Three fractions of eukaryotic DNA, their localization in chromosomes and functions. Chromatin: definition, structure and role in cell division
In a chromatin preparation, DNA usually accounts for 30-40%. This DNA is a double-stranded helical molecule. The DNA of eukaryotic cells is heterogeneous in composition, containing several classes of nucleotide sequences: frequently repeated sequences (>106 times), included in the satellite DNA fraction and not transcribed; a fraction of moderately repetitive sequences (102-105), representing blocks of true genes, as well as short sequences scattered throughout the genome; a fraction of unique sequences that carries information for the majority of cell proteins.
Chromatin
Chromatin consists of DNA in complex with protein. In interphase cells, chromatin can evenly fill the volume of the nucleus or be located in separate clumps (chromocenters). Often it is especially clearly visible at the periphery of the nucleus (parietal, near-membrane chromatin) or forms interweavings of rather thick (about 0.3 µm) and long strands inside the nucleus, forming a semblance of an intranuclear chain.
In interphase, a nucleolus is formed in the zone of the nucleolar organizer. Euchromatin is decondensed, despiralized sections of DNA from which genetic information about the amino acid composition of the protein is read (transcription). Euchromatin - functional active part chromosomes.
Heterochromatin is condensed, spiralized sections of DNA. Heterochromatin is the functionally inactive parts of a chromosome. Heterochromatin is intensely stained with basic dyes, while euchromatin does not have this property and appears as light, unstained areas among clumps of heterochromatin.
The chromatin of interphase nuclei is a DNA-carrying body (chromosomes), which at this time loses its compact shape, loosens, and decondenses. The degree of such chromosome decondensation may vary in the nuclei of different cells. When a chromosome or part of it is completely decondensed, then these zones are called diffuse chromatin. When chromosomes are incompletely loosened, areas of condensed chromatin (sometimes called heterochromatin) are visible in the interphase nucleus. The more diffuse the chromatin of the interphase nucleus, the higher the synthetic processes in it. Chromatin is condensed to its maximum during mitotic cell division, when it is found in the form of dense bodies - chromosomes.
in the working, partially or completely decondensed state, when the processes of transcription and reduplication occur with their participation in the interphase nucleus;
in inactive - in a state of metabolic rest at their maximum condensation, when they perform the function of distributing and transferring genetic material to daughter cells.
Chemically, chromatin preparations are complex complexes of deoxyribonucleoproteins, which include DNA and special chromosomal proteins - histones.
Chromatin proteins
These include histones and non-histone proteins.
Histones are strongly basic proteins. Their alkalinity is related to their enrichment in essential amino acids (mainly lysine and arginine). These proteins do not contain tryptophan. The total histone preparation can be divided into 5 fractions:
H1 (from English histone) - lysine-rich histone,
H2a - moderately lysine-rich histone, H2b - moderately lysine-rich histone,
H4 - arginine-rich histone, H3 - arginine-rich histone,
Histones are synthesized on polysomes in the cytoplasm; this synthesis begins somewhat earlier than DNA reduplication. Synthesized histones migrate from the cytoplasm to the nucleus, where they bind to sections of DNA.
Non-histone proteins are the most poorly characterized fraction of chromatin.
Yamdryshki
Regions of chromosomes where the synthesis of ribosomal ribonucleic acids (rRNA) occurs. They are located inside the cell nucleus and do not have their own membrane membrane, but are clearly visible under light and electron microscopes].
The main function of the nucleolus is the synthesis of ribosomal RNA and ribosomes, on which the synthesis of polypeptide chains is carried out in the cytoplasm. In the cell genome there are special regions, the so-called nucleolar organizers, containing ribosomal RNA (rRNA) genes, around which nucleoli are formed. In the nucleolus, rRNA is synthesized by RNA polymerase I, its maturation, and the assembly of ribosomal subunits. Proteins that take part in these processes are localized in the nucleolus. Some of these proteins have a special sequence - a signal for nucleolar localization. Electron microscopy makes it possible to identify two main components in the nucleolus: granular (along the periphery) - maturing ribosomal subunits and fibrillar (in the center) - ribonucleoprotein strands of ribosome precursors.
The granular component is represented by grains (diameter 10-20 nm), consisting of ribonucleoprotein particles (ribosomal subunits). The fibrillar part consists of dense thin electron-dense filaments (diameter 5-8 nm), forming a compact mass. These fibers are concentrated around lighter cores of less dense material(fibrillar centers). It is believed that the fibrillar material is RNA (ribosomal RNA), and the fibrillar centers consist of DNA and correspond in structure to chromatin grains.
The amorphous component is stained pale and contains areas of nucleolar organizers with specific RNA-binding proteins and large DNA loops that are actively involved in the transcription of ribosomal RNA. The fibrillar and granular components form a nucleolar filament (nucleoneme), the thickness of which is 60-80 nm.
The main function of the nucleolus is the synthesis of ribosomes. In the cell genome there are special regions, the so-called nucleolar organizers, containing ribosomal RNA (rRNA) genes, around which nucleoli are formed. In the nucleolus, rRNA is synthesized by RNA polymerase I, its maturation, and the assembly of ribosomal subunits. The proteins involved in these processes are localized in the nucleolus.
Removal of histone H1 from transcriptionally active chromatin 1*2* . IN early experiences J. Bonner (USA) showed that DNA in chromatin is a much worse matrix than free DNA. Based on these observations, it has been proposed that histones are transcriptional repressors.
L. N. Ananyeva and Yu. V. Kozlov Our laboratory set out to find out whether all histones have an inhibitory effect or only some of them. To do this, histones were removed from the chromatin of mouse Ehrlich ascitic cancer cells by extraction with NaCl solutions with gradually increasing concentrations. The resulting preparations served as a template for RNA synthesis. Transcription was carried out in the presence of RNA polymerase from Escherichia coli, E. coli, which was taken in excess, and a mixture of nucleoside triphosphates. In the range of 0.4-0.6 M NaCl, a sharp decondensation of the material occurred, manifested in the swelling of the nuclear gel and even the dissolution of DNP (if further mechanical processing was carried out). This was shown to selectively remove histone HI. Simultaneously with the decondensation of chromatin, there was a sharp increase in its matrix activity (Fig. 26). A further increase in the salt concentration in the extraction solution led to the removal of other histones and a slight, but not very pronounced, additional increase in matrix activity.
0 t. Thus, hybridizability reveals the percentage of repeating sequences in the synthesized RNA (according to the results obtained by L. N. Ananyeva, Yu. V. Kozlov and the author); b - main parameters of RNA synthesis on different matrices: free DNA, original chromatin (DNP 0) and chromatin from which histone H1 has been removed by extraction with 0.6 M NaCl (DNP 0.6). RNA synthesis was carried out using Escherichia coli RNA polymerase in the presence of labeled nucleoside triphosphates: [ 14 C]-ATP and either [γ- 32 P]-ATP or [γ- 32 P]-GTP. [ 14 C]-UMP was included in the entire RNA, and [γ- 32 P] - only at the beginning of the chain (pp x A- or pp x G). In other words, the inclusion of [ 32 P] provided information about the initiation of synthesis, and [ 14 C] - about the RNA synthesis itself. In some experiments, 3-4 minutes after the start of incubation, rifampicin, an antibiotic, was added to the medium, which prevented the initiation of new RNA chains, but did not affect the synthesis that had already begun, i.e., elongation. 1 - inclusion of [14 C] - UMP, 2 - the same after adding rifampicin; 3 - inclusion of [γ- 32 P]-ATP + GTP; 4 - the same after adding rifampicin. Based on these inclusion curves, it is possible to calculate the main parameters of the RNA polymerase reaction (based on the results obtained by Yu. V. Kozlov and the author)">
Rice. 26. The influence of histone H1 on the template activity of DNA in chromatin. a - the effect of removing proteins from chromatin (1) on the matrix activity (2) of the latter in the presence of exogenous RNA polymerase of Escherichia coli, as well as on the hybridizability of the synthesized RNA (3). Histones and non-histone proteins were extracted with increasing concentrations of NaCl solutions. In the range of 0.4 M NaCl - 0.6 M NaCl, histone H1 was selectively removed. The synthesized RNA was hybridized with excess DNA at intermediate C0 t values. Thus, hybridizability reveals the percentage of repeating sequences in the synthesized RNA (according to the results obtained by L. N. Ananyeva, Yu. V. Kozlov and the author); b - main parameters of RNA synthesis on different matrices: free DNA, original chromatin (DNP 0) and chromatin from which histone H1 has been removed by extraction with 0.6 M NaCl (DNP 0.6). RNA synthesis was carried out using Escherichia coli RNA polymerase in the presence of labeled nucleoside triphosphates: [ 14 C]-UTP and either [γ- 32 P]-ATP or [γ- 32 P]-GTP. [ 14 C]-UMP was included in the entire RNA, and [γ- 32 P] - only at the beginning of the chain (pp x A- or pp x G). In other words, the inclusion of [ 32 P] provided information about the initiation of synthesis, and [ 14 C] - about the RNA synthesis itself. In some experiments, 3-4 minutes after the start of incubation, rifampicin, an antibiotic, was added to the medium, which prevented the initiation of new RNA chains, but did not affect the synthesis that had already begun, i.e., elongation. 1 - inclusion of [14 C] - UMP, 2 - the same after adding rifampicin; 3 - inclusion of [γ- 32 P]-ATP + GTP; 4 - the same after adding rifampicin. Based on these inclusion curves, the main parameters of the RNA polymerase reaction can be calculated (based on the results obtained by Yu. V. Kozlov and the author)
The properties of the synthesized in vitro RNA by hybridization with total mouse DNA. In this case, a fraction of RNA synthesized on repeating DNA sequences was detected. It turned out that in chromatin the transcription of repeated DNA sequences is limited. However, after extraction of chromatin with 0.6 M NaCl, when histone H1 was removed, the hybridization properties of RNA synthesized on the matrix of such chromatin and on the matrix of free DNA became indistinguishable. We hypothesized that histones H2a, H2b, H3 and H4 (then called differently - moderately lysine-rich and arginine-rich histones) are not involved in transcription suppression, but play a purely structural role in chromatin organization, while histone H1 (in the old terminology histone , rich in lysine) is an inhibitor of RNA synthesis. At the same time, it is also a factor causing chromatin condensation (see above).
Later, Yu. V. Kozlov studied the mechanism of transcription inhibition by histone H1, again on a cell-free system with RNA polymerase isolated from E, coli. The effect of histone H1 on the processes of initiation and elongation was studied (see Fig. 26, Table 4). It turned out that it several times reduces the number of RNA chains initiated on the native chromatin matrix. Elongation is especially sharply inhibited: RNA polymerase reads no more than 100-150 bp. DNA and then stops. Meanwhile, on chromatin from which histone H1 has been removed, RNA polymerase reads several thousand nucleotide pairs at a time, and the length of the chains does not differ from the length of the chains synthesized on the free DNA template. True, on free DNA, compared to chromatin from which histone H1 has been removed, the processes of initiation of RNA synthesis occur more efficiently. It was concluded that histone H1, by condensing chromatin, creates obstacles in the path of RNA polymerase and thereby stops RNA synthesis.
* (DNPM is DNP isolated by urea treatment that is fully solubilized but retains histone H1.)
In light of modern data on the solenoid structure of the 300 A-DNP fibril, which depends on the presence of histone H1, this result is easily explained. Indeed, RNA polymerase obviously cannot read more than 100 bp in the solenoid. due to purely topological restrictions.
According to our hypothesis, when the gene was activated, histone H1 should be removed. However, it was not possible to verify it at that time. Only relatively recently has evidence emerged of the loss of histone H1 from chromatin during gene activation. Thus, when actively transcribed regions of chromatin, for example mini-nucleoli, were isolated from a number of organisms, histone H1 was not detected in them. It is not found in yeast either, where all genes are potentially active.
Convincing results were obtained in the laboratory A. D. Mirzabekova V. L. Karpov and O. V. Preobrazhenskaya. They developed a method called “histone shadow hybridization.” To do this, DNA was cross-linked with histones using dimethyl sulfate under conditions where, on average, one histone molecule is cross-linked per DNA segment of about 200-300 bp in length. The DNA was then fragmented and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. After running in the first direction, the histones attached to the DNA were destroyed by proteinase and the already free DNA was accelerated in the second direction. Since during the first round of electrophoresis different histones slowed down the movement of DNA fragments in different ways, after the second run several diagonals were revealed (Fig. 27). Usually three are clearly visible: one corresponding to the initially free DNA, the other to the original DNA complexes with core histones, and the third (bottom) to the original DNA complex with histone H1. The resulting DNA is transferred to a filter and hybridized with a particular sample. If an inactive region of the genome was taken for hybridization, for example, the spacer of the Drosophila ribosomal gene, then the label was associated with all diagonals. If, however, the heat shock gene, which was transcribed in the cells from which chromatin was isolated, was used as a sample, then hybridization with the diagonal corresponding to DNA complexes with histone H1 was sharply weakened or completely absent. In other words, in the nuclei, before attachment, the DNA of the transcribed gene had no contacts with histone H1.
Rice. 27. Loss of histone H1 and core histones upon transcription activation. Experiments were carried out on D. melanogaster culture cells (a, b) under cultivation conditions at 25° (a) and heat shock (b). In case a, there is no expression of heat shock genes; in case b, it is very active. In addition, experiments were carried out on dechorionized embryos (c), where the expression of heat shock genes is at an average level. After the formation of DNA-protein complexes, isolation of DNA fragments, their two-dimensional separation (in the vertical direction after removal of the protein) and transfer to a filter, the same filters were hybridized with different samples: with the regulatory region of the p70 heat shock gene (HS-5") ; with the coding region of the same gene (TS code); with a transcriptionally inactive insertion into the rDNA gene (inactive), three diagonals are visible; 1 - free DNA, 2 - DNA complexes with octamer histones; With histone H1. The weakening or disappearance of DNA-histone complexes upon activation of chromatin is visible (according to the results obtained by A. D. Mirzabekov et al.)
Although all currently available data, taken individually, allow other interpretations, taken together they provide strong evidence in favor of the removal of histone H1 from active chromatin. However, the mechanism of this process is still completely unclear.
Fate of nucleosomes during chromatin activation 2* [ 154-157]. Less clear is the question of the fate of histones H2a, H2b, H3 and H4, which form the core of the nucleosome. In the above experiments Yu. V. Kozlova their presence had virtually no effect on the transcription of DNA by RNA polymerase from E. coli. When studying the products of eukaryotic chromatin hydrolysis, many authors found that nucleosomes contain DNA of active genes, i.e., the latter are also organized into nucleosomes. Data obtained from large experimental material A. D. Mirzabekova et al. show that nucleosomes containing actively transcribed DNA are fundamentally constructed in the same way as nucleosomes containing inactive DNA, although some DNA-histone contacts in them are changed.
Experiments were also carried out on hybridization with histone shadows, which were discussed in the previous section (see Fig. 27). Preparations with diagonals were prepared from Drosophila cells in which heat shock genes either did not work at all, that is, they were turned off, or they worked at a low level, or, finally, they were stimulated by heat shock to active transcription. In all cases, the control sample was the DNA of the ribosomal gene spacer, which hybridized in all three diagonals, including the diagonal derived from DNA complexes with core histones.
The heat shock gene also hybridized normally with this diagonal from cells where it was not transcribed. However, with material obtained from cells with moderate transcription of the heat shock gene, hybridization of the second diagonal was significantly reduced. Finally, if the diagonals were obtained from cells with very active synthesis of heat shock mRNA, then the second diagonal was not visible at all during hybridization (as was the third), and only the diagonal was revealed that corresponded to DNA not cross-linked with histones. The general conclusion drawn from studies using The method of DNA-protein cross-linking was that during transcription, RNA polymerase moving along the DNA chain reversibly collides nucleosomes with DNA and transcribes virtually naked DNA. If the level of transcription is low and there are few RNA polymerases crawling along the DNA, then nucleosomes have time to form again in the area through which the RNA polymerase has already passed. If transcription is active, then the nucleosomal structure of the DNA does not have time to be restored, and the DNA is generally devoid of histones. At the same time, it is postulated that the organization of nucleosomes in active chromatin is practically no different from that in inactive chromatin. Recently, A.D. Mirzabekov et al. reproduced experiments on the attachment of histones to DNA using a different method, by treating isolated nuclei with platinum preparations. This method is milder than dimethyl sulfate. Fundamentally the same results were obtained.
Along with this body of data, there are studies in which the authors come to slightly different conclusions. W. Garrard and A. Worsel(USA), studying the state of active genes in chromatin using nuclease hydrolysis and electron microscopy, came to the conclusion that nucleosomes remain in active chromatin, but undergo structural changes such as turning around, turning into half-nucleosomes. As a result, the periodicity in electropherograms of hydrolysates with micrococcal nuclease is ~200 bp. replaced by a periodicity of ~100 bp. With electron microscopy, the number of beads doubles and their sizes decrease. It is assumed that RNA polymerase can pass through such unfolded nucleosomes.
This possibility is also supported by the data obtained T. Koller(Switzerland). He developed original method studying nucleosomes. The cells are treated with psoralen, a substance that binds to DNA and then cross-links two strands of DNA together with UV light. However, if DNA is part of nucleosomes, its reaction with psoralen does not occur. Therefore, if DNA isolated from treated cells is denatured in the presence of formaldehyde (it prevents the renaturation of DNA), then upon electron microscopy, alternating bubbles (two strands of denatured DNA) corresponding to nucleosomes connected to each other by single strands (cross-linked, not capable of denaturation) are visible on the DNA. DNA) corresponding to internucleosomal linkers. First, actively transcribed ribosomal RNA genes, which are part of extrachromosomal structures and therefore easily analyzed using electron microscopy, were studied. In them, vesicles corresponding to nucleosomes are not visible, i.e., in all likelihood, histones are completely removed from the transcribed regions. Interestingly, in the non-transcribed regions, the spacers, DNA bubbles (nucleosomes) are clearly visible.
However, different results were obtained on SV40 minichromosomes, which are transcribed by RNA polymerase II rather than RNA polymerase I like the ribosomal RNA genes (Fig. 28). Transcriptionally active mini-chromosomes are identified due to the presence of growing RNA chains (usually one or two) on them. Such mini-chromosomes make up 1-2% of all mini-chromosomes isolated from the cell. They, however, contain the same number of vesicles as inactive minichromosomes, and their sizes are the same in both cases. The most interesting thing is that the RNA chains extend both from the linkers and directly from the vesicles, i.e., RNA polymerase apparently transcribes nucleosomes. These data support the unfolding of nucleosomes and their transcription by RNA polymerase.
All the above results are not direct, and therefore final decision The question of the fate of nucleosomes during transcription should be answered by future experiments.
Histone modification and histone variants: association with active chromatin. Back in the early 60s, Allfrey (USA) showed that histones can undergo various modifications. Thus, histone HI is phosphorylated at the ε-amino groups of lysines. Histones H3 and H4 are acetylated on the same groups. There are a number of other modifications (methylation, ADP - ribosylation, ubiquitination, etc.).
It was immediately assumed that enzymatic modifications of histones could affect the structure of chromatin and its activity. Indeed, when lysine is phosphorylated, one positive charge in a histone is replaced by a negative one; when appealing, a positive charge is lost, etc. It is thanks to such changes in charge that modified histones can be separated from unmodified ones when performing gel electrophoresis in an acetate buffer with urea. Thus, in high-resolution electrophoresis, histone H4 gives not one, but four bands, corresponding to molecules that are not acetylated and acetylated at one, two, and three lysine residues. In different tissues the ratio between fractions changes. Histones H3, H2a, H2b and H1 are divided into several fractions (different degrees of acetylation and phosphorylation).
Unfortunately, there are still no good methods for separating transcriptionally active and inactive chromatin and therefore it is difficult to attribute altered histone forms to one or another chromatin state. The most interesting data in this direction were obtained by the same W. Allfrey(USA). During the hydrolysis of active chromatin, he isolated unusual particles that sedimented in the sucrose gradient more slowly than ordinary nucleosomes and, in the author's opinion, corresponded to unfolded nucleosomes. These particles, called A particles, contained all the core histones. Unlike normal nucleosomes, the SH groups of histone H3 in A particles were accessible to a number of chemical reagents, and because of this, A particles could be separated from nucleosomes by fractionation on chloromercuribenzoate columns (an SH group binding reagent). A-particles contain an increased content of acetylated forms of histones. The author suggests that histone acetylation upon chromatin activation leads to the unfolding of nucleosomal particles, and this, in turn, increases the availability of SH groups of histone H3.
Some histones are encoded by more than one type of gene. As a result, there are several variants of these histones that differ slightly in their amino acid sequence. Sometimes in the process of ontogenesis there is a natural replacement of one histone subclass with another. It remains unclear, however, whether this has any regulatory significance. The issue, obviously, can also be resolved after the development of adequate methods for isolating transcriptionally active chromatin.
A special position is occupied by histone H1. There are options for it that differ sharply in their structural organization. This option is, for example, histone H5, which replaces a significant part of histone H1 in the nuclei of avian erythrocytes. In all likelihood, this substitution is an important factor in the complete shutdown of transcription in the nuclei of erythrocytes. IN ordinary cells There is a variant of histone H1 - histone H1 0. Its content constitutes a small fraction of the total histone H1. There are a number of contradictory data that H1 0 is associated with active genes or, conversely, with stably turned off genes. The question remains open.
HMG proteins may be involved in organizing active chromatin 1* . In addition to histones, chromatin contains many non-histone proteins whose function is unknown. Among them, obviously, there should be structural proteins, enzymes that ensure the processes of replication, transcription, etc., and regulatory proteins. E. Jones(Great Britain) attempted to isolate protein components present in sufficiently large quantities to allow their analysis and identification. He really managed to isolate new class nuclear proteins, which he called “a group of proteins with high mobility” ( high mobility group), or HMG proteins. The name depended on the high mobility of these proteins during gel electrophoresis. The HMG protein fraction breaks down into a number of individual components. Among them, the most representative and well characterized are HMG-1, HMG-2, HMG-14 and HMG-17.
HMG proteins have a low molecular weight. They are enriched with both basic and dicarboxylic amino acids. The content of HMG proteins is approximately 7% of the content of histones. In kernels from different types cells it can vary. In this context, we are most interested in the proteins HMG-14 and HMG-17, for which evidence has been obtained for a possible role in transcription activation. H. Weintraub(USA) showed that nuclear extraction with 0.35 M NaCl, which extracts HMG proteins, changes some properties of active chromatin, which are restored when HMG-14 and HMG-17 are added to the chromatin. G. Dixon(Canada) discovered these proteins in the composition of nucleosomes released from chromatin at the early stages of hydrolysis by nuclease, which, according to his data, were enriched in the DNA of tractionally active genes.
ends [ 32 P] and then hybridized with hnRNA from L cells. Hybrids were detected by gel filtration. 1 - CH-2 DNA; 2 - CH-3 DNA; 3 - total DNA of the cell (according to the results obtained by V.V. Bakaev et al.)">
Rice. 29. Possible connection of HMG proteins (14 and 17) with active chromatin. a - detection of subnucleosomes in chromatin hydrolysates using micrococcal nuclease. At different stages of hydrolysis, certain fractions of subnucleosomes appear. Electrophoresis was carried out in polyacrylamide gel under nondenaturing conditions. Staining with ethidium bromide, fluorography on the right column; b - use of two-dimensional electrophoresis to determine the protein composition of CH2 and CH3 subnucleosomes. Chromatin labeled with [14 C] protein was hydrolyzed with micrococcal nuclease and separated by two-dimensional electrophoresis (1st direction - non-dissociating medium, 2nd direction - sodium dodecyl sulfate solution), after which autoradiography was performed to identify proteins. The letters indicate HMG proteins that at the time of the experiment had not yet been identified with the known ones. Now we know that A is HMG-1, B is HMG-2, E is HMG-14, G is HMG-17, the HMG proteins F and H are not clearly identified, probably H also corresponds to HMG-17. It can be seen that HMG proteins are part of mononucleosomes (MH-2 and MH-3) and subnucleosomes CH-2 (HMG-17) and CH-3 (HMG-14); c - demonstration of DNA enrichment of CH-2 and CH-3 transcribed sequences. DNA isolated from the CH-2 and CH-3 bands of L cells was labeled at the 5" ends [32 P] and then hybridized with hnRNA from L cells. Hybrids were detected by gel filtration. 1 - CH-2 DNA; 2 - CH-3 DNA; 3 - total DNA of the cell (according to the results obtained by V.V. Bakaev et al.)
V. V. Bakaev in our laboratory came to the conclusion about the role of HMG proteins in transcription using a different experimental approach. During electrophoretic analysis of chromatin hydrolysates, he revealed, in addition to nucleosomes and oligonucleosomes, minor components with greater mobility. They were called subnucleosomes and, obviously, were products of further breakdown of the nucleosome (Fig. 29, Table 5). Subnucleosome CH-7 corresponded to a nucleosome that had lost one molecule each of H2a and H2b and contained DNA shortened by 40 bp; CH-6 corresponded to a DNA complex 30-40 bp long. with histone H1, which is cleaved from MH-2 during its transformation into MH-1. CH-4 contained a DNA segment and a pair of histones H2a and H2b (the product of the reaction MH-1→CH-7→CH-4). Two subnucleosomes, CH-3 and CH-2, consisted of short DNA and HMG proteins (HMG-14 and HMG-17). It could be assumed that they are linker regions associated with the HMG-14 and HMG-17 proteins, which pass into solution upon digestion of the corresponding nucleosomes. CH-2 and CH-3 were collected, DNA was isolated from them, end-labeled, and its hybridization with nuclear RNA was studied. It turned out that DNA from CH-2 and CH-3 hybridizes much more efficiently with nuclear RNA than total cell DNA fragmented to the same size.
It was therefore concluded that the DNA associated with the HMG-14 and HMG-17 proteins likely originated from transcriptionally active chromatin.
All of these data, obtained independently, suggested that HMG-14 and HMG-17 are somehow associated with gene activation. The mechanism of activation was, however, completely unclear. HMG-14 and HMG-17 could not be the primary factors turning on the gene, since they lack specificity. One might think that they are involved in maintaining the “open conformation” of active chromatin.
In subsequent years, skepticism emerged regarding the role of HMG-14 and HMG-17 in chromatin activation. In particular, recently A. D. Mirzabekov et al. using the method of hybridization with protein tissues, we obtained data on the depletion of active chromatin of HMG-14 and HMG-17. Since, however, all of the above data are indirect, in general the question of the role of HMG proteins remains open and requires further study.
Topoisomerase I and proteins tightly bound to DNA are part of transcriptionally active chromatin 1* . S. Elgin (USA), followed by a number of other authors, showed that transcriptionally active chromatin contains topoisomerase I, an enzyme that relaxes supercoiled DNA. This was first demonstrated on cytological preparations of Drosophila polytene chromosomes using fluorescent antibodies to topoisomerase I. This enzyme introduces a single-strand break into the DNA and covalently binds to the resulting 5" end of the DNA. This allows the DNA to rotate freely at the break site. Then the fragment is cleaved off, and the phosphodiester bond in DNA is restored. By molecular weight, topoisomerase I, or topo I for short, is heterogeneous. The heaviest component has a molecular weight. 135 kDa, and the most richly represented - 80 kDa. When it is cleaved by proteinases, shorter polypeptides are formed, which nevertheless retain enzymatic activity.
The antibiotic captothecin is a topo I inhibitor, and when cells are treated with it, the enzyme forms covalent crosslinks with DNA in the place where it was at the time of contact with the antibiotic. The location of such crosslinks can be easily determined by mapping using a hybridization tag. In this way, it was discovered that topo I is present exclusively in transcribed regions of the genome, i.e., most likely, it works in cooperation with RNA polymerase II, removing local DNA twists that occur during transcription.
Another protein component detected in the transcribed regions of the genome is a set of proteins tightly bound to DNA (DBP), which corresponds to the transcribed DNA of the cell (see Section 3.4).
S. V. Razin and V. V. Chernokhvostov An attempt was made to characterize complexes of DNA with PBP in detail. DNA fragments of 1–2 kb in length associated with PBP were purified and subjected to equilibrium ultracentrifugation in a CsCl density gradient. Their buoyant density turned out to be equal 1.7 g/cm3, i.e., it corresponded to the buoyant density of free DNA containing no protein. In experiments designed to explain this paradox, it was found that treatment with DRNase leads to a decrease in the buoyant density of the complexes to 1.62-1.65 g/cm3. Approximate calculations based on protein density ( ~ 1.3 g/cm3) and RNA ( ~ 1.9 g/cm3), (show that for each DNA molecule there are about 150 kDa protein and about 200 nucleotides of RNA. The nature of this RNA is unclear, but evidence has been obtained of its homogeneity and unique nucleotide sequence.
Thus, much about DNA-PBP complexes remains mysterious, but in all likelihood they play a significant role in the organization of the transcription machinery. Their research is currently ongoing.
DNA demethylation in transcribed genes 2*. One more important feature active chromatin is the demethylation of certain sections of DNA. In inactive genes, most of the cytidyl residues within the CG sequences are methylated. First B.V. Vanyushin in the laboratory A. N. Belozersky it was demonstrated that DNA in different tissues of the same animal differs in the level of C methylation. Based on this, it was suggested that methylation may have a regulatory role in differentiation. Later, many authors showed that some regions in transcribed DNA are undermethylated. The most widely used analysis method is the comparison of restriction maps obtained with restriction enzymes that recognize the same sequence, such as CGCG or CCGG, but have different sensitivity to methylation. One of the restriction enzymes cleaves both methylated and unmethylated sequences, and the other only cleaves unmethylated ones. Typically, unmethylated sequences are localized in the regulatory region of the gene. The region of the gene itself, its coding part and introns is equally methylated in both working and silent genes.
When methylated DNA is introduced into cells, its expression in the cell is significantly reduced compared to unmethylated DNA. Data have been obtained according to which, during DNA replication, the state of DNA methylation is reproduced: if one of the chains is methylated, then the newly formed chain is also methylated in the same place.
At one time it seemed that methylation-demethylation of cytidine in the CG sequences of the regulatory region is the main mechanism of gene inactivation-activation. However, in Lately A number of data have emerged that contradict this hypothesis. Thus, fully CG methylated SV40 DNA is actively expressed. At the same time, methylcytosine is not detected at all in Drosophila DNA. It is possible that demethylation of C is a consequence of gene activation and only perpetuates the state of transcriptional activity. Here, as in other areas of studying gene activation, new experiments are needed.
To isolate chromatin, use is made of its ability to become dissolved during extraction with aqueous solutions of low ionic strength.
Histone proteins make up from 40 to 80% of all proteins that make up isolated chromatin (the rest are membrane components, RNA, carbohydrates, lipids, glycoproteins)
Structurally, chromatin (nucleohistone) is a filamentous complex molecule of deoxyribonucleoprotein ( DNP), which consist of DNA associated with histones.
Thickness of DNP filamentous fibrils: 10-30 nm
Molecular weight of DNA chromatin: 7-9*10^6
DNA concentration in the interphase nucleus: up to 100 mg/ml.
On average, the interphase core contains about 2 m of DNA, which is localized in a spherical core with an average diameter of about 10 μm. This means that such a huge mass of DNA must be packed with a packing ratio of 1*10^3 – 1*10^4
Eukaryotic cells contain only 5-7 types of histone molecules.
The interaction of histones with DNA occurs due to salt or ionic bonds and is nonspecific with respect to the composition and sequence of nucleotides in DNA.
Basic properties of histones:
1) These are alkaline proteins, the properties of which are determined by the relatively high content of such basic amino acids as lysine and arginine (due to the presence of positive charges on the amino groups of lysine and arginine)
2) Relatively small molecular weight.
Histones H3 and H4 classified as arginine rich H2A and H2B classified as proteins moderately enriched in lysine. Histones H1 enriched with lysine.
In histone H1, the most variable is N-terminus, which communicates with other histones, a C-terminus, rich in lysine, interacts with DNA.
Histones are synthesized in the cytoplasm, transported to the nucleus and bind to DNA during its replication in the S-period, i.e. the syntheses of histones and DNA are synchronized.
Levels of DNA compaction:
1) Nucleosomal
2) Nucleomeric
3) Chromomeric
4) Lame
5) Chromatid
1) Histone folding.
Histone types: H1, H2A, H2B, H3, H4.
Histones are positively charged and interact well with negatively charged DNA.
Histone H1 is separated in a solution of 0.6 M NaCl. All histones are separated in a 2M solution.
The N-terminus of the histone interacts with other histones and is variable. The C-terminus is conserved and interacts with DNA.
The most conserved histones are H3 and H4.
Nucleosome– a discrete particle of chromatin. The entire haploid human genome contains 1.5 * 10^7 nucleosomes.
The folding coefficient with histones is K=7, DNA is shortened by 7 times.
One winding= 146 base pairs. Between nucleosomes ( linker) 54 base pairs. Period equal to 200 nucleotides.
The laying of almost two turns of DNA along the periphery of the nucleosome core is believed to occur due to the interaction of positively charged amino acid residues on the surface of the histone octameter with DNA phosphates.
Histones H3 and H4 turned out to be key in the construction of nucleosomes (see the picture in the notebook!!!)
At the time of DNA synthesis, there is already a pool of synthesized histones of all classes, ready to become part of nucleosomes. Histone genes, which belong to the fraction of moderately repetitive DNA sequences, are represented as multiple copies for each histone. They are activated along with the onset of DNA synthesis, so as the replication fork progresses, new sections of DNA can immediately interact with newly synthesized histones.
Newly synthesized histones and old histones within the preceding nucleosomes do not mix during the formation of nucleosomes during DNA replication. Instead, octameters of histones present before replication remain intact and are transferred to the daughter DNA duplex, while new histones are assembled into completely new core particles on nucleosome-free DNA regions.
2) Fibril (30nm)
Solenoid type of installation: Spiral, zigzag arrangement (one turn - 6 nucleosomes). It is believed that histone H1 ensures interaction between neighboring nucleosomes, not only bringing them closer and connecting them, but also promoting the formation of a cooperative bond between neighboring nucleosomes, resulting in the formation of a tight helix. (packing density – 40)
The chromatin fibril was shown to be 30 nm in diameter. can reversibly change its diameter: become a fibril 10 nm thick if chromatin preparations are transferred to deionized water.
Second type of installation: 25-30 nm nucleomers, globular packing. 40-fold DNA compaction.
Laying of almost two turns of DNA along the periphery of the nucleosome core
Irregular solenoid model: the number of nucleosomes per turn of the helix is not strictly constant, which can lead to alternation of regions with a greater or lesser number of nucleosomes per turn.
3) Chromomeres (60-80 nm)
Loop styling model. Average loop size– 60 kb
Number of loops in socket – 15-80.
The base of the loops is anchored inside the nucleus, at sites of non-histone proteins.
4) Chromonema (100 nm)
In plants it can be seen in interphase nuclei, in animals - in telophase, during the process of decondensation.
5) Chromatid.
Non-histone proteins are also present in the nucleus:
1) SIR (silent information regulator)
proteins interacting with heterochromatin modify the N-terminus of histones, chromatin inactivation.
2) H3-CENPA (centromer protein)
localized in the center, constitutive heterochromatin, stable inactivation.
3) HP1 (heterochromatin protein 1)
4) HP1+H3+methyltransferase+deacetylase (removal of acetyl groups) = suppression of transcription.
5) In the area of the kinetochore, a structural formation is observed - a crown. It contains many proteins, the functions of most of them are unknown.
6) HMG proteins (“Jones proteins”) – High Mobility Group
Major HMG proteins: HMG-1, HMG-2, HMG-14, HMG-17. They are transcription regulators.
Models of chromosome structure:
1) Model of tangled threads
2) Chaotic model
3) Loop model
4) Solenoid model
The genetic material of eukaryotic organisms has a very complex organization. DNA molecules located in the cell nucleus are part of a special multicomponent substance - chromatin.
Definition of the concept
Chromatin is the material of the cell nucleus containing hereditary information, which is a complex functional complex of DNA with structural proteins and other elements that ensure packaging, storage and implementation of the karyotic genome. In a simplified interpretation, this is the substance that chromosomes are made of. The term comes from the Greek "chrome" - color, paint.
The concept was introduced by Fleming back in 1880, but there is still debate about what chromatin is in terms of biochemical composition. The uncertainty concerns a small part of the components that are not involved in the structuring of genetic molecules (some enzymes and ribonucleic acids).
In electron photography of the interphase nucleus, chromatin is visualized as numerous areas of dark matter, which can be small and scattered or combined into large dense clusters.
Chromatin condensation during cell division results in the formation of chromosomes, which are visible even in a conventional light microscope.
Structural and functional components of chromatin
In order to determine what chromatin is at the biochemical level, scientists extracted this substance from cells, transferred it into solution, and in this form studied its component composition and structure. Both chemical and physical methods were used, including electron microscopy technologies. It turned out that chemical composition 40% of chromatin is represented by long DNA molecules and almost 60% by various proteins. The latter are divided into two groups: histones and non-histones.
Histones are a large family of basic nuclear proteins that bind tightly to DNA, forming the structural skeleton of chromatin. Their number is approximately equal to the percentage of genetic molecules.
The rest (up to 20%) of the protein fraction consists of DNA-binding and spatially modifying proteins, as well as enzymes involved in the processes of reading and copying genetic information.
In addition to the basic elements, ribonucleic acids (RNA), glycoproteins, carbohydrates and lipids are found in small quantities in chromatin, but the question of their association with the DNA packaging complex is still open.
Histones and nucleosomes
The molecular weight of histones varies from 11 to 21 kDa. A large number of residues of the basic amino acids lysine and arginine give these proteins positive charge, promoting the formation of ionic bonds with oppositely charged phosphate groups of the DNA double helix.
There are 5 types of histones: H2A, H2B, H3, H4 and H1. The first four types are involved in the formation of the main structural unit of chromatin - the nucleosome, which consists of a core (protein core) and DNA wrapped around it.
The nucleosome core is represented by an octamer complex of eight histone molecules, which includes the H3-H4 tetramer and the H2A-H2B dimer. A DNA section of about 146 nucleotide pairs is wound onto the surface of the protein particle, forming 1.75 turns, and passes into a linker sequence (approximately 60 bp) connecting the nucleosomes to each other. The H1 molecule binds to linker DNA, protecting it from the action of nucleases.
Histones can undergo various modifications, such as acetylation, methylation, phosphorylation, ADP-ribosylation, and interaction with ubiquitin protein. These processes affect the spatial configuration and packing density of DNA.
Non-histone proteins
There are several hundred varieties of non-histone proteins with various properties and functions. Their molecular weight varies from 5 to 200 kDa. A special group consists of site-specific proteins, each of which is complementary to a specific region of DNA. This group includes 2 families:
- “zinc fingers” – recognize fragments 5 nucleotide pairs long;
- homodimers – characterized by a helix-turn-helix structure in the fragment associated with DNA.
The best studied are the so-called high mobility proteins (HGM proteins), which are constantly associated with chromatin. The family received this name due to high speed movement of protein molecules in an electrophoresis gel. This group occupies the majority of the non-histone fraction and includes four main types of HGM proteins: HGM-1, HGM-14, HGM-17 and HMO-2. They perform structural and regulatory functions.
Non-histone proteins also include enzymes that provide transcription (the process of synthesis of messenger RNA), replication (doubling of DNA) and repair (elimination of damage in the genetic molecule).
Levels of DNA compaction
The peculiarity of the chromatin structure is such that it allows DNA strands with a total length of more than a meter to fit into a nucleus with a diameter of about 10 microns. This is possible thanks to a multi-stage packaging system of genetic molecules. General scheme compactization includes five levels:
- nucleosomal filament with a diameter of 10–11 nm;
- fibril 25–30 nm;
- loop domains (300 nm);
- 700 nm thick fiber;
- chromosomes (1200 nm).
This form of organization ensures a reduction in the length of the original DNA molecule by 10 thousand times.
A thread with a diameter of 11 nm is formed by a number of nucleosomes connected by linker sections of DNA. In an electron micrograph, such a structure resembles beads strung on a fishing line. The nucleosome filament folds into a spiral like a solenoid, forming a fibril 30 nm thick. Histone H1 is involved in its formation.
The solenoid fibril folds into loops (otherwise known as domains), which are anchored to the supporting intranuclear matrix. Each domain contains from 30 to 100 thousand base pairs. This level of compaction is characteristic of interphase chromatin.
A structure 700 nm thick is formed by the helicalization of a domain fibril and is called a chromatid. In turn, the two chromatids form the fifth level of DNA organization - a chromosome with a diameter of 1400 nm, which becomes visible at the stage of mitosis or meiosis.
Thus, chromatin and chromosome are forms of packaging of genetic material that depend on life cycle cells.
Chromosomes
A chromosome consists of two identical sister chromatids, each of which is formed by one supercoiled DNA molecule. The halves are connected by a special fibrillar body called a centromere. At the same time, this structure is a constriction that divides each chromatid into arms.
Unlike chromatin, which is a structural material, a chromosome is a discrete functional unit, characterized not only by structure and composition, but also by a unique genetic set, as well as a certain role in the implementation of the mechanisms of heredity and variability at the cellular level.
Euchromatin and heterochromatin
Chromatin in the nucleus exists in two forms: less spiralized (euchromatin) and more compact (heterochromatin). The first form corresponds to transcriptionally active regions of DNA and is therefore not so tightly structured. Heterochromatin is divided into facultative (can pass from an active form to a dense inactive one, depending on the stage of the cell’s life cycle and the need to implement certain genes) and constitutive (constantly compacted). During mitotic or meiotic division, all chromatin is inactive.
Constitutive heterochromatin is found near centromeres and in the terminal regions of the chromosome. Electron microscopy results show that such chromatin retains high degree condensation not only at the stage of cell division, but also during interphase.
Biological role of chromatin
The main function of chromatin is to pack tightly large quantity genetic material. However, simply placing DNA in the nucleus is not enough for a cell to function. It is necessary that these molecules “work” properly, that is, they can transmit the information contained in them through the DNA-RNA-protein system. In addition, the cell needs to distribute genetic material during division.
The chromatin structure fully meets these tasks. The protein part contains all the necessary enzymes, and the structural features allow them to interact with certain sections of DNA. Therefore, the second important function of chromatin is to ensure all processes associated with the implementation of the nuclear genome.
The following fractions are distinguished in the eukaryotic genome.
1. Unique, i.e. sequences present in one copy or in a few copies. As a rule, these are cistrons - structural genes encoding proteins.
2. Low-frequency repetitions - sequences repeated dozens of times.
3. Intermediate, or mid-frequency, repetitions - sequences repeated hundreds and thousands of times. These include rRNA genes (in humans there are 200 per haploid set, in mice - 100, in cats - 1000, in fish and flowering plants - thousands), tRNA, genes for ribosomal proteins and histone proteins.
4. High-frequency repeats, the number of which reaches 10 million (per genome). The DNA of mice consists of 70% unique sequences, 20% of low- and medium-frequency repeats, and 10% of high-frequency repeats.
Repeats form so-called families, which are understood as a set of sequences that are completely or mostly homologous to each other.
Often, due to significant differences in the nucleotide composition of high-frequency repeats and the rest of the DNA, the former form, when centrifuged in a density gradient of cesium chloride, so-called satellite peaks, which have a higher or lower buoyant density than the rest of the DNA. This fraction of the genome is represented by a small (10...15) number of families of short (5...12 bp) repeats forming extended blocks. Within blocks, groups of repeats of individual families can alternate with each other, so that satellite DNA has a kind of patchwork structure. Hybridization of fractions of high-frequency sequences with DNA directly on chromosome preparations made it possible to establish that this fraction of the genome is localized in regions of constitutive heterochromatin, most often pericentromeric or telomeric. Back in the 30s, it was shown that genetically these areas are inert, that is, they do not contain genes. In reality, such small sequences that make up satellite DNA cannot encode anything other than oligopeptides. Moreover, heterochromatic regions are not transcribed. Thus, in the case of high-frequency DNA sequences, the identity of the molecular organization and genetic properties of eukaryotic chromosomal DNA is revealed. It should be noted that in the vast majority of species this fraction occupies no more than 10% of the genome. Close species, such as mouse and rat, have completely different high-frequency sequences; in the rat, their nucleotide composition does not differ from the main DNA, while the mouse genome contains a clear AT-rich satellite. This means that high-frequency DNA is capable of rapid changes during speciation.
The remaining 90% of the eukaryotic genome, its euchromatic part, is built on the principle of alternation (interpersion) of unique and repeating sequences. Conventionally, there are two main types of interpersions, named after the species in which they were first described: interpersions of the “xenopus” type (found in the clawed frog Xenopus laevis) and the Drosophila type (first described in the fruit fly D. melanogaster). Approximately 50% of the genome Xenopus laevis unique sequences of 800...1200 bp. alternate with repeating ones, the average size of which is 300 bp. In the rest of the genomes of the “xenopus” type, the distances between neighboring repeats significantly exceed 1...2 bp. The xenopus genome structure is widespread, especially among animals. Mammals and humans also belong to this type of genome organization. A feature of the genome of humans and other primates is interspersed high-frequency repeats about 300 bp long. In humans, these repeats contain a site cut by the restriction enzyme Alu I. The number of Alu-like repeats in the human genome reaches 5 × 10 5, and according to some data, even 10 6.
Alu-like sequences in primates are partial duplications (doublings) of the B1 sequence in the rodent genome, first described by G. P. Georgiev and his colleagues.
U D. melanogaster The parameters of interpersion differ sharply from species with the “xenopus” genome type: repeating sequences 5600 bp long. alternate with unique ones, the length of which is at least 13,000 bp.
It is interesting to note that house fly the genome is arranged according to the “xenopus” type. This fact directly indicates that during evolution, very rapid transformations in the nature of sequence alternation are possible in the euchromatic part of the genome. Birds, in terms of interference parameters, occupy an intermediate position between the “xenopus” type and the “Drosophila” type. As research results show recent years, many species of animals and plants cannot be strictly classified into either type based on genome organization. Thus, in the genomes of mammals there are long repeats - several thousand nucleotide pairs; in the genomes of lilies, up to 90% of the DNA can be represented by repeating sequences. For example, the pea genome does not contain unique sequences exceeding 300 bp in length.
Another feature of repeated sequences in eukaryotic genomes is inverted repeats, or palindromes (see below). Under renaturation conditions, they almost instantly form duplex structures. Essentially, palindromes are part of the intermediate repeats. However, some high-frequency repeats in the euchromatic part of the genome, for example members of the Alu families, can occur in both direct and inverted positions. Sometimes other sequences are inserted between the inverted repeats.