not visible by light microscopy.

Condensed chromatin

Condensed chromatin contains an additional histone, H1, which wraps around groups of nucleosomes, thus forming 30-nm-diameter filaments of helical coils of six nucleosomes per turn, which is the structural unit ofthe chromosome.

Interphase

Interphase is considerably longer than the M phase and is the period during which the cell doubles in size and DNA content. a. Interphase is divided into three separate phases (G1, S, and G2) during which specific cellular functions occur.

Karyotype

Karyotype refers to the number and morphology of chromosomes and is characteristic for each species. 1. Haploid number (n) is the number of chromosomes in germ cells (23 in humans). 2. Diploid number (2n) is the number of chromosomes in somatic cells (46 in humans).

Large intergenic noncoding RNAs (lincRNAs)

Large intergenic noncoding RNAs (lincRNAs), more than 200 nucleotides in length, also function in gene regulation. Since each cell of a female possesses two X chromosomes, one of the X chromosomes is transcribed to form lincRNAs that specifically coat that particular X chromosome and prevents the transcription of its genes. Other lincRNAs prevent the transcription of various genes on different chromosomes. Still other lincRNAs compete with certain mRNAs for miRNAs, thereby acting as decoys that protect the mRNAs from the inhibitory actions of miRNAs and facilitating the translation of the mRNA to synthesize a particular protein.

Meiosis

Meiosis is a special form of cell division in germ cells (oogonia and spermatozoa) in which the chromosome number is reduced from diploid (2n) to haploid (n). These events are accomplished via two reduction divisions. 1. This occurs in developing germ cells in preparation for sexual reproduction. Subsequent fertilization results in diploid zygotes. 2. DNA content ofthe original diploid cell is doubled ( 4n) in the S phase preparatory to meiosis. a. This phase is followed by two successive cell divisions that give rise to four haploid cells. b. In addition, recombination of maternal and paternal genes occurs by crossing over and random assortment, yielding the unique haploid genome of the gamete.

Anoikis

Programmed cell death of cells that detach from the substratum to which they are normally anchored Signals from an intercellular matrix are sensed by integrins that form an integral part of anchoring cell-to-extracellular matrix junctions Due to their connections with cell cytoskeleton, integrins are involved in the intrinsic pathway-signaling mechanisms that control apoptosis, DNA damage responses, and the function of death receptors. In metastatic cancer, cells develop mechanisms to survive the anoikis process. This resistance is due to various mechanisms that include changes in the integrin receptor types, activation of antiapoptotic factors, oncogene activation, and growth factor receptor signaling.

Regulatory RNAs include

Regulatory RNAs include micro RNA (miRNA), large intergenic noncoding RNA (lincRNA), and small interfering RNAs (siRNAs).

Telomeres

Repeated DNA sequences at the ends of eukaryotic chromosomes.

There are two recognizable types of heterochromatin:

There are two recognizable types of heterochromatin: constitutive and facultative.

Pyroptosis

is a form of cell death induced by infection with certain microorganisms that generate intense inflammatory reactions. This pathway is uniquely dependent on the caspase-1 enzyme, which is not involved in caspase cascade in apoptotic cell death. Caspase-1 activates the inflammatory cytokines such as IL-1 and IL-18 that mediate intense inflammatory reactions in surrounding tissue.

Entosis [Gr., inside]

is a nonapoptotic cell death process in which one cell can actively internalize a similar cell that became detached from the extracellular matrix. After internalization, the "swallowed" cell remains alive within the host cell until it is either degraded by the lysosomal mechanism or released. Entosis is a specific receptor-regulated process that involves cadherins and the formation of anchoring cell-to-cell junctions between two similar types of cells (i.e., within the epithelium). This process should be distinguished from cell cannibalism, which is a nonspecific process observed in metastatic tumors that involves cancer cells "eating" and killing the immune cells that are directed against them.

Necroptosis

is a regulated caspase-independent cell death mechanism that can be induced in different cell types. It is initiated by the activation of the tumor necrosis factor receptors (TNFRs or death receptors) and the Fas signaling pathway. Although it occurs underregulated conditions, necroptotic cell death is characterized by the same morphologic features as unregulated necrotic death. Necrostatin-1 is a specific inhibitor of necroptosis that significantly reduces ischemic damage in affected tissues.

Autophagy

is a regulated cellular process that enables cells to turn over their contents by lysosomal degradation of their own components. It starts when an intracellular membrane (often part of sER cistern) wraps around an organelle or portion of the cytoplasm, forming a closed double membrane-bound vacuole. This vacuole, called an autophagosome, initially devoid of any lysosomal enzymes, fuses with lysosomes and initiates digestion. For a detailed description of three pathways utilized in autophagy, see pages 41 to 43.

Mitotic catastrophe

is a type of cell death that occurs during mitosis. It results from a combination of cellular damage and malfunction of several cell-cycle checkpoints such as the G1, S, and G2 DNA-damage checkpoints or the spindle-assembly checkpoint (page 85). Failure to arrest the cell cycle before mitosis occurs causes problems with chromosome separation, which triggers the apoptotic pathway and cell death.

Paraptosis

is an alternative, nonapoptotic cell death that may be induced by growth factor receptors (i.e., insulin growth factor [IGF-1] receptor). In contrast to apoptosis, cell death is not mediated by caspases but by mitogen-activated protein kinases (MAPKs). On a cellular level, paraptosis is characterized by the formation of multiple large vacuoles within the cell cytoplasm along with mitochondrial swellings.

tRNA

tRNA is folded into a cloverleaf shape and contains approximately 80 nucleotides, terminating in adenylic acid (where amino acids attach). 1. Each tRNA combines with a specific amino acid that has been activated by an enzyme. 2. One end of the tRNA molecule possesses an anticodon, a triplet of nucleotides that recognizes the complementary codon in mRNA. If recognition occurs, the anticodon ensures that the tRNA transfers its activated amino acid molecule in the proper sequence to the growing polypeptide chain.

Formation of apoptotic bodies

the final step of apoptosis, results in cell breakage (Fig. 3.19a-c). These membrane-bounded vesicles originate from the cytoplas- mic bleb containing organelles and nuclear material. They are rapidly removed without a trace by phagocytotic cells. The removal of apoptotic bodies is so effi cient that no inflammatory response is elicited. Apoptosis occurs more than 20 times faster than mitosis; therefore, it is challenging to find apoptotic cells in a routine H&E preparation (Fig. 3.19d)

The cell's progress through this phase (G1 phase,) is monitored by two checkpoints:

(1) the restriction checkpoint, which is sensitive to the size of the cell, the state of the cell's physiologic processes, and its interactions with extracellular matrix; and (2) the G1 DNA-damage checkpoint, which monitors the integrity of newly replicated DNA. For instance, if the DNA has irreparable damage, then the G1 DNA-damage checkpoint detects the high levels of tumor-suppressing protein p53 and it does not allow the cell to enter the S phase. The cell will then most likely undergo programmed cell death (apoptosis).

Chromatin has several functions that include

1 .folding of the DNA strand into small enough volume to be able to contain it within the nucleus of the cell; 2. protecting the DNA from physical damage during and between cell divisions; 3. controlling the activity of DNA, that is, permitting or preventing its transcription; 4. controlling the precise duplication of the DNA in preparation for cell division; 5. facilitating the repair of DNA in case of replication error or due to physical or chemical insult.

The nucleolus contains four distinct regions.

1. Fibrillar centers are composed of the NORs of the five chromosomes listed above(chromosomes 13, 14, 15, 21, and 22), the ribonucleoprotein (RNP) signal recognition particle, and RNA polymerase I, the enzyme required for the transcription of rRNA. 2. The pars fibrosa is composed of 5-nm fibrils surrounding the fibrillar centers and contains transcriptionally active DNA, ribosomal genes, and a substantial quantity of rRNA. Additionally, the RNP fibrillarin and the phosphoproteins nucleolin are located in the pars fibrosa; these proteins participate in the processing of rRNA precursors to form mature rRNA. 3. The pars granulosa is composed of 15-nm maturing ribosomal precursor particles where 185 rRNA and 285 rRNA subunits are assembled. Ribosomal proteins, manufactured in and imported from the cytoplasm, are combined with rRNA to form the small and large ribosomal subunits that are then individually exported into the cytoplasm, where ribosomal assembly is completed (see Chapter 3, Cytoplasm and Organelles IIIB 1a). Additionally, a protein that resembles guanine nucleotide-binding protein, known as nucleostemin, is located in the pars granulosa. Large quantities of this protein are present in cancer cells and stem cells because it functions in regulating the cell cycle and it also has a direct influence on cell differentiation. 4. Nucleolar matrix is a fiber network participating in the organization of the nucleolus.

Nuclear pores

1. Nuclear pores average 80 nm in diameter and number from dozens to thousands, depending upon metabolic activity ofthe cell; they are associated with the NPC. 2. They are formed by fusion of the inner and outer nuclear membranes. 3. They permit passage of certain molecules in either direction between the nucleus and the cytoplasm via a 9-nm channel opening. 4. NPCs are aided in communicating with each other by the nuclear lamina.

Inner nuclear membrane

1. The inner nuclear membrane is also about 6 nm thick. 2. It faces the nuclear material but is separated from it and is supported on its inner surface by the nuclear lamina, fibrous lamina that is 80 to 300 nm thick and composed primarily of lamins A, B1, B2, and C. These intermediate filament proteins form an orthogonal trellis that binds to transmembrane receptor molecules, such as emerin and various lamina-associated polypeptides traversing the inner nuclear membrane. The various lamins assist in organizing the nuclear envelope, directing the formation of nuclear pore complexes (NPCs), and the organization of perinuclear chromatin. In addition, they are essential during the mitotic events, when they are responsible for the ordered disassembly and reassembly of the nuclear envelope. Phosphorylation of lamins leads to disassembly, and dephosphorylation results in reassembly of the nuclear envelope.

Perinuclear cisterna

1. The perinuclear cisterna is located between the inner and outer nuclear membranes and is 20 to 40 nm wide. 2. It is continuous with the cisterna of the RER. 3. It is perforated by nuclear pores at various locations.

Outer nuclear membrane

1. This membrane is about 6 nanometers (nm) thick. 2. It faces the cytoplasm and is continuous at certain sites with the rough endoplasmic reticulum (RER). 3. A loosely arranged mesh of intermediate filaments (vimentin) surrounds the outer nuclear membrane on its cytoplasmic aspect. 4. Ribosomes stud the cytoplasmic surface of the outer nuclear membrane. These ribosomes synthesize proteins that enter the perinuclear cisterna.

Transformed cells

1. Transformed cells have lost their a bility to respond to regulatory signals controlling the cell cycle, and by this, they may undergo cell division indefinitely, thus becoming cancerous. 2. Vinca alkaloids may arrest these cells in mitosis, whereas drugs that block purine and pyrimidine synthesis may a rrest these cells in the S phase of the cell cycle.

nuclear particles

1. lnterchromatin granules are clusters of irregularly distributed particles (20-25 nm in diameter) that contain RNP and various enzymes. 2. Perichromatin granules (Figure 2.1) are single dense granules (30-50 nm in diameter) surrounded by a less dense halo. They are located at the periphery of heterochromatin and exhibit a substructure of 3-nm packed fibrils. a. Perichromatin granules contain 4.7S RNA and two peptides similar to those found in heterogeneous nuclear RNPs (hnRNPs). b. They may represent messenger RNPs (mRNPs). c. The number of granules increases in liver cells exposed to carcinogens or temperatures above 37°C. 3. The hnRNP particles are complexes of precursor mRNA (pre-mRNA) and proteins and are involved in processing of pre-mRNA. 4. Small nuclear RNPs (snRNPs) are complexes of proteins and small RNAs and are involved in hnRNP splicing or in cleavage reactions

In a karyotype, chromosome pairs are sorted according to their size, shape, and emitted fluorescent color.

A preparation of chromosomes derived from mechanically ruptured, dividing cells that are then fixed, plated on a microscope slide, and stained is called a metaphase spread. In the past, chromosomes were routinely stained with Giemsa stain; however, with the recent development of in situ hybridization techniques, the fluorescent in situ hybridization (FISH) procedure is now more often used to visualize a chromosomal spread. These spreads are observed with fl uorescence microscopes, and computer-controlled cameras are then used to capture images of the chromosome pairs. Image-processing software is used to sort the chromosome pairs according to their morphology to form a karyotype (see Fig. F3.1.1a). A variety of molecular probes that are now commercially available are used in cytogenetic testing to diagnose disorders caused by chromosomal abnormalities such as nondisjunctions, transpositions (see Fig. F3.1.1a), deletions (see Fig. F3.1.1b), and duplications of specific gene sites. Karyotypes are also used for prenatal determination of sex in fetuses and for prenatal screening for certain genetic diseases (see Fig. 1.7).

Gene

A previous definition of a gene as a segment of DNA involved in producing a polypeptide chain has been recently updated to now read as being a union of genomic sequences encoding a coherent set of potentially overlapping functional products.

THE NUCLEUS

A. Structure. The nucleus, the largest organelle of the cell, includes the nuclear envelope, nucleolus, nucleoplasm, and chromatin and contains the genetic material encoded in the deoxyribonucleic acid (DNA) of chromosomes. B. Function. The nucleus directs protein synthesis in the cytoplasm via ribosomal ribonucleic acid (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). All types of RNAs, including regulatory RNAs (noncoding RNAs), are synthesized in the nucleus.

Apoptosis

Apoptosis is programmed cell death whereby cells are removed from tissues in an orderly fashion as a part of normal maintenance or during development A. Cells that undergo programmed cell death have several morphological features. 1. They include chromatin condensation, breaking up of the nucleus, and bleb bing of the plasma membrane. 2. The cell shrinks and is fragmented into membrane-enclosed fragments called apoptotic bodies. B. Apoptotic cells do not pose a threat to surrounding cells, because changes in their plasma membranes make them subject to rapid phagocytosis by macrophages and by neighboring cells. Macrophages that phagocytose apoptotic cells do not release cytokines that initiate the inflammatory response. Further, apoptotic cells may be inhibited by several survival factors produced by certain cells, growth factors, hormones, proteins, etc. C. The signals that induce apoptosis may occur through several mechanisms. 1. Genes that code for enzymes, called caspases, play an important role in the process. 2. Certain cytokines, such as tumor necrosis factor, may also activate caspases that degrade regulatory and structural proteins in the nucleus and cytoplasm, leading to the morphological changes characteristic of apoptosis. D. Defects in the process of programmed cell death contribute to many major diseases. 1. Excessive apoptosis causes extensive nerve cell loss in Alzheimer disease and stroke. 2. Insufficient apoptosis has been linked to cancer and other autoimmune diseases.

Pachytene

At this stage, synapsis is complete. Crossing- over occurs early in this phase and involves transposition of DNA strands between two diff erent chromosomes.

Cyclin E 2

Cdk1 M phase progression Chromatin-associated proteins, histone H1, nuclear lamins, myosin regulatory proteins, centrosomal proteins, transcription factors c-fos/jun, c-myb, oct-1, SWI5; p60src protein kinases, casein kinase II, c-mos protein kinases

Cyclin A 1

Cdk1 S phase through G2 phase and M phase entry Cdc25 phosphatase, cyclin B

Cyclin E

Cdk2 S phase entry ATMa or ATRb protein kinases, tumor-suppressing protein p53

Cyclin A 2

Cdk2 S phase progression Replication protein A, DNA polymerase, mini- chromosome maintenance (Mcm) protein

Cyclin D

Cdk4/6 G1 phase progression Tumor-suppressing protein p53, retinoblastoma susceptibility protein (pRb)

Constitutive heterochromatin

Constitutive heterochromatin contains the same regions of genetically inactive, highly repetitive sequences of DNA that are condensed and consistently packaged in the same regions of the chromosome when compared with other cells. Large amounts of constitutive heterochromatin are found in chromosomes near the centromeres and telomeres.

DNA

DNA is a long double-stranded helical linear molecule composed of multiple nucleotide sequences. It stores the individual's genetic information and acts as a template for the synthesis of RNA. The complete nucleotide sequences of a human are located in the 46 chromosomes of each cell and if stretched out and placed end to end it would measure almost 6 ft in length.

During cell division, the nuclear envelope is______to allow chromosome separation and is later ________ as the daughter cells form.

During cell division, the nuclear envelope is disassembled to allow chromosome separation and is later reassembled as the daughter cells form.

Chromosomes

During mitotic division, chromatin fibers formed from chromatin loop domains attached to a flexible protein scaffold undergo condensation to form chromosomes [Gr., colored bodies]. Each chromosome is formed by two chromatids that are joined together at a point called the centromere (Fig. 3.3b). The double nature of the chromosome is produced in the preceding synthetic (S) phase of the cell cycle (see page 84), during which DNA is replicated in anticipation of the next mitotic division.

Nondisjunction of Chromosomes

During prophase I of meiosis I, chromosome pairs align themselves at the equatorial plate and exchange genetic materials. During anaphase I, the chromosome pairs will separate and begin their migrations to opposite poles. Sometimes, the members of a pair fail to separate, resulting in one daughter cell containing an extra chromosome (n + 1 = 24), whereas the daughter cell at the opposite pole is minus a chromosome (n - 1 = 22). This development is known as nondisjunction. Upon fertilization with a normal gamete containing 23 chromosomes, the resulting zygote will contain either 47 chromosomes (trisomy for that extra chromosome) or 45 chromosomes (monosomy for that missing chromosome). Chromosomes 8, 9, 1 3, 1 8, and 21 are most frequently affected by nondisjunction. Aneuploidy, defined as an abnormal number of chromosomes, can be detected by karyotyping. 1 .Down syndrome (trisomy 21 ) is characterized by mental reta rdation, short stature, stubby appendages, congenital heart malformations, and other defects. 2. Klinefelter syndrome (XXY) is aneuploidy of the sex chromosomes, chara cterized by infertility, va riable degrees of masculinization, and small testes. 3. Turner syndrome (XO) is monosomy of the sex chromosomes, chara cterized by short stature, sterility, and various other abnormalities.

Diplotene

Early in this stage, the synaptonemal complex dissolves, and the chromosomes condense further. Homologous chromosomes begin to separate from each other and appear to be connected by newly formed junctions between chromosomes called chiasmata (sing., chiasma). Sister chromatids still remain closely associated with each other. Chiasmata indicate that crossing-over may have occurred.

Equatorial division (meiosis II)

Equatorial division (meiosis II) begins soon after the completion of meiosis I, following a brief interphase without DNA replication (no S phase). a. The sister chromatids are portioned out between the two daughter cells formed in meiosis I. The two daughter cells then divide, resulting in the distribution of chromosomes into four daughter cells, each containing its own unique recombined genetic material (1CDNA;n). Thus, every gamete contains its own unique set of genetic materials. b. The stages of meiosis II are similar to those of mitosis; thus, the stages are named similarly (prophase II, metaphase II, anaphase II, and telophase II). c. Meiosis II occurs more rapidly than mitosis.

Euchromatin

Euchromatin, constituting approximately 10% of the total chromatin, is transcriptionally active and appears in light micrographs as a lightly stained region of the nucleus. Viewed with the transmission electron microscope (TEM), euchromatin appears as electron-lucent regions among heterochromatins and is composed of 10-nm strings of nucleosomes (see Sections VI and VII in this chapter).

Extended chromatin

Extended chromatin is formed by adjacent nucleosomes. Each nucleosome core is around which the DNA double helix is wrapped two full turns. a. The nucleosome core consists of two copies each of histones H2A, H2B, H3, and H4. Nucleosomes are spaced at intervals of 200 base pairs. b. When viewed with TEM, extended chromatin resembles beads on a string; the beads represent nucleosomes, and the string between adjacent nucleosomes represents linker DNA. Nucleosomes support DNA and regulate its accessibility for replication and transcription as well as for its repair.

Facultative heterochromatin

Facultative heterochromatin is also condensed and is not involved in the transcription process. In contrast to constitutive heterochromatin, facultative heterochromatin is not repetitive and has inconsistent nuclear and chromosomal localization when compared with other cell types. Facultative heterochromatin may undergo active transcription in certain cells (see Barr body description on page 78) due to specific conditions such as explicit cell cycle stages, nuclear localization changes (i.e., migration from the center to the periphery), or the active transcription of only one allele of a gene (monoallelic gene expression)

G-banding

G-banding is observed in chromosomes during mitosis after staining with Giemsa, which is specific for DNA sequences rich in adenine (A) and thymine (T). Banding is thought to represent highly folded DNA loops. G-banding is characteristic for each species and is used to identify particular chromosomes and chromosomal anomalies.

G1 phase

G1 phase (gap one phase) lasts for hours to several days. (a) Occurring after mitosis, it is the period during which the cell grows and proteins are synthesized, restoring the daughter cells to normal volume and size. (b) Certain trigger proteins are synthesized; these proteins enable the cell to reach a threshold (restriction point) and proceed to the S phase. Cells that fail to reach the restriction point become resting cells and enter the G0 phase (gap outside phase), where they may remain for a few days, months, or years eventually to reenter the cell cycle or remain in the G0 phase permanently (see above).

G2 phase

G2 phase (gap two phase) lasts 2 to 4 hours. (a) This phase follows the S phase and extends to mitosis. (b) Centrioles duplicate, and each gives rise to a new, daughter centriole; the cell prepares to divide as the energy required for the completion of mitosis is stored; and RNA and proteins necessary for mitosis are synthesized, including tubulin for the spindle apparatus. Two checkpoints monitor DNA quality: the G2 DNA-damage checkpoint and the unreplicated-DNA checkpoint. The latter checkpoint prevents the progression of the cell into the M phase before DNA synthesis is complete.

Heterochromatin

Heterochromatin is chromatin that is condensed because it is not being transcribed and comprises approximately 90% of the total chromatin in the cell. It is formed from euchromatin that is folded into 30-nm-thick filaments. a. When examined under the light microscope (LM), it appears as basophilic clumps of nucleoprotein. b. Although transcriptionally inactive, recent evidence indicates that heterochromatin functions in maintaining the integrity of chromosomal centromeres and telomeres and, during meiosis, it also has a role in interchromosomal interactions and chromosomal segregation. c. Heterochromatin corresponds to one of two X chromosomes and is therefore present in nearly all somatic cells of female mammals. During interphase, the inactive X chromosome, referred to as the Barr body (or sex chromatin), is visible as a dark-staining body within the nucleus.

Laminopathies

Impairment in nuclear lamina architecture or function is associated with certain genetic diseases (laminopathies) and apoptosis. Mutations in lamin A/C cause tissue-specific diseases that affect striated muscle, adipose tissue, peripheral nerve or skeletal development, and premature aging. Recently, two hereditary forms of Emery-Dreifuss muscular dystrophy (EDMD) have been associated with mutations in either lamins or lamin receptors. The X-linked recessive form of EDMD is caused by mutations of emerin, whereas the autosomal dominant form of EDMD is caused by mutations in lamin A/C. In general, EDMD is characterized by early- onset contractures of major tendons, very slow progressive muscle weakness, muscle wasting in the upper and lower limbs, and cardiomyopathy (weakening of the heart muscle).

MicroRNAs (miRNAs)

MicroRNAs (miRNAs), first discovered in the roundworm in the 1990s, are very small segments of single-stranded RNA molecules of only 19 to 25 nucleotides in length that function to regulate gene expression. Although miRNAs are transcribed from DNA, they are noncoding and are not translated into proteins. Recent research demonstrated the presence of a diverse population of more than 1,000 human miRNAs that regulate developmental and physiological processes. Some miRNAs methylate specific regions of the DNA, thus preventing transcription from taking place, whereas other miRNAs insert into a matching portion of the mRNA strand, which prevents the translation of the mRNA; thus, the miRNA acts to regulate gene expression. It has been estimated that miRNAs may regulate a third or more of human genes. Because each miRNA can control hundreds of gene targets, they may influence most genetic pathways. In addition to functioning in gene expression, miRNAs also act as "central switchboards" of signaling networks that control stem cell homeostasis as well as various disease processes such as fibrosis, metastasis, and the biology of malignant cells.

Mitosis

Mitosis (Figure 2.7; Table 2.1) lasts 1 to 3 hours. It follows the G2 phase and completes the cell cycle. It includes segregation of the replicated chromosomes, division of the nucleus (karyokinesis), and finally division of the cytoplasm (cytokinesis), resulting in the production of two identical daughter cells. It consists of five major stages. The M phase possesses two checkpoints: the spindle-assembly checkpoint, which prevents premature entry into anaphase, and the chromosome-segregation checkpoint, which prevents the process of cytokinesis until all of the chromosomes have been correctly separated.

Nuclear matrix

Nuclear matrix acts as a scaffold that aids in organizing the nucleoplasm 1. Structural components include fibrillar elements, nuclear pore-nuclear lamina complex, residual nucleoli, and a residual RNP network. 2. Functional components are involved in the transcription and processing of mRNA and rRNA, steroid receptor-binding sites, carcinogen-binding sites, heat shock proteins, DNA viruses, viral proteins (T antigen), and perhaps many other functions that are as yet not known. 3. A nucleoplasmic reticulum is continuous with the endoplasmic reticulum of the cytoplasm and the nuclear envelope. It contains nuclear calcium functioning within the nucleus and possesses receptors for inositol 1,4,5-triphosphate, regulating calcium signals within compartments of the nucleus related to gene transcription, protein transport, and perhaps other functions.

Nucleoplasm

Nucleoplasm is the protoplasm within the nuclear envelope, in which the chromosomes and nucleoli are embedded. It is a viscous matrix composed mostly of water, whose viscosity is increased by the various types of macromolecules (some from the NPCs) and ions along with transcriptional process ing apparatus that are suspended or dissolved in it. It is believed by most authors that the nucleoplasm is ordered by the presence of a cytoskeletal-like framework known as the nuclear matrix. Other authors dispute the presence of this structure.

Nucleotides

Nucleotides are composed of a base (purine or pyrimidine), a deoxyribose sugar, and a phosphate group. 1. The purines are adenine (A) and guanine (G). 2. The pyrimidines are cytosine (C) and thymine (T).

Oncogenes

Oncogenes are the result of mutations of certain regulatory genes, called proto-oncogenes, which normally stimulate or inhibit cell proliferation and development. 1. Genetic a ccidents or viruses may lead to the formation of oncogenes. 2. Whatever be their origin, oncogenes dominate the normal a lleles (proto-oncogenes), causing deregulation of cell division, which leads to a cancerous state. 3. Bladder cancer and acute myelogenous leukemia are caused by oncogenes.

RNA

RNA is a linear molecule similar to DNA; however, it is single stranded and contains riboses instead of deoxyribose sugar and uracil (U) instead of thymine (T). RNA is synthesized by transcription of DNA. Transcription is catalyzed by three RNA polymerases: I for rRNA, II for mRNA, and III for tRNA. Some of the noncoding segments of DNA are transcribed to form transfer RNA (tRNA), ribosomal RNA (rRNA), as well as regulatory RNAs. Moreover, other RNAs can act as enzymes, such as ribozymes that catalyze the formation of peptide bonds during protein synthesis.

Reductional division (meiosis I)

Reductional division (meiosis I) occurs after interphase during the cell cycle, when the DNA content is duplicated, whereas the chromosome number (46) remains unchanged, giving the cell a 4CDNA content (considered to be the total DNA content of the cell). a. Prophase I is divided into five stages (leptotene, zygotene, pachytene, diplotene, and diakinesis), which accomplish the following events: (1 ) Chromatin condenses into the visible chromosomes, each containing two chromatids joined at the centromere. (2) Homologous maternal and paternal chromosomes pair via the synaptonemal complex, forming a tetrad. Crossing over (random exchanging of genes between segments of homologous chromosomes) occurs at the chiasmata, thus increasing genetic diversity. (3) The nucleolus and nuclear envelope disappear. b. Metaphase I (1 ) Homologous pairs of chromosomes align on the equatorial plate of the spindle in a random arrangement, facilitating genetic mixing. (2) Spindle fibers from either pole attach to the kinetochore of any one of the chromosome pairs, thus ensuring genetic mixing. c. Anaphase I (1 ) This phase is similar to anaphase in mitosis except in mitosis the two chromatids are pulled apart and each is delivered to each pole of the cell, whereas in anaphase I of meiosis the four chromatids are separated in such a fashion that two chromatids remain held together as they are being pulled to each pole of the cell. (2) Chromosomes migrate to the poles. d. Telophase I is similar to telophase in mitosis in that the nuclear envelope is reestablished and two daughter cells are formed via cytokinesis. (1 ) Each daughter cell now contains 23 chromosomes (n) number but has a 2CDNA content (the diploid amount). (2) Each chromosome is composed of two similar sister chromatids (but not genetically identical following recombination).

Ribosomal RNA

Ribosomal RNA associates with many different proteins (including enzymes) to form ribosomes. 1. rRNA associates with mRNA and tRNA during protein synthesis. 2. rRNA synthesis takes place in the nucleolus and is catalyzed by RNA polymerase I. A single 45S precursor rRNA (pre-rRNA) is formed and processed to form ribosomes as follows (Figure 2.5): a. Pre-rRNA associates with ribosomal proteins and is cleaved into the three sizes (28S, 18S, and 5.8S) of rRNAs present in ribosomes. b. The RNP containing 28S and 5.8S rRNA then combines with 5S rRNA, which is synthesized outside of the nucleolus, to form the large subunit of the ribosome. c. The RNP containing 18S rRNA forms the small subunit of the ribosome

S phase

S phase (synthetic phase) lasts 8 to 12 hours in most cells. (a) DNA is replicated, and histone and non-histone proteins are synthesized, resulting in duplication of the chromosomes. (b) Centrosomes are also duplicated. Presence of the S DNA-damage checkpoint in this phase monitors quality of replicating DNA.

Small interfering RNAs (siRNAs

Small interfering RNAs (siRNAs) are similar to miRNAs; they are 19 to 25 nucleotides in length, but they frequently arise from the genome of RNA viruses that infect a cell (although some siRNAs are transcribed from the cell's own genome). They resemble miRNAs in their mode of action in that they also methylate specific regions of the DNA and thus interfere with the process of transcription.

Zygotene

Synapsis, the close association of homologous chromosomes, begins at this stage and continues throughout pachytene. This process involves the formation of a synaptonemal complex, a tripartite structure that binds the chromosomes together. The synaptonemal complex is often compared to railroad tracks with an additional third rail positioned in the middle between two others. The cross ties in this track are represented by the transverse filaments that bind the scaff old material of both homologous chromosomes together.

The NPC represents protein subunits surrounding the nuclear pore (Figure 2.2).

The NPC is composed of nearly 100 proteins (jointly known as nucleoporins), some of which are arranged in eight fold symmetry around the margin of the pore. The nucleoplasmic side of the pore exhibits a nuclear basket, whereas the cytoplasmic side displays fibers extending into the cytoplasm. A transporter protein is located in the central core and is believed to be responsible for transporting proteins into and out of the nucleus via receptor-mediated transport a. The cytoplasmic ring, located around the cytoplasmic margin of the nuclear pore, is composed of eight subunits, each possessing a cytoplasmic filament composed of a Ran-binding protein (guanosine triphosphate [GTP]-binding protein) extending into the cytoplasm. These fibers may serve as a staging area prior to protein transport. b. The nucleoplasmic ring is located around the nucleoplasmic margin of the nuclear pore and is composed of eight subunits. Extending from this ring into the nucleoplasm is a basket-like structure, the nuclear basket. Attached to the distal end of the nuclear basket is the distal ring. This innermost ring assists in the export of RNA into the cytoplasm. c. The luminal ring is interposed between the cytoplasmic and nucleoplasmic rings. Eight transmembrane proteins project into the lumen of the nuclear pore, anchoring the complex into the pore rim. The lumen may be a gated channel that impedes passive diffusion. A moiety of each of these transmembrane proteins also projects into the perinuclear cistern. d. A structure described by some as the hourglass-shaped transporter or central plug in the center of the luminal ring is believed to be material such as ribosomes or protein complexes that are being transported through the NPC rather than a structural component of the NPC. Thus, the transporter is now referred to as the central plug.

The NPC function

The NPC permits passive movement across the nuclear envelope via a 9- to 1 1 -nm open channel for simple diffusion. Most proteins, regardless of size, pass in either direction only by receptor-mediated transport. These proteins have clusters of certain amino acids known as nuclear localization segments that act as signals for transport.

The cell cycle

The cell cycle varies in length in different types of cells, but is repeated each time a cell divides. It is composed of not only the series of events that prepare the cell to divide into two daughter cells but the process of cell division as well. 1. It is temporarily suspended in nondividing resting cells (e.g., peripheral lymphocytes), which are in the gap outside phase (G0 phase). Such cells may reenter the cycle and begin to divide again. 2. It is permanently interrupted in differentiated cells that do not divide (e.g., most cardiac muscle cells and neurons)

Numenculature of the DNA and Chromosomes

The chromosomal number, 46, is found in most of the somatic cells of the body and is called the diploid (2n) number. To simplify the description of chromosomal number and DNA changes during mitosis and meiosis, we use the lowercase letter (n) for chromosome number and lowercase letter (d) for DNA content. Diploid chromosomes have the (2d) amount of DNA immediately after cell division but have twice that amount—that is, the (4d) amount of DNA—after the S phase (see page 89). As a result of meiosis, eggs and sperm have only 23 chromosomes, the haploid (1n) number, as well as the haploid (1d) amount of DNA. The somatic chromosome number (2n) and the diploid (2d) amount of DNA are reestablished at fertilization by the fusion of the sperm nucleus with the egg nucleus.

Diakinesis

The homologous chromosomes condense and shorten to reach their maximum thickness, the nucleolus disappears, and the nuclear envelope disintegrates.

nuclear envelope

The nuclear envelope surrounds the nuclear material and consists of two parallel membranes separated from each other by a narrow perinuclear cisterna. These membranes fuse at intervals, forming openings called nuclear pores in the nuclear envelope.

The nuclear lamina

The nuclear lamina, a thin, electron-dense intermediate filament network-like layer, resides underneath the nuclear membrane. It addition to its supporting or "nucleoskeletal" function, nuclear lamina is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. If the membranous component of the nuclear envelope is disrupted by exposure to detergent, the nuclear lamina remains, and the nucleus retains its shape. The major components of the lamina, as determined by biochemical isolation, are nuclear lamins, a specialized type of nuclear intermediate filament (see page 63), and lamin-associated proteins. Nuclear lamina is essentially composed of lamin A and lamin C proteins that form intermediate fi laments. These filaments are cross-linked into an orthogonal lattice (see Fig. 3.6), which is attached mainly via lamin B protein to the inner nuclear membrane through its interactions with lamin receptors. The family of lamin receptors includes emerin (34 kDa) that binds both lamin A and B, nurim (29 kDa) that binds lamin A, and a 58 kDa lamin B receptor (LBR) that, as its name suggests, binds lamin B.

Structure of nucleolus

The nucleolus is a nuclear inclusion that is not surrounded by a membrane. It is observed in interphase cells that are actively synthesizing proteins; more than one nucleolus can be present in the nucleus. It contains mostly rRNA and proteins, such as nucleostemin, nucleolin, and fibrillarin, along with a modest amount of DNA. It possesses nucleolar organizer regions (NORs), portions of the chromosomes (in humans, chromosomes 13, 14, 15, 21, and 22) where rRNA genes are located; these regions are involved in reconstituting the nucleolus during the G, phase of the cell cycle.

Function of the nucleolus

The nucleolus is involved in the synthesis of rRNA and its preliminary assembly into ribosome subunit precursors as well as in the primary processing of micro RNAs. The nucleolus also sequesters certain nucleolar proteins, such as nucleostemin, that function as cell cycle checkpoint signaling proteins. These cell cycle regulator proteins remain sequestered in the nucleolus until their release is required for targets in the nucleus and/ or the cytoplasm. Following prophase of the cell cycle, the nucleolus disintegrates because the NORs of chromosomes 13, 14, 15, 21, and 22 are unavailable for transcription. Subsequent to telophase, the NORs unwind and facilitate the reconstitution ofthe nucleolus.

The nucleolus stains

The nucleolus stains intensely with hematoxylin and basic dyes and metachromatically with thionine dyes. The relation of basophilia and metachromasia of the nucleolus to the phosphate groups of the nucleolar RNA is confirmed by predigestion of specimens with ribonuclease (RNAse), which abolishes the staining. As mentioned above, DNA is present in the nucleolus; however, its concentration is below the detection capability of the Feulgen reaction. Thus, when examined in the light mi- croscope, nucleoli appear Feulgen-negative with Feulgen- positive nucleolus-associated chromatin that often rims the nucleolus

restriction checkpoint (or "point of no return")

The restriction checkpoint (or "point of no return") is the most important checkpoint in the cell cycle. At this checkpoint, the cell self-evaluates its own replicative potential before deciding to either enter the S phase and the next round of cell division or to retire and leave the cell cycle. A cell that leaves the cycle in the G1 phase usually begins terminal differentiation by entering the GO phase ("O" stands for " outside" the cycle). Thus, the G1 phase may last for only a few hours (average 9 to 12 hours) in a rapidly dividing cell, or it may last a lifetime in a nondividing cell. This checkpoint is mediated by interactions between the retinoblastoma susceptibility protein (pRb) and a family of essential transcription factors (E2F) with target promoters. In normal cells, proper interaction between pRb and E2F turns off many genes and blocks cell-cycle progression.

Nucleosome

The smallest units of chromatin structure are macromolecular complexes of DNA and histones called nucleosomes. These 10-nm-diameter particles represent the first level of chromatin folding and are formed by the coiling of the DNA molecule around a protein core. This step shortens the DNA molecule by approximately sevenfold relative to the unfolded DNA molecule. The core of the nucleosome consists of eight histone molecules (called an octamer). Two loops of DNA ( approximately 146 nucleotide pairs) are wrapped around the core octamer. The DNA extends between each particle as a 2-nm filament that joins adjacent nucleosomes. When chromatin is extracted from the nucleus, the nucleosomal substructure of chromatin is visible in transmission electron microscopy (TEM) and is often described as "beads on a string" (Fig. 3.3a). In the next step, a long strand of nucleosomes is coiled to produce a 30-nm chromatin fibril. Six nucleosomes form one turn in the coil of the chromatin fibril, which is approximately 40-fold shorter than unfolded DNA. Long stretches of 30-nm chromatin fibrils are further organized into loop domains (containing 15,000 to 100,000 base pairs), which are anchored into a chromosome scaffold or nuclear matrix composed of nonhistone proteins. In heterochromatin, the chromatin fibers are tightly packed and folded on each other; in euchromatin, the chromatin fibrils are more loosely arranged.

Several control factors have been identified.

These include a category of proteins known as cyclins as well as cyclin-dependent kinases (CDKs), which initiate and/or induce progression through the cell cycle. (1 ) During the G1 phase, cyclins D and E bind to their respective CDKs; these complexes enable the cell to enter and advance through the S phase. During both the G1 phase and the S phase, DNA replication is monitored and if errors are detected the cell cycle cannot continue until the errors are corrected. These are known as the G1 DNA damage checkpoint and the S DNA damage checkpoint, respectively. (2) Cyclin A binds to its CDKs, thus enabling the cell to leave the S phase and enter the G2 phase as well as to manufacture cyclin B. There are two further DNA checkpoints in the G2 phase; one is the unreplicated DNA checkpoint that cannot be passed unless all of the DNA was replicated in the S phase, and the G2 damage checkpoint that prevents the continuation of the G2 phase if errors are present in the replicated DNA. (3) Cyclin B binds to its CDK, inducing the cell to leave the G2 phase and enter the M phase. In the beginning of the M phase, the spindle apparatus is monitored, and if the spindle assembly is faulty the cell cannot leave the M phase. This is referred to as the spindle assembly checkpoint. Near the termination of the M phase, the condition of the chromosomes is monitored, and if any of the chromosomes are "sticking" to each other, the cell is not permitted to leave the M phase. This is referred to as the chromosome segregation checkpoint.

Leptotene

This stage is characterized by the condensation of chromatin and by the appearance of chromosomes. Sister chromatids also condense and become connected with each other by meiosis-specific cohesion complexes (Rec8p). At this phase, pairing of homologous chromosomes of maternal and paternal origin is initiated. Homologous pairing can be described as a process in which chromosomes actively search for each other. After finding their mates, they align themselves side by side with a slight space separating them.

The NPC transport mechanism

Transport mechanisms involve a group of transporter proteins, exportins and importins. The function of these transporter proteins is regulated by Ran, a group of GTP-binding proteins. Transporter proteins recognize polypeptide sequences on the proteins that are to be transported in one direction or the other. Exportins recognize polypeptide sequences known as nuclear export sequences and export molecules bearing them into the cytoplasm, whereas importins recognize nuclear localization sequences, and facilitate their import into the nucleus. Transport signals of this type are called nucleocytoplasmic shuttling signals.

Exons are

are regions of the DNA molecule that code for specific RNAs.

Introns are

are regions of the DNA molecule, between exons, that do not code for RNAs.

Anaphase

begins as sister chromatids separate at the centromere and daughter chromosomes move to the opposite poles ofthe cell. (1 ) The spindle elongates. (2) In the later stages of anaphase, a cleavage furrow begins to form around the cell as the contractile ring, a band of actin filaments, contracts

Prophase

begins when the duplicated chromosomes condense and become visible. Each chromosome is composed of two sister chromatids (future daughter chromosome) attached to each other at the centromere and the cohesins. A number of proteins assemble on each chromatid in the vicinity of the centromere, forming a kinetochore on the opposing aspects of each chromatid. Thus, there will be two kinetochores, one on each chromatid, facing opposite poles of the cell. During prophase, the nucleolus and nuclear envelope begin to dissipate.

Prometaphase

begins when the nuclear envelope disappears, allowing the chromosomes to disperse apparently randomly in the cytoplasm. Sister chromatids are held together by cohesisn, a group of binding proteins, and their compressed condition is maintained by the proteins condensin.

The DNA double helix..

consists of two complementary DNA strands held together by hydrogen bonds between the base pairs A-T and G-C.

Chromatids are

identical copies of each other if they are part of the same chromosome

Two major periods comprise the cell cycle:

interphase (interval between cell divisions) and M phase (mitosis, the period of cell division).

A gene

is a segment of the DNA molecule, located in a specific region of a chromosome. It is responsible not only for the formation of a single RNA molecule but also for the regulatory sequences that control the expression of a particular trait. In certain viruses, a gene may be composed of RNA rather than DNA.

A codon is

is a sequence of three bases in the DNA molecule that codes for a single amino acid.

Decrease in cell volume

is achieved by shrinking of the cytoplasm. The cytoskeletal elements become reorganized in bundles parallel to the cell surface. Ribosomes become clumped within the cytoplasm, the rER forms a series of concentric whorls, and most of the endocytotic vesicles fuse with the plasma membrane.

Loss of mitochondrial function

is caused by changes in the permeability of the mitochondrial membrane channels. The integrity of the mitochondrion is breached, the mitochondrial transmembrane potential drops, and the electron-transport chain is disrupted. Proteins from the mitochondrial intermembrane space, such as cytochrome c and SMAC/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point [pI]) are released into the cytoplasm to activate a cascade of proteolytic enzymes called caspases that are responsible for dismantling the cell. The regulated release of cytochrome c and SMAC/DIABLO suggests that mitochondria, under the influence of Bcl-2 proteins (see page 92), are the decision makers for initiating apoptosis. Thus, many researchers view mitochondria either as the "headquarters for the leader of a crack suicide squad" or as a "high-security prison for the leaders of a military coup."

Telophase

is characterized by each set of chromosomes reaching the pole, a deepening of the cleavage furrow; the midbody (containing overlapping polar microtubules) is now between the newly forming daughter cells. (1) Micro tubules in the midbody are depolymerized, facilitating cytokinesis and formation of two identical daughter cells. (2) The nuclear envelope is reestablished around the condensed chromosomes in the daughter cells, and nucleoli reappear. Nucleoli arise from the specific nucleolar organizing regions (called secondary constriction sites), which are carried on five separate chromosomes in humans (see Section III Nucleolus in this chapter). (3) The daughter nuclei gradually enlarge, and the condensed chromosomes disperse to form the typical interphase nucleus with heterochromatin and euchromatin. (4) It appears that at the end of cytokinesis the mother centriole of the duplicated pair moves from the newly forming nuclear pole to the intercellular bridge. This event is necessary to initiate disassembly of the midbody microtubules and complete the separation of the daughter cells. If this event fails, DNA replication is arrested at one of the G1 checkpoints during the next interphase.

A genome

is the complete set of hereditary information that an individual possesses. These are classified into two categories, genes and non coding segments of the DNA (or RNA in some viruses). In fact, only about 2% of the genome is composed of genes (which code for proteins/ polypeptides), whereas most ofthe remainder is no nco ding, in that they do not code for proteins/ polypeptides but possess regulatory or other functions.

Metaphase

is the phase during which the duplicated condensed chromosomes align at the equatorial plate of the mitotic spindle and become attached to kinetochore microtubules at their kinetochore. If this connection is not stable, anaphase-promoting complex interferes with cyclin E, and the cell cannot progress through metaphase. The centrosome contains centrioles and a pericentriolar cloud of material containing y-tubulin rings. It is the principal microtubule-organizing center (MTOC) of the cell. As centrosomes migrate to opposite poles of the cell, they set up two MTOCs, one at each pole of the dividing cell, and each MTOC gives rise to three sets of microtubules that will compose the spindle apparatus of the cell: Astral microtubules arise from the MTOCs in a spoke-like fashion and they ensure that the MTOCs maintain their correct location at the opposite poles of the cell near the cell membrane, and in this fashion astral microtubules facilitate the proper orientation of the spindle apparatus. Polar microtubules arise from each MTOC, grow toward each other, and meet near the equator of the cell. They function in ensuring that the two MTOCs are kept apart from each other and maintain their respective locations. Kinetochore microtubules emanate from each MTOC and grow toward the chromosomes. Once they reach each chromosome, they attach to the kinetochores of the sister chromatids and, during anaphase, begin the process of dragging sister chromatids to opposing poles of the cell.

mRNA

mRNA carries the genetic code to the cytoplasm to direct protein synthesis (Figure 2.4). 1. This single-stranded molecule consists of hundreds to thousands of nucleotides. 2. mRNA contains codons that are complementary to the DNA codons from which it was transcribed, including one start codon (AUG) for initiating protein synthesis and one of three stop codons (UAA, UAG, or UGA) for terminating protein synthesis. 3. mRNA is synthesized in the following series of steps. a. RNA polymerase II recognizes a promoter on a single strand of the DNA molecule and binds tightly to it. b. The DNA helix unwinds about two turns, separating the DNA strands and exposing the codons that act as the template for synthesis of the complementary RNA molecule. c. RNA polymerase II moves along the DNA strand and promotes base pairing between DNA and complementary RNA nucleotides. d. When RNA polymerase II recognizes a chain terminator (stop codons-UAA, UAG, or UGA) on the DNA molecule, it terminates its association with the DNA and is released to repeat transcription. e. The primary transcript, pre-mRNA after the introns are removed, associates with proteins to form hnRNP. f. Exons are spliced through several steps, involving spliceosomes producing an mRNP. g. Proteins are removed as the mRNP enters the cytoplasm, resulting in functional mRNA. h. RNA segments remaining from the transcription process as introns were once thought to be degraded and recycled because they were believed to have no function. However, recent evidence shows that these RNA segments may become modified to perform regulatory functions that parallel regulatory proteins related to development, gene expression, and evolution.

The stages of meiosis are_____________

meiosis I (reductional division) and meiosis II (equatorial division).

miRNAs have been shown to repress_____

miRNAs have been shown to repress certain cancer-related genes. Additionally, miRNAs are known to depress angiogenesis, which may become useful in clinically restricting cancer growth. Thus, it is expected that miRNAs may prove useful in the diagnosis and treatment of cancer. Certain other miRNAs appear have been shown to regulate differentiation (i.e., repressing adipocyte formation, an understanding that may lead to a clinical treatment modality for obesity). Still other miRNAs repress certain cancer-related genes, such as lung cancer where human lung cancer cells treated with miRNA had reduced the rate of their proliferation, slowed greatly their capability to migrate, and reduced their invasive a bilities. Additionally, the treated cells had a greater rate of apoptosis; thus, it is expected that miRNAs may prove useful in the diagnosis and treatment of cancer.

DNA fragmentation

occurs in the nucleus and is an irreversible event that commits the cell to die. DNA fragmentation is a result of Ca-dependent and Mg- dependent activation of nuclear endonucleases. These enzymes selectively cleave DNA, generating small oligonucleosomal fragments. Nuclear chromatin then aggregates, and the nucleus may divide into several discrete fragments bounded by the nuclear envelope.

necrosis

or accidental cell death, is a pathologic process. It occurs when cells are exposed to an unfavorable physical or chemical environment (e.g., hypothermia, hypoxia, radiation, low pH, cell trauma) that causes acute cellular injury and damage to the plasma membrane. Under physiologic conditions, damage to the plasma membrane may also be initiated by viruses, or proteins called perforins. Rapid cell swelling and lysis are two characteristic features of this process. Necrotic cell death is often associated with extensive surrounding tissue damage and an intense inflammatory response

Cells undergoing apoptosis show the following characteristic morphologic and biochemical features (see Fig. 3.18):

• DNA fragmentation • Decrease in cell volume • Loss of mitochondrial function • Membrane blebbing • Formation of apoptotic bodies

Transport through the NPC largely depends on the size of the molecules:

• Large molecules (such as large proteins and macromolecular complexes) depend on the presence of an attached signal sequence called the nuclear localization signal (NLS) for passage through the pores. Labeled NLS proteins destined for the nucleus then bind to a soluble cytosolic receptor called a nuclear import receptor (importin) that directs them from the cytoplasm to an appropriate NPC. They are then actively transported through the pore by a GTP energy-dependent mechanism. An export of proteins and RNA from the nucleus is similar to the import mechanism into the nucleus. Proteins that possess the nuclear export sequence (NES) bind in the nucleus to exportin (a protein that moves molecules from nucleus into cytoplasm) and to a GTP molecule. Protein-exportin-GTP complexes pass through NPC into the cytoplasm where GTP is hydrolyzed and the NES protein is released. The NPC transports proteins and all forms of RNA, as well as ribosomal subunits in their fully folded configurations. • Ions and smaller water-soluble molecules (less than 9 Da) may cross the water-filled channels of the NPC by simple diffusion. Th is process is nonspecific and does not require nuclear signal proteins. The effective size of the pore is about 9 nm for substances that cross by diffusion rather than the 70- to 80-nm measurement of the pore boundary. However, even the smaller nuclear proteins that are capable of diffusion are selectively transported, presumably because the rate is faster than simple diffusion.

Heterochromatin is disposed in three locations (Fig. 3.2):

• Marginal chromatin is found at the periphery of the nucleus (the structure light microscopists formerly referred to as the nuclear membrane actually consists largely of marginal chromatin). • Karyosomes are discrete bodies of chromatin irregular in size and shape that are found throughout the nucleus. • Nucleolar-associated chromatin is chromatin found in association with the nucleolus.

A simple microscopic evaluation of the nucleus provides a great deal of information about cell well-being. Evaluation of nuclear size, shape, and structure plays an important role in tumor diagnosis. For instance, dying cells have visible nuclear alterations. These include:

• pyknosis, or condensation of chromatin leading to shrinkage of the nuclei (they appear as dense basophilic masses); and • karyorrhexis, or fragmentations of nuclei (these changes usually are preceded by pyknosis). • karyolysis, or the disappearance of nuclei due to complete dissolution of DNA by increased activity of DNAase;