Heterochromatin is a region of chromosomes that is constantly in a compact state.
Euchromatin is loosely packed (decondensed) regions of chromosomes.
In the pericenromeric regions of chromosomes and the short arms of acrocentric chromosomes, heterochromatin is stained, designated as structural, which is constantly detected during mitotic cell division and in the interphase nucleus. Another type of heterochromatin is facultative, which arises by compaction of euchromatic regions and contains genes involved in protein metabolism. The condensation of the optional region is reversible, expressed in decondensation.
Chromosomes consist of DNA (approx. 40%) and proteins (approx. 60%), forming a nucleoprotein complex. Proteins are divided into two groups: histone and non-histone. Histones are represented by five molecules: H1, H2A, H2B, H3 and H4. Histone proteins make up 40 to 80% of all chromosome proteins. They are made up of small (+) charged molecules. The main amino acids arginine and lysine predominate in them. Due to their structure, histone proteins combine with (–) charged DNA to form a DNA-histone complex. This complex is called chromatin. Gis. proteins perform the function of specifically packaging a huge DNA molecule into a compact chromosome structure. Histones prevent the biological information contained in DNA from being read. This is their regulatory role. In addition, these proteins perform a structural function, ensuring the spatial organization of DNA in chromosomes
The number of non-histone protein fractions exceeds 100. Among them are enzymes for RNA synthesis and processing, DNA reduplication and repair. Acidic proteins of chromosomes also perform structural and regulatory roles. In addition to DNA and proteins, chromosomes also contain RNA, lipids, polysaccharides, and metal ions. Chromosome RNA is represented partly by transcription products that have not yet left the site of synthesis. Some fractions have a regulatory function. The regulatory role of chromosome components is to “prohibit” or “permit” the copying of information from the DNA molecule.
In different regions of chromosomes, DNA differs in composition and properties.
Centromeric DNA is located in the region of primary constrictions. Telomeres contain special DNA that prevents chromosome shortening during replication. In the zones of secondary constrictions there are DNA sections responsible for the synthesis of rRNA. The main part of the DNA responsible for the synthesis of numerous messenger RNAs is located in the chromosome arms.
While maintaining continuity in a number of cell generations, chromatin changes its structure depending on the period and phase of the cell cycle. organization. In interphase, under light microscopy, it is detected in the form of clumps scattered in the nucleoplasm of the nucleus. During the transition of a cell to mitosis, especially in metaphase, chromatin takes on the appearance of clearly visible individual intensely colored bodies - chromosomes.
Interphase and metaphase forms of existence of chromatin are regarded as two polar variants of its structural organization, connected in the mitotic cycle by mutual transitions. The most common point of view is that chromatin (chromosome) is a spiral thread. In this case, several levels of chromatin spiralization (compactization) are distinguished
Nucleosomal thread . This level of chromatin organization is provided by four types of nucleosomal histones: H2A, H2B, H3, H4. They form puck-shaped protein bodies - cores, consisting of eight molecules (two molecules of each type of histones)
Chromatin fibril. Further compaction of the nucleosomal strand is ensured by the HI piston, which, connecting to the linker DNA and two neighboring protein bodies, brings them closer to each other. The result is a more compact structure, possibly built like a solenoid. This chromatin fibril, also called elementary, has a diameter of 20-30 nm
Interphase chromonema . The next level of structural organization of genetic material is due to the folding of the chromatin fibril into loops. Non-histone proteins, which are capable of recognizing specific nucleotide sequences of extranucleosomal DNA, distant from each other at a distance of several thousand nucleotide pairs, apparently take part in their formation. These proteins bring these areas together to form loops from fragments of the chromatin fibril located between them. As a result of this packaging, a chromatin fibril with a diameter of 20-30 nm is transformed into a structure with a diameter of 100-200 nm, called an interphase chromonema .
Individual sections of the interphase chromonema undergo further compaction, forming structural blocks that unite neighboring loops with the same organization
Lampbrush chromosomes found in oocytes of fish, amphibians, reptiles and birds at the diplotene stage. Each of the two chromosomes bivalently consists of two chromatids, therefore, during their conjugation, extended four-chromatid structures are formed. Each chromatid consists of a tightly twisted axial strand with lateral loops extending from it, formed by a single DNA double helix. These loops probably represent DNA freed from proteins to allow transcription to occur. L-type chromosomes sch." are transcribed more actively than ordinary records. This is due to the need to accumulate significant amounts of gene products in oocytes.
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Structure and chemistry of chromatin
Chromatin is a complex mixture of substances from which eukaryotic chromosomes are built. The main components of chromatin are DNA and chromosomal proteins, which include histones and non-histone proteins that form highly ordered structures in space. The ratio of DNA and protein in chromatin is ~1:1, and the bulk of chromatin protein is represented by histones. The term “X” was introduced by W. Flemming in 1880 to describe intranuclear structures stained with special dyes.
Chromatin- the main component of the cell nucleus; it is quite easy to obtain from isolated interphase nuclei and from isolated mitotic chromosomes. To do this, they use its ability to go into a dissolved state during extraction with aqueous solutions with low ionic strength or simply deionized water.
Chromatin fractions obtained from different objects have a fairly uniform set of components. It was found that the total chemical composition of chromatin from interphase nuclei differs little from chromatin from mitotic chromosomes. The main components of chromatin are DNA and proteins, the bulk of which are histones and non-histone proteins.
Slide3 . There are two types of chromatin: heterochromatin and euchromatin. The first corresponds to chromosome regions condensed during interphase; it is functionally inactive. This chromatin stains well and is what can be seen in a histological specimen. Heterochromatin is divided into structural (these are sections of chromosomes that are constantly condensed) and facultative (can decondensate and turn into euchromatin). Euchromatin corresponds to chromosome regions that decondense during interphase. This is working, functionally active chromatin. It does not stain and is not visible on the histological specimen. During mitosis, all euchromatin is condensed and incorporated into chromosomes.
On average, about 40% of chromatin is DNA and about 60% is proteins, among which specific nuclear histone proteins make up from 40 to 80% of all proteins that make up the isolated chromatin. In addition, the chromatin fractions include membrane components, RNA, carbohydrates, lipids, and glycoproteins. The question of how much these minor components are included in the chromatin structure has not yet been resolved. Thus, RNA may be transcribed RNA that has not yet lost its connection with the DNA template. Other minor components may refer to substances from co-precipitated fragments of the nuclear membrane.
PROTEINS are a class of biological polymers present in every living organism. With the participation of proteins, the main processes that ensure the vital functions of the body take place: respiration, digestion, muscle contraction, transmission of nerve impulses.
Proteins are polymers, and amino acids are their monomer units.
Amino acids - these are organic compounds containing in their composition (in accordance with the name) an amino group NH2 and an organic acidic group, i.e. carboxyl, COOH group.
A protein molecule is formed as a result of the sequential connection of amino acids, while the carboxyl group of one acid interacts with the amino group of a neighboring molecule, resulting in the formation of a peptide bond - CO-NH- and the release of a water molecule. Slide 9
Protein molecules contain from 50 to 1500 amino acid residues. The individuality of a protein is determined by the set of amino acids that make up the polymer chain and, no less important, by the order of their alternation along the chain. For example, the insulin molecule consists of 51 amino acid residues.
Chemical composition of histones. Features of physical properties and interaction with DNA
Histones- relatively small proteins with a very large proportion of positively charged amino acids (lysine and arginine); The positive charge helps histones bind tightly to DNA (which is highly negatively charged) regardless of its nucleotide sequence. The complex of both classes of proteins with the nuclear DNA of eukaryotic cells is called chromatin. Histones are a unique characteristic of eukaryotes and are present in huge quantities per cell (about 60 million molecules of each type per cell). Histone types fall into two main groups - nucleosomal histones and H1 histones, forming a family of highly conserved core proteins consisting of five large classes - H1 and H2A, H2B, H3 and H4. Histone H1 is larger (about 220 amino acids) and has proven to be less conserved during evolution. The size of histone polypeptide chains ranges from 220 (H1) to 102 (H4) amino acid residues. Histone H1 is highly enriched in Lys residues, histones H2A and H2B are characterized by a moderate Lys content, and the polypeptide chains of histones H3 and H4 are rich in Arg. Within each class of histones (with the exception of H4), several subtypes of these proteins are distinguished based on amino acid sequences. This multiplicity is especially characteristic of mammalian H1 histones. In this case, there are seven subtypes called H1.1-H1.5, H1o and H1t. Histones H3 and H4 belong to the most conserved proteins. This evolutionary conservation suggests that almost all of their amino acids are important for the function of these histones. The N-terminal part of these histones can be reversibly modified in the cell due to acetylation of individual lysine residues, which removes the positive charge of lysines.
The core region of the histone tail.
Beads on the A string
Short range interaction
Linker histones
30 nm fiber
Chromonema fiber
Long Range Fiber Interactions
nucleosome chromatin histone
The role of histones in DNA folding is important for the following reasons:
1) If chromosomes consisted only of stretched DNA, it is difficult to imagine how they could replicate and separate into daughter cells without getting tangled or broken.
2) In an extended state, the DNA double helix of each human chromosome would cross the cell nucleus thousands of times; Thus, histones pack a very long DNA molecule in an orderly manner into a core several micrometers in diameter;
3) Not all DNA is folded in the same way, and the way a region of the genome is packaged into chromatin likely affects the activity of the genes contained in that region.
In chromatin, DNA extends as a continuous double-stranded strand from one nucleosome to the next. Each nucleosome is separated from the next by a section of linker DNA, which varies in size from 0 to 80 nucleotide pairs. On average, repeating nucleosomes have a nucleotide spacing of about 200 nucleotide pairs. In electron micrographs, this alternation of the histone octamer with coiled DNA and linker DNA gives the chromatin a “beads on a string” appearance (after treatments that unfold higher-order packaging).
Methylation As a covalent modification of histones, it is more complex than any other, since it can occur at both lysines and arginines. Additionally, unlike any other modification in group 1, the effects of methylation can be either positive or negative on transcriptional expression depending on the position of the residue in the histone (Table 10.1). Another level of complexity arises from the fact that there can be multiple methylation states at each residue. Lysines can be mono-(me1), di-(me2) or tri-(me3) methylated, whereas arginines can be mono-(me1) or di-(me2) methylated.
Phosphorylation is the best known PTM, since it has long been understood that kinases regulate signal transmission from the cell surface through the cytoplasm and into the nucleus, leading to changes in gene expression. Histones were among the first proteins to be discovered to be phosphorylated. By 1991, it was discovered that when cells were stimulated to proliferate, so-called “immediate-early” genes were induced and became transcriptionally active and functioned to stimulate the cell cycle. This increased gene expression correlates with phosphorylation of histone H3 (Mahadevan et al., 1991). Serine 10 residue of histone H3 (H3S10) has been shown to be an important phosphorylation site for transcription from yeast to humans and appears to be particularly important in Drosophila (Nowak and Corces, 2004).
Ubiquitination the process of attaching a “chain” of ubiquitin molecules to a protein (see Ubiquitin). In U., the C-terminus of ubiquitin joins the side lysine residues in the substrate. The polyubiquitin chain is attached at a strictly defined moment and is a signal indicating that the protein is subject to degradation.
Histone acetylation plays an important role in modulating chromatin structure upon transcriptional activation, increasing the accessibility of chromatin to the transcription machinery. It is believed that acetylated histones are less tightly bound to DNA and therefore it is easier for the transcription machine to overcome the resistance of chromatin packaging. In particular, acetylation may facilitate access and binding of transcription factors to their recognition elements on DNA. Enzymes that carry out the process of histone acetylation and deacetylation have now been identified, and we will probably soon learn more about how this relates to transcription activation.
It is known that acetylated histones are a sign of transcriptionally active chromatin.
Histones are the most biochemically studied proteins.
Nucleosome organization
The nucleosome is the elementary packaging unit of chromatin. It consists of a DNA double helix wrapped around a specific complex of eight nucleosomal histones (histone octamer). The nucleosome is a disc-shaped particle with a diameter of about 11 nm, containing two copies of each of the nucleosomal histones (H2A, H2B, H3, H4). The histone octamer forms a protein core around which double-stranded DNA is wrapped twice (146 DNA base pairs per histone octamer).
The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10-20 nm from each other.
Data on the structure of nucleosomes were obtained using low- and high-resolution X-ray diffraction analysis of nucleosome crystals, intermolecular protein-DNA cross-links, and DNA cleavage within nucleosomes using nucleases or hydroxyl radicals. A. Klug constructed a model of a nucleosome, according to which DNA (146 bp) in the B-form (a right-handed helix with a pitch of 10 bp) is wound around a histone octamer, in the central part of which histones H3 and H4 are located, and on the periphery - H2a and H2b. The diameter of such a nucleosome disk is 11 nm, and its thickness is 5.5 nm. The structure, consisting of a histone octamer and DNA wound around it, is called the nucleosomal core particle. Core particles are separated from each other by segments of linker DNA. The total length of the DNA segment included in the animal nucleosome is 200 (+/-15) bp.
Histone polypeptide chains contain several types of structural domains. The central globular domain and flexible protruding N- and C-terminal regions enriched in basic amino acids are called arms. The C-terminal domains of polypeptide chains involved in histone-histone interactions inside the core particle are predominantly in the form of an alpha helix with an extended central helical region, along which one shorter helix is laid on both sides. All known sites of reversible post-translational modifications of histones that occur throughout the cell cycle or during cell differentiation are localized in the flexible basic domains of their polypeptide chains (Table I.2). Moreover, the N-terminal arms of histones H3 and H4 are the most conserved regions of the molecules, and histones in general are one of the most evolutionarily conserved proteins. Genetic studies of the yeast S. cerevisiae have shown that small deletions and point mutations in the N-terminal portions of histone genes are accompanied by profound and diverse changes in the phenotype of yeast cells, indicating the importance of the integrity of histone molecules in ensuring the proper functioning of eukaryotic genes. In solution, histones H3 and H4 can exist in the form of stable tetramers (H3) 2 (H4) 2, and histones H2A and H2B - in the form of stable dimers. A gradual increase in ionic strength in solutions containing native chromatin leads to the release first of H2A/H2B dimers and then of H3/H4 tetramers.
The fine structure of nucleosomes in crystals was clarified in the work of K. Lueger et al. (1997) using high-resolution X-ray diffraction analysis. It has been established that the convex surface of each histone heterodimer in the octamer is surrounded by DNA segments 27-28 bp long, located at an angle of 140 degrees relative to each other, which are separated by linker regions 4 bp long.
Levels of DNA compaction: nucleosomes, fibrils, loops, mitotic chromosome
The first level of DNA compaction is nucleosomal. If chromatin is exposed to nucleases, it and DNA are broken down into regularly repeating structures. After nuclease treatment, a fraction of particles with a sedimentation rate of 11S is isolated from chromatin by centrifugation. 11S particles contain about 200 base pairs of DNA and eight histones. Such a complex nucleoprotein particle is called Nucleosome. In it, histones form a protein core, on the surface of which DNA is located. The DNA forms a section that is not connected to the core proteins - a Linker, which, connecting two neighboring nucleosomes, passes into the DNA of the next nucleosome. They form “beads,” globular formations about 10 nm, sitting one after another on elongated DNA molecules. The second level of compaction is 30 nm fibril. The first, nucleosomal, level of chromatin compaction plays a regulatory and structural role, ensuring DNA packaging density by 6-7 times. In mitotic chromosomes and in interphase nuclei, chromatin fibrils with a diameter of 25-30 nm are detected. A solenoid type of nucleosome packing is distinguished: a thread of densely packed nucleosomes with a diameter of 10 nm forms turns with a helical pitch of about 10 nm. There are 6-7 nucleosomes per turn of such a superhelix. As a result of such packing, a spiral-type fibril with a central cavity appears. Chromatin in the nuclei has 25-nm fibrils, which consists of close globules of the same size - Nucleomers. These nucleomers are called superbeads (“superbeads”). The main chromatin fibril with a diameter of 25 nm is a linear alternation of nucleomers along a compacted DNA molecule. As part of the nucleomer, two turns of the nucleosomal fibril are formed, with 4 nucleosomes in each. The nucleomeric level of chromatin packing ensures a 40-fold compaction of DNA. Nuclesomal and nucleomeric (superbid) levels of chromatin DNA compaction are carried out by histone proteins. Loop domains of DNA-Tthird level structural organization of chromatin. At higher levels of chromatin organization, specific proteins bind to specific sections of DNA, which forms large loops, or domains, at the binding sites. In some places there are clumps of condensed chromatin, rosette-like formations consisting of many loops of 30 nm fibrils connecting at a dense center. The average size of rosettes reaches 100-150 nm. Rosettes of chromatin fibrils - Chromomeres. Each chromomere consists of several nucleosome-containing loops that are connected at a single center. Chromomeres are connected to each other by sections of nucleosomal chromatin. This loop-domain chromatin structure ensures structural compaction of chromatin and organizes the functional units of chromosomes - replicons and transcribed genes.
Using the neutron scattering method, it was possible to determine the shape and exact dimensions of nucleosomes; to a rough approximation, it is a flat cylinder or washer with a diameter of 11 nm and a height of 6 nm. Located on a substrate for electron microscopy, they form “beads” - globular formations of about 10 nm, in single file, sitting tandemly on elongated DNA molecules. In fact, only the linker regions are elongated; the remaining three-quarters of the DNA length are helically arranged along the periphery of the histone octamer. The histone octamer itself is believed to have a rugby ball-like shape, consisting of a (H3·H4)2 tetramer and two independent H2A·H2B dimers. In Fig. Figure 60 shows a diagram of the location of histones in the core part of the nucleosome.
Composition of centromeres and telomeres
Today almost everyone knows what chromosomes are. These nuclear organelles, in which all genes are localized, constitute the karyotype of a given species. Under a microscope, chromosomes look like uniform, elongated dark rod-shaped structures, and the picture you see is unlikely to seem an intriguing sight. Moreover, preparations of chromosomes of a great many living creatures living on Earth differ only in the number of these rods and modifications of their shape. However, there are two properties that are common to chromosomes of all species.
Five stages of cell division (mitosis) are usually described. For simplicity, we will focus on three main stages in the behavior of the chromosomes of a dividing cell. At the first stage, gradual linear compression and thickening of chromosomes occurs, then a cell division spindle consisting of microtubules is formed. In the second, the chromosomes gradually move toward the center of the nucleus and line up along the equator, probably to facilitate the attachment of microtubules to the centromeres. In this case, the nuclear membrane disappears. At the last stage, the halves of the chromosomes - chromatids - separate. It seems that microtubules attached to the centromeres, like a tugboat, pull the chromatids towards the poles of the cell. From the moment of divergence, the former sister chromatids are called daughter chromosomes. They reach the spindle poles and come together in a parallel pattern. The nuclear envelope is formed.
A model explaining the evolution of centromeres.
Up- centromeres (gray ovals) contain a specialized set of proteins (kinetochores), including histones CENH3 (H) and CENP-C (C), which in turn interact with spindle microtubules (red lines). In different taxa, one of these proteins evolves adaptively and in concert with the divergence of the primary DNA structure of centromeres.
At the bottom- changes in the primary structure or organization of centromeric DNA (dark gray oval) can create stronger centromeres, resulting in more attached microtubules.
Telomeres
The term “telomere” was proposed by G. Möller back in 1932. In his view, it meant not only the physical end of the chromosome, but also the presence of a “terminal gene with a special function of sealing the chromosome,” which made it inaccessible to harmful influences (chromosomal rearrangements, deletions, the action of nucleases, etc.). The presence of the terminal gene was not confirmed in subsequent studies, but the function of the telomere was precisely determined.
Later another function was discovered. Since the normal replication mechanism does not work at the ends of chromosomes, the cell has another pathway that maintains stable chromosome sizes during cell division. This role is performed by a special enzyme, telomerase, which acts like another enzyme, reverse transcriptase: it uses a single-stranded RNA template to synthesize the second strand and repair the ends of chromosomes. Thus, telomeres in all organisms perform two important tasks: they protect the ends of chromosomes and maintain their length and integrity.
A model of a protein complex of six telomere-specific proteins that forms on the telomeres of human chromosomes has been proposed. The DNA forms a t-loop, and the single-stranded overhang inserts into the double-stranded DNA region located distally (Fig. 6). The protein complex allows cells to distinguish telomeres from chromosome (DNA) breakpoints. Not all telomere proteins are part of a complex that is abundant at telomeres but absent in other regions of the chromosomes. The protective properties of the complex stem from its ability to influence the structure of telomeric DNA in at least three ways: determining the structure of the telomere tip itself; participate in the formation of a t-loop; control the synthesis of telomeric DNA by telomerase. Related complexes have also been found on the telomeres of some other eukaryotic species.
Up -telomere at the time of chromosome replication, when its end is accessible to the telomerase complex, which carries out replication (doubling of the DNA strand at the very tip of the chromosome). After replication, telomeric DNA (black lines) together with the proteins located on it (shown as multi-colored ovals) forms t - Ploop (bottom of the picture ).
Time of DNA compaction in the cell cycle and the main factors stimulating processes
Let us recall the structure of chromosomes (from a biology course) - they are usually displayed as a pair of letters X, where each chromosome is a pair, and each has two identical parts - the left and right chromatids. This set of chromosomes is typical for a cell that has already begun its division, i.e. cells in which the DNA duplication process has taken place. The doubling of the amount of DNA is called the synthetic period, or S-period, of the cell cycle. They say that the number of chromosomes in a cell remains the same (2n), and the number of chromatids in each chromosome is doubled (4c - 4 chromatids per pair of chromosomes) - 2n4c. During division, one chromatid from each chromosome will enter the daughter cells and the cells will receive the full diploid set of 2n2c.
The state of the cell (more precisely, its nucleus) between two divisions is called interphase. There are three parts in interphase - presynthetic, synthetic and postsynthetic periods.
Thus, the entire cell cycle consists of 4 time periods: mitosis proper (M), presynthetic (G1), synthetic (S) and postsynthetic (G2) periods of interphase (Fig. 19). The letter G - from the English Gap - interval, interval. In the G1 period, which occurs immediately after division, cells have a diploid DNA content per nucleus (2c). During the G1 period, cell growth begins mainly due to the accumulation of cellular proteins, which is determined by an increase in the amount of RNA per cell. During this period, the cell begins to prepare for DNA synthesis (S-period).
It was found that suppression of protein or mRNA synthesis in the G1 period prevents the onset of the S period, since during the G1 period the synthesis of enzymes necessary for the formation of DNA precursors (for example, nucleotide phosphokinases), RNA and protein metabolism enzymes occurs. This coincides with an increase in RNA and protein synthesis. At the same time, the activity of enzymes involved in energy metabolism sharply increases.
In the next S-period, the amount of DNA per nucleus doubles and the number of chromosomes accordingly doubles. In different cells in the S period, different amounts of DNA can be found - from 2c to 4c. This is due to the fact that cells are studied at different stages of DNA synthesis (those that have just started synthesis and those that have already completed it). The S period is a key period in the cell cycle. Without DNA synthesis, not a single case of cells entering mitotic division is known.
The postsynthetic (G2) phase is also called premitotic. The last term emphasizes its great importance for passing through the next stage - the stage of mitotic division. In this phase, the synthesis of mRNA necessary for the passage of mitosis occurs. Somewhat earlier, the rRNA of the ribosomes, which determine cell division, is synthesized. Among the proteins synthesized at this time, tubulins, the proteins of the microtubules of the mitotic spindle, occupy a special place.
At the end of the G2 period or in mitosis, as mitotic chromosomes condense, RNA synthesis drops sharply and completely stops during mitosis. Protein synthesis during mitosis decreases to 25% of the initial level and then in subsequent periods reaches its maximum in the G2 period, generally repeating the nature of RNA synthesis.
In the growing tissues of plants and animals there are always cells that are, as it were, outside the cycle. Such cells are usually called G0-period cells. These cells are the so-called resting cells, which have temporarily or permanently stopped reproducing. In some tissues, such cells can remain for a long time without particularly changing their morphological properties: they retain, in principle, the ability to divide, turning into cambial stem cells (for example, in hematopoietic tissue). More often, the loss (even if temporary) of the ability to divide is accompanied by the appearance of the ability to specialize and differentiate. Such differentiating cells exit the cycle, but under special conditions they can enter the cycle again. For example, most liver cells are in the G0 period; they do not participate in DNA synthesis and do not divide. However, when part of the liver is removed from experimental animals, many cells begin preparation for mitosis (G1 period), proceed to DNA synthesis and can divide mitotically. In other cases, for example, in the epidermis of the skin, after leaving the cycle of reproduction and differentiation, the cells function for some time and then die (keratinized cells of the integumentary epithelium).
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Lecture No. 2.13.9.11. “Stages of the formation of cell theory. Cell as a structural unit of living things"
Stages of development of cell theory:
1) 1665 – R. Hooke gave the name of the cell – “cellula”
2) 1839 - Schleiden and Schwann proposed a new cage. theory
Cell – structural unit of plants and animals
The process of cell formation determines their growth and development
1858 – Virchow added to the cage. theory
"Every cell of a cell"
3) modern cage. theory
The cell is the basic structural and functional unit of all living things.
Cells of one multicellular organism are similar in structure, composition and important manifestations of life activity
Reproduction – division of the original mother cell
The cells of a multicellular organism, according to their functions, form tissues → organs → organ systems → organism
General plan of the structure of a eukaryotic cell.
Three main components of a cell:
1)cytoplasmic membrane (plasmalemma)
A lipid bilayer and one layer of proteins sit on the surface of the lipid layer or are immersed in it.
Functions:
Demarcation
Transport
Protective
Receptor (signal)
2)cytoplasm:
a) hyaloplasm (a colloidal solution of proteins, phospholipids and other substances. Can be a gel or sol)
Functions of hyaloplasm:
Transport
Homeostatic
Metabolism
Creating optimal conditions for the functioning of organelles
b) Organelles – permanent components of the cytoplasm that have a specific structure and execution def. functions.
Classification of organelles:
○ by localization:
Nuclear (nucleoli and chromosomes)
Cytoplasmic (ER, ribosomes)
○ by structure:
Membrane:
a) single-membrane (lysosomes, ER, Golgi apparatus, vacuoles, peroxisomes, spherosomes)
b) double-membrane (plastids, mitochondria)
Non-membrane (ribosomes, microtubules, myofibrils, microfilaments)
○ by purpose:
General (found in all cells)
Special (found in certain cells - plastids, cilia, flagella)
○ by size:
Visible under a light microscope (ER, Golgi apparatus)
Invisible under a light microscope (ribosomes)
Inclusions- non-permanent components of the cell that have a specific structure and execution def. functions.
3)core
Single membrane.
EPS (Endoplasmic reticulum, reticulum).
A system of interconnected cavities and tubules connected to the outer nuclear membrane.
Rough (granular). There are ribosomes→ protein synthesis
Smooth (agranular). Synthesis of fats and carbohydrates.
Functions:
1) delimiting
2) transport
3)removal of toxic substances from the cell
4) synthesis of steroids
Golgi apparatus (lamellar complex).
Stacks of flattened tubules and cisterns, called dictosomes.
Dictosoma– a stack of 3-12 flattened discs called cisternae (up to 20 dictos)
Functions:
1) concentration, release and compaction of intercellular secretion
2) accumulation of glyco- and lipoproteins
3) accumulation and removal of substances from the cell
4) formation of the cleavage furrow during mitosis
5) formation of primary lysosomes
Lizsoma.
A vesicle surrounded by a single membrane and containing hydrolytic enzymes.
Functions:
1) digestion of absorbed material
2) destruction of bacteria and viruses
3) autolysis (destruction of cell parts and dead organelles)
4)removal of whole cells and intercellular substance
Peroxisome.
Vesicles surrounded by a single membrane containing peroxidase.
Functions- oxidation of org. substances
Spherosome.
Oval organelles surrounded by a single membrane containing fat.
Functions– synthesis and accumulation of lipids.
Vacuoles.
Cavities in the cytoplasm of cells bounded by a single membrane.
In plants (cell sap - dissolution of organic and inorganic substances) and single cell. animals (digestive, contractile - osmoregulation and excretion)
Double membrane.
Core.
1)membrane (karyolemma):
Two membranes permeated with pores
Between the membranes there is a perenuclear space
The external membrane is connected to the ER
Functions - protective and transport
2)nuclear pores
3)nuclear juice:
According to physical state close to hyaloplasm
Chemically it contains more nucleic acids
4)nucleoli:
Non-membrane components of the nucleus
There may be one or more
Formed in specific areas of chromosomes (nucleolar organizers)
Functions:
rRNA synthesis
tRNA synthesis
Ribosome formation
5)chromatin– DNA strands + protein
6)chromosome– highly spiralized chromatin, containing genes
7)viscous karyoplasm
Ultrastructure of chromosomes.
Chromosome → 2 chromatids (connected in the centromere region) → 2 hemichromatids → chromonema → microfibrils (30-45% DNA + protein)
Satellite- a region of a chromosome separated by a secondary constriction.
Telomere– terminal region of chromosome
Types of chromosomes depending on the position of the centromere:
1) equal arm (metocentric)
2) unequal shoulders (submetacentric)
3) rod-shaped (acrocentric)
Karotype– a set of data on the number, shape and size of chromosomes.
Idiogram– graphical construction of a karyotype
Properties of chromosomes:
1)constancy of number
In one species, the number of chromosomes is always constant.
2)pairing– in somatic cells, each chromosome has its own pair (homologous chromosomes)
3)individuality– each chromosome has its own characteristics (size, shape...)
4)continuity– each chromosome from a chromosome
Functions of chromosomes:
1) storage of hereditary information
2)transmission of hereditary information
3)implementation of hereditary information
Mitochondria.
1)consists of 2 membranes:
External (smooth, inside has protrusions - cristae)
External (rough)
2) Inside the space is filled with a matrix in the room. There are:
Ribosomes
Proteins - enzymes
Functions:
1)ATP synthesis
2) synthesis of mitochondrial proteins
3) synthesis of nucleons. acids
4) synthesis of carbohydrates and lipids
5) formation of mitochondrial ribosomes
Plastids.
1) double-membrane organelles
2) inside the stroma, in ct. located tillakoids → grana
3) in the stroma:
Ribosomes
Carbohydrates
Based on color they are divided into:
1) chloroplasts (green, chlorophyll). Photosynthesis.
2) chromoplasts:
Yellow (xanthophyll)
Red (lycopectin)
Orange (carotene)
Coloring of fruits, leaves and roots.
3) leucoplasts (colorless, do not contain pigments). Stock of proteins, fats and carbohydrates.
Non-membrane.
Ribosome
1)consists of rRNA, protein and magnesium
2) two subunits: large and small
Function - protein synthesis
Chromatin called the complex mixture of substances from which eukaryotic chromosomes are built. The main components of chromatin are DNA, histones and non-histone proteins, which form highly ordered structures in space. The ratio of DNA and protein in chromatin is ~1:1, and the bulk of chromatin protein is represented by histones. Histones form a family of highly conserved core proteins that are divided into five large classes called H1, H2A, H2B, H3 and H4. The size of histone polypeptide chains ranges from ~ 220 (H1) and 102 (H4) amino acid residues. Histone H1 is highly enriched in residues Lys, histones H2A and H2B are characterized by a moderate Lys content, the polypeptide chains of histones H3 and H4 are rich Arg. Within each class of histones (with the exception of H4), several subtypes of these proteins are distinguished based on amino acid sequences. This multiplicity is especially characteristic of mammalian H1 histones. In this case, there are seven subtypes called H1.1–H1.5, H1 o and H1t.
Rice. I.2. Schematic representation of the loop-domain level of chromatin compaction
A– fixation of the chromomere loop on the nuclear matrix using MAR/SAR sequences and proteins; b– “rosettes” formed from a chromometer loop; V– condensation of rosette loops with the participation of nucleosomes and nucleomers
An important result of the interaction of DNA with proteins in chromatin is its compaction. The total length of DNA contained in the nucleus of human cells approaches 1 m, while the average diameter of the nucleus is 10 µm. The length of a DNA molecule contained in one human chromosome is on average ~4 cm. At the same time, the length of a metaphase chromosome is ~4 µm. Consequently, the DNA of human metaphase chromosomes is compacted in length by at least 10 4 times. The degree of DNA compaction in interphase nuclei is much lower and uneven in individual genetic loci. From a functional point of view, there are euchromatin And heterochromatin . Euchromatin is characterized by less compaction of DNA compared to heterochromatin, and actively expressed genes are mainly localized in it. Currently, there is a widespread belief that heterochromatin is genetically inert. Since its true functions cannot be considered established today, this point of view may change as knowledge about heterochromatin accumulates. Already, actively expressed genes are found in it.
Heterochromatization of certain chromosome regions is often accompanied by suppression of the transcription of genes present in them. Extended sections of chromosomes and even entire chromosomes can be involved in the process of heterochromatization. Accordingly, it is believed that the regulation of eukaryotic gene transcription mainly occurs at two levels. In the first of these, compaction or decompactization of DNA in chromatin can lead to long-term inactivation or activation of extended sections of chromosomes or even entire chromosomes during the ontogenesis of the organism. More fine regulation of transcription of activated chromosome regions is achieved at the second level with the participation of non-histone proteins, including numerous transcription factors.
Structural organization of chromatin and chromosomes in eukaryotes. The question of the structural organization of chromatin in interphase nuclei is currently far from being resolved. This is due, first of all, to the complexity and dynamism of its structure, which easily changes even with minor exogenous influences. Most of the knowledge about the structure of chromatin was obtained in vitro on preparations of fragmented chromatin, the structure of which differs significantly from that in native nuclei. In accordance with the common point of view, there are three levels of structural organization of chromatin in eukaryotes: 1 ) nucleosome fibril ; 2) solenoid , ornucleomer ; 3) loop domain structure , includingchromomeres .
Nucleosome fibrils. Under certain conditions (at low ionic strength and in the presence of divalent metal ions), it is possible to observe regular structures in isolated chromatin in the form of extended fibrils with a diameter of 10 nm, consisting of nucleosomes. These fibrillar structures, in which nucleosomes are arranged like beads on a string, are considered to be the lowest level of eukaryotic DNA packaging in chromatin. The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10–20 nm from each other. Nucleosomes contain four pairs of histone molecules: H2a, H2b, H3 and H4, as well as one histone molecule H1. Data on the structure of nucleosomes are mainly obtained using three methods: low- and high-resolution X-ray diffraction analysis of nucleosome crystals, intermolecular protein-DNA cross-links, and cleavage of DNA within nucleosomes using nucleases or hydroxyl radicals. Based on such data, A. Klug constructed a model of the nucleosome, according to which DNA (146 bp) in B-shape(a right-handed helix with a pitch of 10 bp) is wound around a histone octamer, in the central part of which histones H3 and H4 are located, and in the periphery - H2a and H2b. The diameter of such a nucleosome disk is 11 nm, and its thickness is 5.5 nm. The structure consisting of a histone octamer and DNA wound around it is called nucleosomal kó moat particles. TO ó moat particles are separated from each other by segments linker DNA. The total length of the DNA segment included in the animal nucleosome is 200 (15) bp.
Histone polypeptide chains contain several types of structural domains. The central globular domain and flexible protruding N- and C-terminal regions enriched in basic amino acids are called shoulders(arm). C-terminal domains of polypeptide chains involved in histone–histone interactions within the ó ry particles are predominantly in the form of an -helix with an extended central spiral section, along which one shorter spiral is laid on both sides. All known sites of reversible post-translational modifications of histones that occur throughout the cell cycle or during cell differentiation are localized in the flexible basic domains of their polypeptide chains (Table I.2). Moreover, the N-terminal arms of histones H3 and H4 are the most conserved regions of the molecules, and histones in general are one of the most evolutionarily conserved proteins. Using genetic studies of the yeast S. cerevisiae It was found that small deletions and point mutations in the N-terminal parts of histone genes are accompanied by profound and diverse changes in the phenotype of yeast cells. This indicates the extreme importance of the integrity of histone molecules in ensuring the proper functioning of eukaryotic genes.
In solution, histones H3 and H4 can exist in the form of stable tetramers (H3) 2 (H4) 2, and histones H2A and H2B - in the form of stable dimers. A gradual increase in ionic strength in solutions containing native chromatin leads to the release first of H2A/H2B dimers and then of H3/H4 tetramers.
Further refinement of the fine structure of nucleosomes in crystals was recently carried out in the work of K. Lueger et al. (1997) using high-resolution X-ray diffraction analysis. It was found that the convex surface of each histone heterodimer in the octamer is surrounded by DNA segments 27–28 bp long, located at an angle of 140° relative to each other, which are separated by linker regions 4 bp long.
In accordance with modern data, the spatial structure of DNA as part of ó rovy particles are somewhat different from the B-form: the DNA double helix is twisted by 0.25–0.35 bp/turn of the double helix, which leads to the formation of a helix pitch equal to 10.2 bp/turn (in B -forms in solution – 10.5 bp/turn). Stability of the histone complex in the composition of ó The formation of a particle is determined by the interaction of their globular parts; therefore, the removal of flexible arms under conditions of mild proteolysis is not accompanied by destruction of the complex. The N-terminal arms of histones apparently ensure their interaction with specific DNA regions. Thus, the N-terminal domains of histone H3 contact DNA regions at the entrance to the ó first particle and exits it, while the corresponding domain of histone H4 binds to the internal part of the DNA of the nucleosome.
The high-resolution nucleosome structure studies mentioned above show that the central part of the 121-bp DNA segment. within the nucleosome forms additional contacts with histone H3. In this case, the N-terminal parts of the polypeptide chains of histones H3 and H2B pass through the channels formed by the minor grooves of the adjacent DNA supercoils of the nucleosome, and the N-terminal part of histone H2A contacts the minor groove of the outer part of the DNA supercoil. Taken together, high-resolution data show that DNA within the core particles of nucleosomes bends around histone octamers unevenly. Curvature is disrupted at sites where DNA interacts with the histone surface, and such breaks are most noticeable at distances of 10–15 and 40 bp. from the center of the DNA supercoil.
Chromatin is a complex mixture of substances from which eukaryotic chromosomes are built. The main components of chromatin are DNA and chromosomal proteins, which include histones and non-histone proteins that form highly ordered structures in space. The ratio of DNA and protein in chromatin is ~1:1, and the bulk of chromatin protein is represented by histones. The term “X” was introduced by W. Flemming in 1880 to describe intranuclear structures stained with special dyes.
Chromatin- the main component of the cell nucleus; it is quite easy to obtain from isolated interphase nuclei and from isolated mitotic chromosomes. To do this, they use its ability to go into a dissolved state during extraction with aqueous solutions with low ionic strength or simply deionized water.
Chromatin fractions obtained from different objects have a fairly uniform set of components. It was found that the total chemical composition of chromatin from interphase nuclei differs little from chromatin from mitotic chromosomes. The main components of chromatin are DNA and proteins, the bulk of which are histones and non-histone proteins.
Slide 3. There are two types of chromatin: heterochromatin and euchromatin. The first corresponds to chromosome regions condensed during interphase; it is functionally inactive. This chromatin stains well and is what can be seen in a histological specimen. Heterochromatin is divided into structural (these are sections of chromosomes that are constantly condensed) and facultative (can decondensate and turn into euchromatin). Euchromatin corresponds to chromosome regions that decondense during interphase. This is working, functionally active chromatin. It does not stain and is not visible on the histological specimen. During mitosis, all euchromatin is condensed and incorporated into chromosomes.
On average, about 40% of chromatin is DNA and about 60% is proteins, among which specific nuclear histone proteins make up from 40 to 80% of all proteins that make up the isolated chromatin. In addition, the chromatin fractions include membrane components, RNA, carbohydrates, lipids, and glycoproteins. The question of how much these minor components are included in the chromatin structure has not yet been resolved. Thus, RNA may be transcribed RNA that has not yet lost its connection with the DNA template. Other minor components may refer to substances from co-precipitated fragments of the nuclear membrane.
PROTEINS are a class of biological polymers present in every living organism. With the participation of proteins, the main processes that ensure the vital functions of the body take place: respiration, digestion, muscle contraction, transmission of nerve impulses.
Proteins are polymers, and amino acids are their monomer units.
Amino acids - these are organic compounds containing in their composition (in accordance with the name) an amino group NH2 and an organic acidic group, i.e. carboxyl, COOH group.
A protein molecule is formed as a result of the sequential connection of amino acids, while the carboxyl group of one acid interacts with the amino group of a neighboring molecule, resulting in the formation of a peptide bond - CO-NH- and the release of a water molecule. Slide 9
Protein molecules contain from 50 to 1500 amino acid residues. The individuality of a protein is determined by the set of amino acids that make up the polymer chain and, no less important, by the order of their alternation along the chain. For example, the insulin molecule consists of 51 amino acid residues.
Chemical composition of histones. Features of physical properties and interaction with DNA
Histones- relatively small proteins with a very large proportion of positively charged amino acids (lysine and arginine); The positive charge helps histones bind tightly to DNA (which is highly negatively charged) regardless of its nucleotide sequence. The complex of both classes of proteins with the nuclear DNA of eukaryotic cells is called chromatin. Histones are a unique characteristic of eukaryotes and are present in huge quantities per cell (about 60 million molecules of each type per cell). Histone types fall into two main groups - nucleosomal histones and H1 histones, forming a family of highly conserved core proteins consisting of five large classes - H1 and H2A, H2B, H3 and H4. Histone H1 is larger (about 220 amino acids) and has proven to be less conserved during evolution. The size of histone polypeptide chains ranges from 220 (H1) to 102 (H4) amino acid residues. Histone H1 is highly enriched in Lys residues, histones H2A and H2B are characterized by a moderate Lys content, and the polypeptide chains of histones H3 and H4 are rich in Arg. Within each class of histones (with the exception of H4), several subtypes of these proteins are distinguished based on amino acid sequences. This multiplicity is especially characteristic of mammalian H1 histones. In this case, there are seven subtypes called H1.1-H1.5, H1o and H1t. Histones H3 and H4 belong to the most conserved proteins. This evolutionary conservation suggests that almost all of their amino acids are important for the function of these histones. The N-terminal part of these histones can be reversibly modified in the cell due to acetylation of individual lysine residues, which removes the positive charge of lysines.
The core region of the histone tail.
Beads on the A string
Short range interaction
Linker histones
30 nm fiber
Chromonema fiber
Long Range Fiber Interactions
nucleosome chromatin histone
The role of histones in DNA folding is important for the following reasons:
- 1) If chromosomes consisted only of stretched DNA, it is difficult to imagine how they could replicate and separate into daughter cells without getting tangled or broken.
- 2) In an extended state, the DNA double helix of each human chromosome would cross the cell nucleus thousands of times; Thus, histones pack a very long DNA molecule in an orderly manner into a core several micrometers in diameter;
- 3) Not all DNA is folded in the same way, and the way a region of the genome is packaged into chromatin likely affects the activity of the genes contained in that region.
In chromatin, DNA extends as a continuous double-stranded strand from one nucleosome to the next. Each nucleosome is separated from the next by a section of linker DNA, which varies in size from 0 to 80 nucleotide pairs. On average, repeating nucleosomes have a nucleotide spacing of about 200 nucleotide pairs. In electron micrographs, this alternation of the histone octamer with coiled DNA and linker DNA gives the chromatin a “beads on a string” appearance (after treatments that unfold higher-order packaging).
Methylation As a covalent modification of histones, it is more complex than any other, since it can occur at both lysines and arginines. Additionally, unlike any other modification in group 1, the effects of methylation can be either positive or negative on transcriptional expression depending on the position of the residue in the histone (Table 10.1). Another level of complexity arises from the fact that there can be multiple methylation states at each residue. Lysines can be mono-(me1), di-(me2) or tri-(me3) methylated, whereas arginines can be mono-(me1) or di-(me2) methylated.
Phosphorylation is the best known PTM, since it has long been understood that kinases regulate signal transmission from the cell surface through the cytoplasm and into the nucleus, leading to changes in gene expression. Histones were among the first proteins to be discovered to be phosphorylated. By 1991, it was discovered that when cells were stimulated to proliferate, so-called “immediate-early” genes were induced and became transcriptionally active and functioned to stimulate the cell cycle. This increased gene expression correlates with phosphorylation of histone H3 (Mahadevan et al., 1991). Serine 10 residue of histone H3 (H3S10) has been shown to be an important phosphorylation site for transcription from yeast to humans and appears to be particularly important in Drosophila (Nowak and Corces, 2004).
Ubiquitination the process of attaching a “chain” of ubiquitin molecules to a protein (see Ubiquitin). In U., the C-terminus of ubiquitin joins the side lysine residues in the substrate. The polyubiquitin chain is attached at a strictly defined moment and is a signal indicating that the protein is subject to degradation.
Histone acetylation plays an important role in modulating chromatin structure upon transcriptional activation, increasing the accessibility of chromatin to the transcription machinery. It is believed that acetylated histones are less tightly bound to DNA and therefore it is easier for the transcription machine to overcome the resistance of chromatin packaging. In particular, acetylation may facilitate access and binding of transcription factors to their recognition elements on DNA. Enzymes that carry out the process of histone acetylation and deacetylation have now been identified, and we will probably soon learn more about how this relates to transcription activation.
It is known that acetylated histones are a sign of transcriptionally active chromatin.
Histones are the most biochemically studied proteins.
Nucleosome organization
The nucleosome is the elementary packaging unit of chromatin. It consists of a DNA double helix wrapped around a specific complex of eight nucleosomal histones (histone octamer). The nucleosome is a disc-shaped particle with a diameter of about 11 nm, containing two copies of each of the nucleosomal histones (H2A, H2B, H3, H4). The histone octamer forms a protein core around which double-stranded DNA is wrapped twice (146 DNA base pairs per histone octamer).
The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10-20 nm from each other.
Data on the structure of nucleosomes were obtained using low- and high-resolution X-ray diffraction analysis of nucleosome crystals, intermolecular protein-DNA cross-links, and DNA cleavage within nucleosomes using nucleases or hydroxyl radicals. A. Klug constructed a model of a nucleosome, according to which DNA (146 bp) in the B-form (a right-handed helix with a pitch of 10 bp) is wound around a histone octamer, in the central part of which histones H3 and H4 are located, and on the periphery - H2a and H2b. The diameter of such a nucleosome disk is 11 nm, and its thickness is 5.5 nm. The structure, consisting of a histone octamer and DNA wound around it, is called the nucleosomal core particle. Core particles are separated from each other by segments of linker DNA. The total length of the DNA segment included in the animal nucleosome is 200 (+/-15) bp.
Histone polypeptide chains contain several types of structural domains. The central globular domain and flexible protruding N- and C-terminal regions enriched in basic amino acids are called arms. The C-terminal domains of polypeptide chains involved in histone-histone interactions inside the core particle are predominantly in the form of an alpha helix with an extended central helical region, along which one shorter helix is laid on both sides. All known sites of reversible post-translational modifications of histones that occur throughout the cell cycle or during cell differentiation are localized in the flexible basic domains of their polypeptide chains (Table I.2). Moreover, the N-terminal arms of histones H3 and H4 are the most conserved regions of the molecules, and histones in general are one of the most evolutionarily conserved proteins. Genetic studies of the yeast S. cerevisiae have shown that small deletions and point mutations in the N-terminal portions of histone genes are accompanied by profound and diverse changes in the phenotype of yeast cells, indicating the importance of the integrity of histone molecules in ensuring the proper functioning of eukaryotic genes. In solution, histones H3 and H4 can exist in the form of stable tetramers (H3) 2 (H4) 2, and histones H2A and H2B - in the form of stable dimers. A gradual increase in ionic strength in solutions containing native chromatin leads to the release first of H2A/H2B dimers and then of H3/H4 tetramers.
The fine structure of nucleosomes in crystals was clarified in the work of K. Lueger et al. (1997) using high-resolution X-ray diffraction analysis. It has been established that the convex surface of each histone heterodimer in the octamer is surrounded by DNA segments 27-28 bp long, located at an angle of 140 degrees relative to each other, which are separated by linker regions 4 bp long.
Levels of DNA compaction: nucleosomes, fibrils, loops, mitotic chromosome
The first level of DNA compaction is nucleosomal. If chromatin is exposed to nucleases, it and DNA are broken down into regularly repeating structures. After nuclease treatment, a fraction of particles with a sedimentation rate of 11S is isolated from chromatin by centrifugation. 11S particles contain about 200 base pairs of DNA and eight histones. Such a complex nucleoprotein particle is called Nucleosome. In it, histones form a protein core, on the surface of which DNA is located. The DNA forms a section that is not connected to the core proteins - a Linker, which, connecting two neighboring nucleosomes, passes into the DNA of the next nucleosome. They form “beads,” globular formations about 10 nm, sitting one after another on elongated DNA molecules. The second level of compaction is 30 nm fibril. The first, nucleosomal, level of chromatin compaction plays a regulatory and structural role, ensuring DNA packaging density by 6-7 times. In mitotic chromosomes and in interphase nuclei, chromatin fibrils with a diameter of 25-30 nm are detected. A solenoid type of nucleosome packing is distinguished: a thread of densely packed nucleosomes with a diameter of 10 nm forms turns with a helical pitch of about 10 nm. There are 6-7 nucleosomes per turn of such a superhelix. As a result of such packing, a spiral-type fibril with a central cavity appears. Chromatin in the nuclei has 25-nm fibrils, which consists of close globules of the same size - Nucleomers. These nucleomers are called superbeads (“superbeads”). The main chromatin fibril with a diameter of 25 nm is a linear alternation of nucleomers along a compacted DNA molecule. As part of the nucleomer, two turns of the nucleosomal fibril are formed, with 4 nucleosomes in each. The nucleomeric level of chromatin packing ensures a 40-fold compaction of DNA. Nuclesomal and nucleomeric (superbid) levels of chromatin DNA compaction are carried out by histone proteins. Loop domains of DNA-third level structural organization of chromatin. At higher levels of chromatin organization, specific proteins bind to specific sections of DNA, which forms large loops, or domains, at the binding sites. In some places there are clumps of condensed chromatin, rosette-like formations consisting of many loops of 30 nm fibrils connecting at a dense center. The average size of rosettes reaches 100-150 nm. Rosettes of chromatin fibrils - Chromomeres. Each chromomere consists of several nucleosome-containing loops that are connected at a single center. Chromomeres are connected to each other by sections of nucleosomal chromatin. This loop-domain chromatin structure ensures structural compaction of chromatin and organizes the functional units of chromosomes - replicons and transcribed genes.
Using the neutron scattering method, it was possible to determine the shape and exact dimensions of nucleosomes; to a rough approximation, it is a flat cylinder or washer with a diameter of 11 nm and a height of 6 nm. Located on a substrate for electron microscopy, they form “beads” - globular formations of about 10 nm, in single file, sitting tandemly on elongated DNA molecules. In fact, only the linker regions are elongated; the remaining three-quarters of the DNA length are helically arranged along the periphery of the histone octamer. The histone octamer itself is believed to have a rugby ball-like shape, consisting of a (H3·H4)2 tetramer and two independent H2A·H2B dimers. In Fig. Figure 60 shows a diagram of the location of histones in the core part of the nucleosome.
Composition of centromeres and telomeres
Today almost everyone knows what chromosomes are. These nuclear organelles, in which all genes are localized, constitute the karyotype of a given species. Under a microscope, chromosomes look like uniform, elongated dark rod-shaped structures, and the picture you see is unlikely to seem an intriguing sight. Moreover, preparations of chromosomes of a great many living creatures living on Earth differ only in the number of these rods and modifications of their shape. However, there are two properties that are common to chromosomes of all species.
Five stages of cell division (mitosis) are usually described. For simplicity, we will focus on three main stages in the behavior of the chromosomes of a dividing cell. At the first stage, gradual linear compression and thickening of chromosomes occurs, then a cell division spindle consisting of microtubules is formed. In the second, the chromosomes gradually move toward the center of the nucleus and line up along the equator, probably to facilitate the attachment of microtubules to the centromeres. In this case, the nuclear membrane disappears. At the last stage, the halves of the chromosomes - chromatids - separate. It seems that microtubules attached to the centromeres, like a tugboat, pull the chromatids towards the poles of the cell. From the moment of divergence, the former sister chromatids are called daughter chromosomes. They reach the spindle poles and come together in a parallel pattern. The nuclear envelope is formed.
A model explaining the evolution of centromeres.
Up- centromeres (gray ovals) contain a specialized set of proteins (kinetochores), including histones CENH3 (H) and CENP-C (C), which in turn interact with spindle microtubules (red lines). In different taxa, one of these proteins evolves adaptively and in concert with the divergence of the primary DNA structure of centromeres.
At the bottom- changes in the primary structure or organization of centromeric DNA (dark gray oval) can create stronger centromeres, resulting in more attached microtubules.
Telomeres
The term “telomere” was proposed by G. Möller back in 1932. In his view, it meant not only the physical end of the chromosome, but also the presence of a “terminal gene with a special function of sealing the chromosome,” which made it inaccessible to harmful influences (chromosomal rearrangements, deletions, the action of nucleases, etc.). The presence of the terminal gene was not confirmed in subsequent studies, but the function of the telomere was precisely determined.
Later another function was discovered. Since the normal replication mechanism does not work at the ends of chromosomes, the cell has another pathway that maintains stable chromosome sizes during cell division. This role is performed by a special enzyme, telomerase, which acts like another enzyme, reverse transcriptase: it uses a single-stranded RNA template to synthesize the second strand and repair the ends of chromosomes. Thus, telomeres in all organisms perform two important tasks: they protect the ends of chromosomes and maintain their length and integrity.
A model of a protein complex of six telomere-specific proteins that forms on the telomeres of human chromosomes has been proposed. The DNA forms a t-loop, and the single-stranded overhang inserts into the double-stranded DNA region located distally (Fig. 6). The protein complex allows cells to distinguish telomeres from chromosome (DNA) breakpoints. Not all telomere proteins are part of a complex that is abundant at telomeres but absent in other regions of the chromosomes. The protective properties of the complex stem from its ability to influence the structure of telomeric DNA in at least three ways: determining the structure of the telomere tip itself; participate in the formation of a t-loop; control the synthesis of telomeric DNA by telomerase. Related complexes have also been found on the telomeres of some other eukaryotic species.
Up -telomere at the time of chromosome replication, when its end is accessible to the telomerase complex, which carries out replication (doubling of the DNA strand at the very tip of the chromosome). After replication, telomeric DNA (black lines) together with the proteins located on it (shown as multi-colored ovals) forms a t-loop ( bottom of the picture).
Time of DNA compaction in the cell cycle and the main factors stimulating processes
Let us recall the structure of chromosomes (from a biology course) - they are usually displayed as a pair of letters X, where each chromosome is a pair, and each has two identical parts - the left and right chromatids. This set of chromosomes is typical for a cell that has already begun its division, i.e. cells in which the DNA duplication process has taken place. The doubling of the amount of DNA is called the synthetic period, or S-period, of the cell cycle. They say that the number of chromosomes in a cell remains the same (2n), and the number of chromatids in each chromosome is doubled (4c - 4 chromatids per pair of chromosomes) - 2n4c. During division, one chromatid from each chromosome will enter the daughter cells and the cells will receive the full diploid set of 2n2c.
The state of the cell (more precisely, its nucleus) between two divisions is called interphase. There are three parts in interphase - presynthetic, synthetic and postsynthetic periods.
Thus, the entire cell cycle consists of 4 time periods: mitosis proper (M), presynthetic (G1), synthetic (S) and postsynthetic (G2) periods of interphase (Fig. 19). The letter G - from the English Gap - interval, interval. In the G1 period, which occurs immediately after division, cells have a diploid DNA content per nucleus (2c). During the G1 period, cell growth begins mainly due to the accumulation of cellular proteins, which is determined by an increase in the amount of RNA per cell. During this period, the cell begins to prepare for DNA synthesis (S-period).
It was found that suppression of protein or mRNA synthesis in the G1 period prevents the onset of the S period, since during the G1 period the synthesis of enzymes necessary for the formation of DNA precursors (for example, nucleotide phosphokinases), RNA and protein metabolism enzymes occurs. This coincides with an increase in RNA and protein synthesis. At the same time, the activity of enzymes involved in energy metabolism sharply increases.
In the next S-period, the amount of DNA per nucleus doubles and the number of chromosomes accordingly doubles. In different cells in the S period, different amounts of DNA can be found - from 2c to 4c. This is due to the fact that cells are studied at different stages of DNA synthesis (those that have just started synthesis and those that have already completed it). The S period is a key period in the cell cycle. Without DNA synthesis, not a single case of cells entering mitotic division is known.
The postsynthetic (G2) phase is also called premitotic. The last term emphasizes its great importance for passing through the next stage - the stage of mitotic division. In this phase, the synthesis of mRNA necessary for the passage of mitosis occurs. Somewhat earlier, the rRNA of the ribosomes, which determine cell division, is synthesized. Among the proteins synthesized at this time, tubulins, the proteins of the microtubules of the mitotic spindle, occupy a special place.
At the end of the G2 period or in mitosis, as mitotic chromosomes condense, RNA synthesis drops sharply and completely stops during mitosis. Protein synthesis during mitosis decreases to 25% of the initial level and then in subsequent periods reaches its maximum in the G2 period, generally repeating the nature of RNA synthesis.
In the growing tissues of plants and animals there are always cells that are, as it were, outside the cycle. Such cells are usually called G0-period cells. These cells are the so-called resting cells, which have temporarily or permanently stopped reproducing. In some tissues, such cells can remain for a long time without particularly changing their morphological properties: they retain, in principle, the ability to divide, turning into cambial stem cells (for example, in hematopoietic tissue). More often, the loss (even if temporary) of the ability to divide is accompanied by the appearance of the ability to specialize and differentiate. Such differentiating cells exit the cycle, but under special conditions they can enter the cycle again. For example, most liver cells are in the G0 period; they do not participate in DNA synthesis and do not divide. However, when part of the liver is removed from experimental animals, many cells begin preparation for mitosis (G1 period), proceed to DNA synthesis and can divide mitotically. In other cases, for example, in the epidermis of the skin, after leaving the cycle of reproduction and differentiation, the cells function for some time and then die (keratinized cells of the integumentary epithelium).
