Chromatin: definition, structure and role in cell division. Three fractions of eukaryotic DNA, their localization in chromosomes and functions Structural and functional components of chromatin

Chromatin, the main component of the cell nucleus, is fairly 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. In this case, sections of chromatin swell and turn into a gel. In order to convert such drugs into real solutions, strong mechanical influences are required: shaking, stirring, additional homogenization. This, of course, leads to partial destruction of the original chromatin structure, crushing it into small fragments, but practically does not change its chemical composition.

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 and mitotic chromosomes differs little from each other. The main components of chromatin are DNA and proteins, the bulk of which are histones and non-histone proteins (see Table 3).

Table 3. Chemical composition of chromatin. Protein and RNA contents are given relative to DNA

On average, about 40% of chromatin is DNA and about 60% is proteins, including specific nuclear proteins - histones, make up from 40 to 80% of all proteins that make up the isolated chromatin. In addition, the chromatin fraction includes 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, for example, RNA may be transcribed RNA that has not yet lost its connection with the DNA template. Other minor components may represent substances from coprecipitated fragments of the nuclear membrane.

Structurally, chromatin is a filamentous complex of deoxyribonucleoprotein (DNP) molecules, which consist of DNA associated with histones (see Fig. 57). Therefore, another name for chromatin has taken root - nucleohistone. It is due to the association of histones with DNA that very labile, variable nucleic acid-histone complexes are formed, where the DNA:histone ratio is approximately one, i.e. they are present in equal weight quantities. These filamentous DNP fibrils are elementary chromosomal or chromatin filaments, the thickness of which, depending on the degree of DNA packaging, can range from 10 to 30 nm. These DNP fibrils can, in turn, be further compacted to form higher levels of DNP structuring, up to the mitotic chromosome. The role of some non-histone proteins is precisely in the formation of high levels of chromatin compaction.

DNA chromatin

In a chromatin preparation, DNA usually accounts for 30-40%. This DNA is a double-stranded helical molecule, similar to pure isolated DNA in aqueous solutions. This is evidenced by many experimental data. Thus, when chromatin solutions are heated, an increase in the optical density of the solution is observed, the so-called hyperchromic effect associated with the breaking of internucleotide hydrogen bonds between DNA chains, similar to what happens when pure DNA is heated (melted).

The question of the size and length of DNA molecules in chromatin is important for understanding the structure of the chromosome as a whole. Using standard DNA isolation methods, chromatin has a molecular weight of 7-9 x 10 6, which is significantly less than the molecular weight of DNA from Escherichia coli (2.8 x 10 9). Such a relatively low molecular weight of DNA from chromatin preparations can be explained by mechanical damage to DNA during the process of chromatin isolation. If DNA is isolated under conditions that exclude shaking, homogenization and other influences, it is possible to obtain very long DNA molecules from cells. The length of DNA molecules from the nuclei and chromosomes of eukaryotic cells can be studied using the light-optical autoradiography method, just as it was studied on prokaryotic cells.

It was discovered that within chromosomes the length of individual linear (unlike prokaryotic chromosomes) DNA molecules can reach hundreds of micrometers and even several centimeters. Thus, DNA molecules ranging from 0.5 mm to 2 cm were obtained from different objects. These results showed that there is a close agreement between the calculated length of DNA per chromosome and autoradiographic observation.

After mild lysis of eukaryotic cells, the molecular weights of DNA can be directly determined by physicochemical methods. It has been shown that the maximum molecular weight of a Drosophila DNA molecule is 41 x 10 9, which corresponds to a length of about 2 cm. In some yeasts, there is a DNA molecule per chromosome with a molecular weight of 1 x 10 8 -10 9, which measures about 0.5 mm .

Such long DNA is a single molecule, and not several shorter ones, stitched together in single file using protein bonds, as some researchers believed. This conclusion was reached after it turned out that the length of DNA molecules does not change after treatment of drugs with proteolytic enzymes.

The total amount of DNA included in the nuclear structures of cells, in the genome of organisms, varies from species to species, although in microorganisms the amount of DNA per cell is significantly lower than in invertebrates, higher plants and animals. Thus, a mouse has almost 600 times more DNA per nucleus than E. coli. When comparing the amount of DNA per cell in eukaryotic organisms, it is difficult to discern any correlation between the degree of complexity of the organism and the amount of DNA per nucleus. Such different organisms as flax, sea urchin, perch (1.4-1.9 pg) or char and bullfish (6.4 and 7 pg) have approximately the same amount of DNA.

There are significant fluctuations in the amount of DNA in large taxonomic groups. Among higher plants, the amount of DNA in different species can differ hundreds of times, just as among fish, the amount of DNA in amphibians differs by tens of times.

Some amphibians have 10-30 times more DNA in their nuclei than in human nuclei, although the genetic constitution of humans is incomparably more complex than that of frogs. Therefore, it can be assumed that the “excess” amount of DNA in lower organized organisms is either not associated with the fulfillment of a genetic role, or the number of genes is repeated one or another number of times.

Table 4. DNA content in the cells of some objects (pg, 10 -12 g)

It turned out to be possible to resolve these issues by studying the kinetics of the reaction of renaturation or DNA hybridization. If fragmented DNA molecules in solutions are subjected to thermal denaturation and then incubated at a temperature slightly lower than that at which denaturation occurs, then the original double-stranded structure of DNA fragments is restored due to the reunification of complementary chains - renaturation. For DNA viruses and prokaryotic cells, it was shown that the rate of such renaturation directly depends on the size of the genome; the larger the genome, the greater the amount of DNA per particle or cell, the more time is needed for the random approach of complementary chains and the specific reassociation of a larger number of DNA fragments different in nucleotide sequence (Fig. 53). The nature of the DNA reassociation curve of prokaryotic cells indicates the absence of repeated base sequences in the prokaryotic genome; all sections of their DNA carry unique sequences, the number and diversity of which reflect the degree of complexity of the genetic composition of the objects and, consequently, their general biological organization.

A completely different picture of DNA reassociation is observed in eukaryotic organisms. It turned out that their DNA contains fractions that renature at a much higher rate than would be expected based on the size of their genome, as well as a fraction of DNA that renatures slowly, like the unique DNA sequences of prokaryotes. However, eukaryotes require significantly more time to renature this fraction, which is associated with the overall large size of their genome and the large number of different unique genes.

In that part of eukaryotic DNA that is characterized by a high rate of renaturation, two subfractions are distinguished: 1) a fraction with highly or frequently repeated sequences, where similar DNA sections can be repeated 10 6 times; 2) a fraction of moderately repetitive sequences that occur 10 2 -10 3 times in the genome. Thus, in mice, the fraction of DNA with frequently repeated sequences includes 10% of the total amount of DNA per genome and 15% is accounted for by the fraction with moderately repeated sequences. The remaining 75% of all mouse DNA is represented by unique regions corresponding to a large number of different non-repeating genes.

Fractions with highly repeated sequences may have a different buoyant density than the bulk of DNA and can therefore be isolated in pure form as so-called fractions satellite DNA. In the mouse, this fraction has a density of 1.691 g/ml, and the main part of the DNA is 1.700 g/ml. These density differences are determined by differences in nucleotide composition. For example, in a mouse there are 35% G and C pairs in this fraction, and 42% in the main DNA peak.

As it turned out, satellite DNA, or the fraction of DNA with frequently repeated sequences, is not involved in the synthesis of the main types of RNA in the cell and is not associated with the process of protein synthesis. This conclusion was made based on the fact that none of the cell RNA types (tRNA, mRNA, rRNA) hybridizes with satellite DNA. Consequently, these DNAs do not contain sequences responsible for the synthesis of cellular RNA, i.e. satellite DNAs are not templates for RNA synthesis and are not involved in transcription.

There is a hypothesis that highly repetitive sequences that are not directly involved in protein synthesis may carry information that plays an important structural role in the maintenance and functioning of chromosomes. These may include numerous sections of DNA associated with the core proteins of the interphase nucleus (see below), sites at the origin of replication or transcription, as well as sections of DNA that regulate these processes.

Using the method of hybridization of nucleic acids directly on chromosomes ( in situ) the localization of this fraction was studied. To do this, RNA labeled with 3H-uridine was synthesized on isolated satellite DNA using bacterial enzymes. Then the cytological preparation with chromosomes was subjected to such treatment that DNA denaturation occurs (elevated temperature, alkaline environment, etc.). After this, 3H-labeled RNA was placed on the preparation and hybridization between DNA and RNA was achieved. Autoradiography revealed that most of the label is localized in the zone of primary constrictions of chromosomes, in the zone of their centromeric regions. The mark was also detected in other regions of the chromosomes, but very weakly (Fig. 54).

Over the past 10 years, great strides have been made in studying centromeric DNA, especially in yeast cells. So do S. cerevisiae Centromeric DNA consists of repeating regions of 110 bp. It consists of two conserved regions (I and III) and a central element (II), enriched in AT base pairs. Drosophila chromosomes have a similar centromere DNA structure. Human centromeric DNA (alphoid satellite DNA) consists of a tandem of 170 bp monomers organized into groups of dimers or pentamers, which in turn form large sequences of 1-6 x 10 3 bp. This largest unit is repeated 100-1000 times. Special centromeric proteins are complexed with this specific centromeric DNA and are involved in the formation kinetochore, a structure that ensures the connection of chromosomes with spindle microtubules and in the movement of chromosomes in anaphase (see below).

DNA with highly repetitive sequences has also been found in telomeric regions chromosomes of many eukaryotic organisms (from yeast to humans). Repeats are most often found here, which include 3-4 guanine nucleotides. In humans, telomeres contain 500-3000 TTAGGG repeats. These sections of DNA perform a special role - to limit the ends of the chromosome and prevent its shortening during the process of repeated replication.

It was recently found that highly repetitive DNA sequences of interphase chromosomes bind specifically to lamin proteins underlying the nuclear envelope and participate in the anchoring of extended decondensed interphase chromosomes, thereby determining the order in the localization of chromosomes in the volume of the interphase nucleus.

It has been suggested that satellite DNA may be involved in the recognition of homologous regions of chromosomes during meiosis. According to other assumptions, regions with frequently repeated sequences play the role of separators (spacers) between various functional units of chromosomal DNA, for example, between replicons (see below).

As it turned out, the fraction of moderately repeating (from 10 2 to 10 5 times) sequences belongs to a variegated class of DNA regions that play an important role in the processes of creating the protein synthesis apparatus. This fraction includes ribosomal DNA genes, which can be repeated 100 to 1000 times in different species. This fraction includes many times repeated regions for the synthesis of all tRNAs. Moreover, some structural genes responsible for the synthesis of certain proteins can also be repeated many times, represented by many copies. These are the genes for chromatin proteins - histones, repeated up to 400 times.

In addition, this fraction includes DNA sections with different sequences (100-400 nucleotide pairs each), also repeated many times, but scattered throughout the genome. Their role is not yet completely clear. It has been suggested that such DNA sections may represent acceptor or regulatory regions of different genes.

So, the DNA of eukaryotic cells is heterogeneous in composition, containing several classes of nucleotide sequences: frequently repeated sequences (> 10 6 times), included in the satellite DNA fraction and not transcribed; a fraction of moderately repetitive sequences (10 2 -10 5), 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.

Based on these ideas, the differences in the amount of DNA that are observed in different organisms become clear: they may be associated with an unequal proportion of certain classes of DNA in the genome of organisms. So, for example, in an amphibian Amphiuma(which has 20 times more DNA than humans) repeating sequences account for up to 80% of the total DNA, in onions - up to 70, in salmon - up to 60%, etc. The true wealth of genetic information should be reflected by the fraction of unique sequences. We must not forget that in a native, non-fragmented DNA molecule of the chromosome, all regions that include unique, moderately and frequently repeated sequences are linked into a single giant covalent DNA chain.

DNA molecules are heterogeneous not only in areas of different nucleotide sequences, but also differ in their synthetic activity.

Eukaryotic DNA replication

The bacterial chromosome replicates as one structural unit, having one replication start point and one termination point. Thus bacterial circular DNA is one replicon. From the starting point, replication proceeds in two opposite directions, so that as DNA is synthesized, a so-called replication eye is formed, bounded on both sides by replication forks, which is clearly visible during electron microscopic examination of viral and bacterial replicating chromosomes.

In eukaryotic cells, the replication organization is of a different nature - polyreplicon. As already mentioned, with the pulsed inclusion of 3 HT, a multiple label appears in almost all mitotic chromosomes. This means that simultaneously there are many replication sites and many autonomous replication origins in the interphase chromosome. This phenomenon was studied in more detail using autoradiography of labeled molecules isolated from DNA (Fig. 55). If the cells were pulse-labeled with 3 HT, then in a light microscope on the autographs of isolated DNA one can see areas of reduced silver in the form of dotted lines. These are small stretches of DNA that have managed to replicate, and between them there are sections of unreplicated DNA that did not leave an autoradiograph and therefore remains invisible. As the time of contact of 3 NT with the cell increases, the size of such segments increases, and the distance between them decreases. From these experiments, the rate of DNA replication in eukaryotic organisms can be accurately calculated. The speed of movement of the replication fork turned out to be 1-3 kb. per minute in mammals, about 1 kb. per minute in some plants, which is much lower than the rate of DNA replication in bacteria (50 kb per minute). In the same experiments, the polyreplicon structure of the DNA of eukaryotic chromosomes was directly proven: along the length of the chromosomal DNA, along it, there are many independent replication sites - replicons. According to the distance between the midpoints of adjacent tagging replicons, i.e. Based on the distance between two adjacent replication starting points, the size of individual replicons can be determined. On average, the replicon size of higher animals is about 30 µm or 100 kb. Therefore, there should be 20,000-30,000 replicons in the haploid set of mammals. In lower eukaryotes, the replicons are smaller, about 40 kb. Thus, in Drosophila there are 3500 replicons per genome, and in yeast – 400. As mentioned, DNA synthesis in a replicon occurs in two opposite directions. This can be easily proven by autoradiography: if cells, after a pulse label, are allowed to continue synthesizing DNA for some time in a medium without 3 HT, then its inclusion in DNA will decrease, a dilution of the label will occur, and on the autoradiograph it will be possible to see a symmetrical pattern on both sides of the replicated region , reducing the number of grains of reduced silver.

The replicating ends or forks in a replicon stop moving when they meet the forks of neighboring replicons (at a terminal point common to neighboring replicons). At this point, replicated sections of neighboring replicons are combined into single covalent chains of two newly synthesized DNA molecules. The functional division of chromosome DNA into replicons coincides with the structural division of DNA into domains or loops, the bases of which, as already mentioned, are held together by protein bonds.

Thus, all DNA synthesis on a single chromosome occurs through independent synthesis on many individual replicons, followed by joining the ends of adjacent DNA segments. The biological meaning of this property becomes clear when comparing DNA synthesis in bacteria and eukaryotes. Thus, a bacterial monoreplicon chromosome with a length of 1600 microns is synthesized at a speed of about half an hour. If a centimeter-long DNA molecule of a mammalian chromosome were also replicated as a monoreplicon structure, it would take about a week (6 days). But if such a chromosome contains several hundred replicons, then its complete replication will take only about an hour. In fact, DNA replication time in mammals is 6-8 hours. This is due to the fact that not all replicons of an individual chromosome are turned on at the same time.

In some cases, the simultaneous inclusion of all replicons or the appearance of additional replication origins is observed, which makes it possible to complete the synthesis of all chromosomes in a minimally short time. This phenomenon occurs early in the embryogenesis of some animals. It is known that when crushing the eggs of clawed frogs Xenopus laevis DNA synthesis takes only 20 minutes, whereas in somatic cell culture this process lasts about a day. A similar picture is observed in Drosophila: in the early embryonic stages, the entire DNA synthesis in the nucleus takes 3.5 minutes, and in tissue culture cells – 600 minutes. At the same time, the size of replicons in culture cells turned out to be almost 5 times greater than in embryos.

DNA synthesis occurs unevenly along the length of an individual chromosome. It was found that in an individual chromosome, active replicons are assembled into groups, replicative units, which include 20-80 origins of replication. This followed from the analysis of DNA autographs, where exactly such blocking of replicating segments was observed. Another basis for the idea of the existence of blocks or clusters of replicons or replication units were experiments with the inclusion of a thymidine analogue, 5'-bromodeoxyuridine (BrdU), into DNA. The inclusion of BrdU in interphase chromatin leads to the fact that during mitosis, areas with BrdU condense to a lesser extent (insufficient condensation) than those areas where thymidine was included. Therefore, those regions of mitotic chromosomes in which BrdU is included will be weakly stained during differential staining. This makes it possible to determine the sequence of BrdU incorporation using synchronized cell cultures, i.e. sequence of DNA synthesis along the length of one chromosome. It turned out that the inclusion of the precursor in large sections of the chromosome occurs. The inclusion of different sections occurs strictly sequentially during the S-period. Each chromosome is characterized by high stability of the replication order along its length and has its own specific replication pattern.

Clusters of replicons, combined into replication units, are associated with nuclear matrix proteins (see below), which, together with replication enzymes, form the so-called. clusterosomes are zones in the interphase nucleus in which DNA synthesis occurs.

The order in which replication units are activated may likely be determined by the chromatin structure at these regions. For example, zones of constitutive heterochromatin (near the centromere) are usually replicated at the end of the S-period; also, at the end of the S-period, part of the facultative heterochromatin doubles (for example, the X chromosome of female mammals). The sequence of replication of chromosome sections correlates especially clearly in time with the pattern of differential coloring of chromosomes: R-segments belong to early-replicating segments, G-segments correspond to chromosome sections with late replication. C-segments (centromeres) are the sites of latest replication.

Since in different chromosomes the size and number of different groups of differentially colored segments are different, this creates a picture of the asynchronous beginning and end of replication of different chromosomes as a whole. In any case, the sequence of the beginning and end of replication of individual chromosomes in the set is not random. There is a strict sequence of chromosome reproduction relative to the other chromosomes in the set.

The duration of the replication process of individual chromosomes does not directly depend on their size. Thus, large human chromosomes of group A (1-3) are labeled throughout the entire S-period, as well as shorter chromosomes of group B (4-5).

Thus, DNA synthesis in the eukaryotic genome begins almost simultaneously on all chromosomes of the nucleus at the beginning of the S-period. But at the same time, sequential and asynchronous inclusion of different replicons occurs both in different parts of the chromosomes and in different chromosomes. The replication sequence of a particular genome region is strictly determined genetically. This last statement is proven not only by the pattern of inclusion of the label in different segments of the S-period, but also by the fact that there is a strict sequence of appearance of peaks in the sensitivity of certain genes to mutagens during the S-period.

1. Types of chromatin

2. Genes, spacers

3. Sequence of nucleotides in DNA

4. Spatial organization of DNA

1. During rest between acts of division, certain sections of chromosomes and entire chromosomes remain compact. These regions of chromatin are called heterochromatin. It paints well.

After nuclear division, chromatin loosens and in this form is called euchromatin. Heterochromatin is inactive in relation to transcription, and in relation to DNA replication it behaves differently than euchromatin.

Facultative heterochromatin is heterochromatic only at times. It is informative, i.e. it contains genes. When it enters the euchromatic state, these genes may become available for transcription. Of two homologous chromosomes, one may be heterochromatic. This facultative heterochromatization is tissue specific and does not occur in certain tissues.

Constitutive heterochromatin always heterochromatic. It consists of repeatedly repeated sequences of bases, is uninformative (does not contain genes) and is therefore always inactive in relation to transcription. You can see him And during nuclear fission. He's dating:

Most often at the centromere;

At the ends of chromosomes (including satellites);

Near the organizer of the nucleolus;

Near the 5S-RNA gene.

Heterochromatin, primarily facultative, during interphase can unite into an intensely stained chromocenter, which is in most cases located at the edge of the cell nucleus or nucleolus.

2. Each chromosome is continuous double helix of DNA, which in higher organisms consists of more than 10 8 base pairs. In the chromosomes of higher plants and animals, each DNA double helix (2 nm in diameter) has a length of one to several centimeters. As a result of repeated twisting, it is packaged into a chromatid several micrometers long.

Genes are linearly distributed along this double helix, which together make up up to 25% of the DNA.

Geneis the functional unit of DNA, containing information for the synthesis of a polypeptide or RNA. The average gene length is about 1000 base pairs. The sequence of bases in each gene is unique.

Between the genes are spacers- uninformative DNA stretches of varying lengths (sometimes more than 20,000 base pairs), which are important for regulating the transcription of a neighboring gene.

Transcribed spacers are terminated during transcription along with the gene, and their complementary copies appear in pre-i-RNA on either side of the gene copy. Even within the gene itself there are (only in eukaryotes and their viruses) non-informative sequences, the so-called introns, which are also transcribed. During processing, all copies of introns and most copies of spacers are excised by enzymes.

Non-transcriptable spacers occur between genes for histones, as well as between genes for rRNA.

Redundant genes are represented by a large number (up to 10 4 or more) identical copies. This is genes:

For tRNA;

5S-RNA and histones;

For products synthesized in large quantities.

The copies are located directly next to each other and are resolved by identical spacers. In the sea urchin, the genes for histones H4, H2b, H2a and Hi lie one after the other, and this gene sequence is repeated in DNA more than 100 times.

3. Repeating sequences - These are sequences of nucleotides present multiple times in DNA. Moderately repetitive sequences - sequences with an average length of 300 base pairs with 10 2 -10 4 repetitions. These include redundant genes, as well as most spacers.

Highly repetitive sequences with 10 5 -10 6 repetitions form constitutive heterochromatin. They always uninformative. These are mostly short sequences, most often 7-10 are found in them and only rarely - only 2 (for example, AT) or, conversely, over 300 nucleotide pairs. They cluster together, with one repeating sequence immediately following the other. Highly repetitive chromatin DNAs are called “satellite DNAs” because of their behavior during analytical fractionation procedures. About 75% of all chromatin is not involved in transcription: these are highly repetitive sequences and non-transcriptable spacers.

4. In isolated chromatin sections of the DNA double helix wrap around histone molecules, so that a first-order superhelix appears here. Complexes of DNA with histone are called nucleosomes. They have the shape of a disk or lens and dimensions are about 10 x 10 x 5 nm. One nucleosome included:

8 molecules histones:

Central tetramer of two H3 and two H4 molecules; and separately two H 2a and H 2 b;

A section of DNA (about 140 base pairs) that forms approximately 1.25 turns of a helix and is tightly bound to the central tetramer.

Between the nucleosomes there are sections of a helix of 30-100 base pairs without a superhelical structure; Histone binds here Hi

In stitched chromatin The DNA is further shortened by a little-understood further coiling (higher-order supercoil) that is apparently fixed by histone Hi (and some non-histone proteins). During the transition to interphase, euchromatin loosens as some of the higher order supercoils unwind. This probably occurs as a result of conformational changes in histones and weakening of interactions between Hi molecules. Chromatin structures 10-25 nm thick (main chromatin threads or helices) are also visible during interphase.

1. Types of chromatin

2. Genes, spacers

3. Sequence of nucleotides in DNA

4. Spatial organization of DNA

1. During rest between acts of division, certain sections of chromosomes and entire chromosomes remain compact. These regions of chromatin are called heterochromatin. It paints well.

Most often at the centromere;

At the ends of chromosomes (including satellites);

Near the organizer of the nucleolus;

Near the 5S-RNA gene.

Heterochromatin, primarily facultative, during interphase can unite into an intensely stained chromocenter, which is in most cases located at the edge of the cell nucleus or nucleolus.

Genes are linearly distributed along this double helix, which together make up up to 25% of the DNA.

Between the genes are spacers- uninformative DNA stretches of varying lengths (sometimes more than 20,000 base pairs), which are important for regulating the transcription of a neighboring gene.

Non-transcriptable spacers occur between genes for histones, as well as between genes for rRNA.

Redundant genes are represented by a large number (up to 10 4 or more) identical copies. This is genes:

For tRNA;

5S-RNA and histones;

For products synthesized in large quantities.

8 molecules histones:

Central tetramer of two H3 and two H4 molecules; and separately two H 2a and H 2 b;

A section of DNA (about 140 base pairs) that forms approximately 1.25 turns of a helix and is tightly bound to the central tetramer.

Between the nucleosomes there are sections of a helix of 30-100 base pairs without a superhelical structure; Histone binds here Hi

Transcriptionally active chromatin - genes that transmit their information through synthesis RNA, as a result of further despiralization, it loosens even more. According to some data, in the corresponding sections of the DNA helix, histone Hi is either absent or chemically altered, for example, phosphorylated.

Nucleosome structure also changes or is completely destroyed (in genes for r-RNA in the nucleolus). The double helix unwinds in certain places. These processes apparently involve certain non-histone proteins that accumulate in transcribed regions of DNA.

Question 38. Set of chromosomes

/. Genome. Cell ploidy

2. Polytene chromosomes

1. The entire fund of genetic information of each cell nucleus - genome- distributed among a certain constant number of chromosomes (n). This number is specific to each species or subspecies. In horse roundworm it is 1, in corn - 10, in humans - 23, in algae Netrium digitus - about 600. Chromosomes of the same set are different according to the following criteria:

size;

Picture of a chromometer;

The position of the constrictions;

Depending on the multiplicity of chromosome set - ploidy- cells divide:

To haploid;

Diploid;

Polyploid.

Haploid are called cells that contain a single set of chromosomes (“), for example, sex cells.

If cells contain a double set of chromosomes (2 P), they diploid, since genetic information is presented twice in them. Almost all somatic cells of higher plants and animals are diploid. They contain one paternal and one maternal set of chromosomes.

IN polyploid cells have several sets of chromosomes (4 P, 8 P, 16 P, etc.). These cells are often particularly metabolically active, such as many liver cells in mammals.

Haploid cells are formed from diploid cells as a result of meiosis, and diploid cells are formed from haploid cells as a result of fertilization.

Polyploid cells arise from diploid ones through endomitosis - prematurely interrupted nuclear division: after complete replication and separation of chromatids, the daughter chromosomes remain in one cell nucleus, instead of being distributed between two nuclei. This process can be repeated many times.

Anomalies during the formation of germ cells can lead to polyploidy of the entire organism. At incomplete replication Some parts of the genome, such as heterochromatin, do not replicate and remain diploid after endomitosis, in contrast to other parts that become polyploid.

Gene amplification - this is multiple super-replication when only certain genes are replicated and become polyploid (genes for rRNA in the nucleolus).

Chromosomes diploid nucleus can be grouped in pairs, two homologous chromosomes. Most of them (the so-called autosomes) pairwise identical. Only two sex chromosomes that determine the sex of an individual are not the same in males - these are the X and Y chromosomes (heterochromosomes). Most of the Y chromosome is occupied by constitutive heterochromatin. Females have two X chromosomes. However, in butterflies, birds and a number of other animals the situation is the other way around: males have the XX set, females have the XY set.

2. Polytene chromosomes(giant chromosomes) contain many times more DNA than normal ones. They do not change their shape throughout the division cycle and reach a length of up to 0.5 mm and a thickness of 25 microns. They are found, for example, in the salivary glands of dipterans (flies and mosquitoes), in the macronucleus of ciliates and in the ovary tissues of beans. Most often they are visible in the haploid number, since homologous chromosomes are closely paired. Polythenia occurs as a result of endoreplication. Compared to endomitosis, this is an even more reduced division process - after replication, the chromatids are not separated (the process is repeated many times). Wherein different stretches of DNA are multiplied to varying degrees:

Centromere areas - insignificant;

Most informative areas are approximately 1000 times;

Some - more than 30,000 times.

That's why polytene chromosomes They are bundles of countless chromatids that are not completely separated. Chromatids are stretched, homologous chromomeres form dark disks closely located along the chromosome. These discs are separated by lighter stripes. Probably, on the chromatid, one disk and one intermediate stripe form, in addition to the spacer, one gene (less often several genes), which, apparently, is located in the disk. Polytene chromosomes are extremely poor in heterochromatin.

On polytene chromosomes separate disks sometimes swell into poufs(Balbiani rings). There, homologous chromatids separate from each other, homologous chromomeres move apart, and a loose structure of transcriptionally active chromatin appears. Puffs contain less histone Hi than discs and instead contain the enzyme RNA polymerase (which indicates RNA synthesis). There is also little histone Hi in the intermediate bands, but there is RNA polymerase and, possibly, at least minor synthesis occurs RNA.

In a chromatin preparation, DNA usually accounts for 30-40%. This DNA is a double-stranded helical molecule. Chromatin DNA has a molecular weight of 7-9*10 6 . Such a relatively small mass of DNA from the preparations can be explained by mechanical damage to the DNA during the process of chromatin isolation.

The total amount of DNA included in the nuclear structures of cells, in the genome of organisms, varies from species to species. When comparing the amount of DNA per cell in eukaryotic organisms, it is difficult to discern any correlation between the degree of complexity of the organism and the amount of DNA per nucleus. Different organisms, such as flax, sea urchin, perch (1.4-1.9 pg) or char and bullfish (6.4 and 7 pg), have approximately the same amount of DNA.

Some amphibians have 10-30 times more DNA in their nuclei than in human nuclei, although the genetic constitution of humans is incomparably more complex than that of frogs. Consequently, it can be assumed that the “excess” amount of DNA in lower organized organisms is either not associated with the fulfillment of a genetic role, or the number of genes is repeated one or another number of times.

Satellite DNA, or the fraction of DNA with frequently repeated sequences, may be involved in the recognition of homologous regions of chromosomes during meiosis. According to other assumptions, these regions play the role of separators (spacers) between various functional units of chromosomal DNA.

As it turned out, the fraction of moderately repeating (from 10 2 to 10 5 times) sequences belongs to a variegated class of DNA regions that play an important role in metabolic processes. This fraction includes ribosomal DNA genes, repeatedly repeated sections for the synthesis of all tRNAs. Moreover, some structural genes responsible for the synthesis of certain proteins can also be repeated many times, represented by many copies (genes for chromatin proteins - histones).

So, the DNA of eukaryotic cells is heterogeneous in composition and contains several classes of nucleotide sequences:

frequently repeated sequences (>10 6 times), included in the satellite DNA fraction and not transcribed;

a fraction of moderately repetitive sequences (10 2 -10 5), 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.

The DNA of a prokaryotic organism is one giant cyclic molecule. The DNA of eukaryotic chromosomes is linear molecules consisting of replicons of different sizes arranged in tandem (one after another). The average replicon size is about 30 microns. Thus, the human genome should contain more than 50,000 replicons, DNA sections that are synthesized as independent units. These replicons have a starting point and a terminal point for DNA synthesis.

Let's imagine that in eukaryotic cells, each of the chromosomal DNA, like in bacteria, is one replicon. In this case, at a synthesis rate of 0.5 microns per minute (for humans), the reduplication of the first chromosome with a DNA length of about 7 cm should take 140,000 minutes, or about three months. In fact, due to the polyreplicon structure of DNA molecules, the entire process takes 7-12 hours.

Removal of histone H1 from transcriptionally active chromatin 1*2* . Early experiments by 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 sample of a transcriptionally inactive insertion into the rDNA gene (inactive). Three diagonals are visible in the inactive gene; 1 - free DNA, 2 - DNA complexes with octamer histones; 3 - DNA complexes With histone H1. The weakening or disappearance of DNA-histone complexes upon chromatin activation 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 somewhat 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 an original method for 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 of the above results are not direct, and therefore future experiments should provide a final solution to the question of the fate of nucleosomes during transcription.

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. Alfrey(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 normal 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 actually managed to isolate a new class of nuclear proteins, which he called the “high mobility group of proteins” ( 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. It can vary in nuclei from different types of cells. 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 activation mechanism 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. In terms of 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*. Another important feature of 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 methylation sensitivities. 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, recently 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.