NUCLEUS Animal cells contain DNA in nucleus (contains ~ 98% of cell DNA) and mitochondrion. Both compartments are surrounded by an envelope (double membrane). Nuclear DNA represents some linear molecules and mitochondrial DNA represents circular molecules. All eukaryotic cells contain at least one nucleus. The contents of the nucleus are present as a viscous, amorphous mass of material enclosed by a complex nuclear envelope. Nucleus consists of: Chromosomes, which are present as extended nucleoprotein fibers, called chromatin; Nuclear matrix, which is a proteincontaining fibrillar network; Nucleolus (or some nucleoli) that are responsible for synthesis of rrna and assembling of ribosomes; Nucleoplasm (or karyoplasm) the fluid substance in which the solutes of nucleus are Fig. 1. Position of nucleus in the cell dissolved. CHROMATIN The eukaryotic cell nucleus is typically ~1mm in diameter. It contains a large amount of DNA (a total length of 1-2 meters), which must be efficiently packaged in such a way as to guarantee access to genetic information. Thus, each DNA molecule is packed forming chromatin, a densely staining material initially recognized in two different forms: highly condensed heterochromatin and more diffuse euchromatin. Chemically chromatin is organized from 30% DNA + 40% histones + 25% non-histones + 5% RNA. Chromatin fragments contain DNA (a negatively charged polymer) in complex with highly positively charged (basic) proteins called histones, and much smaller amounts of other DNAbinding proteins, collectively referred to as non-histone proteins. Human histone genes are represented as a family of moderately repeated sequences with variations in the structure, organization, and regulation of the different copies. The histones organize DNA into a regular repeating structure, the basic unit of which is the nucleosome, which wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. There are 5 classes of histones: H1, H2A, H2B, H3, H4. They contain Leucine, Lysine, Arginine and other basic amino acids. The quantity of histones is the same in all tissues and they have no tissuespecificity. They induce the tertiary structure of DNA (DNP). The non-histones are acid Fig. 2. Various stages in the condensation of chromatin 1
Nucleus. PL5. proteins, are very heterogeneous and comprise: enzymes (for replication, repair, transcription, RNA processing, biogenesis of ribosomes), site-specific proteins, scaffold proteins. More active tissues contain larger quantity and diversity of non-histones. Euchromatin represents the active part of chromatin; contain structural genes that are transcribed. It is less compacted and replicates early in the S period of interphase. Heterochromatin is tightly condensed and contains inactive sequences which are not transcribed. It replicates late in the S period. There are two types of heterochromatin: constitutive and facultative. Facultative heterochromatin contains genes that in some condition, in some tissues may be active, so it can be transformed into euchromatin. It contains coding but inactive sequences. Facultative heterochromatin assures cell differentiation, sexual differentiation, and control of ontogenesis. Constitutive heterochromatin contains repetitive sequences, which always remain condensed. It is represented by centromeres, telomeres, satellites, spacers between genes. Constitutive heterochromatin is the same in all cells. LEVELS OF DNA CONDENSATION I. Nucleosomal. Decondensed chromatin viewed in the electron microscope resembles beads on a string. Each bead is a nucleosome containing 146 bp of DNA wrapped around the core histone octamer, and sealed by a single molecule of linker histone (H1) bound at the point where the DNA enters and exits. H1 also binds to the linker DNA (the string connecting one bead to the next) (Fig. 2, 4). The histone octamer is composed of two molecules each of the four core histones: 2xH2A, 2xH2B, 2xH3 and 2xH4. These are small, basic proteins, which have been highly conserved during evolution. Histone H1, the linker histone, is not part of the nucleosome core (Fig. 5). It is very rich in lysine residues, less well conserved than the core histones, and is larger. The length of DNA in the core particle Fig. 4. Schematic diagram of nucleosome core is invariable (146 bp), but the average length of the linker DNA varies between species and tissues, giving rise to a characteristic repeat length (the average length of DNA in a nucleosome). II. Chromatin folding (Solenoid). Most of the chromatin in the nucleus is in the form of a highly condensed filament about 30-nm in diameter (the 30-nm filament). Formation of these highly condensed filaments is dependent on H1 (one H1 molecule per nucleosome). The condensed 30-nm filament is the result of solenoidal (helical) folding of the beads-on-a- Fig. 6. The solenoidal model of chromatin Fig. 3. Structure of nucleus Fig. 5. Binding of histone H1 to the linker DNA 2
string nucleosomal (10 nm) filament, having 6-12 nucleosomes per turn (Fig. 6). Nucleus. PL5. III. Chromatin Loops. The interphase chromosomes are organized into loops of chromatin filament attached to the nuclear skeleton (nuclear matrix) at their bases and projecting into the interior of the nucleus (Fig. 7). Each loop may contain a gene or related cluster of genes whose expression may in principle be regulated at the level of loop structure. The regions of DNA, which interact with matrix, are called MARs (matrix attachment regions) or SARs (scaffold attachment regions), while. The loops contain an average of 40 000-80 000 bp. IV. Metaphase Chromosomes. When the nuclear membrane breaks down during mitosis, chromatin is reorganized to form metaphase chromosomes in which a chromosomal metaphase scaffold is folded to form a quite regular helical coil, to which chromatin loops are attached at their bases (Fig. 8). Sister chromatids are usually of opposite helical handedness. The organization of DNA into the organelle, which is a chromosome, is most obvious at metaphase when the chromosome is condensed into a highly structured body. At this point in the cell cycle important chromosomal elements are visible: two arms (short p and long q), centromere and telomere. The telomeres are special sequences responsible for preventing the shortening of chromosomes during replication, protect linear molecules of DNA against actions of exonucleases and prevent joining of different chromosomes. Structurally, the telomeres are rich in tandemly repeated G/C sequences with a total length of 3 20 kb. The centromere is the genetic element (formed by constitutive heterochromatin) responsible for chromosome Fig. 8. Structure of a chromosome Fig. 7. Attachment of solenoid fiber to scaffold segregation during cell division. It is visible at metaphase as a constriction in mammalian chromosomes and is the site of attachment of spindle microtubules to the proteins that make up the kinetochore. Centromere contains A/T rich repetitive sequences. Within centromeres, H3 histone is substituted by CENP-A histone. Chromatine activation: Gene activity depends on: the stage of ontogenetic period, type of cell, environment. It also depends on the level of DNA condensation. Transcription is associated with nucleosomal level only. Transcriptionally active chromatin regions have core histones undergoing high rates of acetylation and deacetylation. Histone acetylation (which is a type of post-translational modification of histones) is generally linked to gene activation. The enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group (functional group COCH 3 ) are called histone acetyltransferases (HAT), which can also acetylate non-histone proteins, such as transcription factors and nuclear receptors to facilitate gene expression. Removal of the acetyl group by histone deacetylases (HDACs) condenses DNA structure, thereby preventing transcription. Another common way to block DNA and inhibit gene transcription is DNA methylation, which involves the addition of a methyl group (CH 3 ) to cytosine or adenine. 3
So, the post-translational modifications of histones include: Methylation of H3 (Lys4) active expression of a gene; Methylation of H3 (Lys9) diminishing of transcription; Histone acetylation assisting the transcription, gene activation; Histone deacetylation chromatin condensation, inactivation of transcription; Phosphorylation of H1 chromatin supercoiling Dephosphorylation of H1 chromatin decondensation. Nucleus. PL5. NUCLEAR ENVELOPE Nuclear envelope is a double membrane that surrounds the nucleus during most of the cell's lifecycle. The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum (ER), having numerous ribosomes attached to the surface. The outer membrane is also continuous with the inner nuclear membrane since the two layers are fused together at numerous tiny holes called nuclear pores that perforate the nuclear envelope. These pores regulate the selective passage of molecules between the nucleus and cytoplasm, The space between the outer and inner membranes is termed the perinuclear space and is connected with the lumen of the rough ER. Structural support is provided to the nuclear envelope by two different networks of intermediate filaments. Along the inner surface of the nucleus, one of these networks is organized into the nuclear lamina (made of fibrous proteins nuclear lamins and membrane associated proteins), which binds to chromatin, integral membrane proteins, and other nuclear components. The nuclear lamina is also thought to play a role in directing materials inside the nucleus toward the nuclear pores for export and in the disintegration of the nuclear envelope during cell division and its subsequent reformation at the end of the process. The other intermediate filament network is located on the outside of the outer nuclear membrane and is not organized in such a systemic way as the nuclear lamina. The amount of traffic that must pass through the nuclear envelope on a continuous basis in order for the eukaryotic cell to function properly is considerable. RNA and ribosomal subunits must be constantly transferred from the nucleus where they are made to the cytoplasm, and histones, gene regulatory proteins, DNA and RNA polymerases, and other substances required for nuclear activities must be imported from the cytoplasm. An active mammalian cell can synthesize about 20,000 ribosome subunits per minute, and at certain points in the cell cycle, as many as 30,000 histones per minute are required by the nucleus. In order for such a tremendous number of molecules to pass through the nuclear envelope in a timely manner, the nuclear pores must be highly efficient at selectively allowing the passage of materials to and from the nucleus. Nuclear Pores It is generally thought that a protein structure called the nuclear pore complex (NPCs) that surrounds each pore plays a key role in allowing the active transport of a selected set of large molecules into and out of the nucleus. The nuclear pore proteins are called nucleoporins, which are not only engaged in nucleocytoplasmic transport, but also in transcription regulation, kinetochore organization and other cellular events. Given the various functions of nucleoporins, it is not surprising to find them involved in a wide variety of human disease. 4
Nucleus. PL5. In addition to their role in nuclear transport, nuclear pores are important as sites where the outer membrane and inner membrane of the nuclear envelope are fused together. Due to this fusion, the membranes can be considered continuous with one another although they have different biochemical characteristics and can function in distinctive ways. Since the outer nuclear membrane is also continuous with the membrane of the endoplasmic reticulum (ER), both it and the inner nuclear membrane can exchange membranous materials with the ER. This capability enables the nuclear envelope to grow bigger or smaller when necessary to accommodate the dynamic contents of the nucleus. KARYOPLASM The karyoplasm or nucleoplasm or nuclear sap is a highly viscous liquid contained within the nucleus that surrounds the chromosomes and other subnuclear organelles. A network of fibers known as the nuclear matrix (scaffold, skeleton) can also be found in the nucleoplasm. Nuclear Scaffold The skeleton maintains the overall size and shape of the nucleus. The matrix acts as a structural attachment site for the DNA loops during the interphase: evolutionary highly conserved 300-1000 bp long DNA sequences, referred to as SARs (Scaffold Associated Regions), have been identified that define the base of DNA loops, anchoring them to specific proteins (SAPs - Scaffold Associated Proteins). By means of such chromosomal attachment sites, the matrix might help to organize chromosomes, localize genes, and regulate DNA transcription and replication within the nucleus. NUCLEOLUS Nucleolus is a part of nucleus responsible for biogenesis of ribosomes. It is the place of: transcription of ribosomal genes and synthesis of precursor rrna (45S), followed by processing of 45S rrna and formation of 3 types of rrna: 5.8S + 18S + 28S. As a result of assembling the RNP is formed: rrna 18S + 33 ribosomal proteins = 40S RNP (small ribosomal subunit) and rrna 28S + rrna 5.8S +rrna 5S + 49 ribosomal proteins =60S RNP (large ribosomal subunit). Sequences of DNA containing ribosomal genes (tandem repeats of rrna genes) form the nucleolar organizer region (NOR). The human genome contains more than 200 clustered copies of the rrna genes on five different chromosomes (13, 14, 15, 21, 22). Transcription of rrna genes is executed by RNA-polymerase I (rrna 45S) and III (for rrna 5S). Further processing is needed to generate the 18S RNA, 5.8S and 28S RNA molecules. This processing involves the function of a 5
Nucleus. PL5. class of small nucleolar RNAs (snornas) which are complexed with proteins and exist as smallnucleolar-ribonucleoproteins (snornps). Once the rrna subunits are processed, they are ready to be assembled into larger ribosomal subunits. Fig. 9. Scheme of ribosome biogenesis. 6