Functions of the centromere. Methods of dividing somatic and germ cells

By the middle of the last century, numerous cytological studies showed the decisive role of the centromere in the morphology of chromosomes. It was later discovered that the centromere, together with the kinetochore (a structure consisting mainly of proteins), is responsible for the correct segregation of chromosomes into daughter cells during cell division. The guiding role of the centromere in this process is obvious: after all, it is to it that the division spindle is attached, which, together with the cell centers (poles), constitutes the cell division apparatus. Due to the contraction of the spindle strands, chromosomes move toward the cell poles during division.

Five stages of cell division (mitosis) are usually described. For simplicity, we will focus on three main stages in the behavior of the chromosomes of a dividing cell (Fig. 2). At the first stage, gradual linear compression and thickening of chromosomes occurs, then a cell division spindle consisting of microtubules is formed. In the second, the chromosomes gradually move toward the center of the nucleus and line up along the equator, probably to facilitate the attachment of microtubules to the centromeres. In this case, the nuclear membrane disappears. At the last stage, the halves of the chromosomes - chromatids - separate. It seems that microtubules attached to the centromeres, like a tugboat, pull the chromatids towards the poles of the cell. From the moment of divergence, the former sister chromatids are called daughter chromosomes. They reach the spindle poles and come together in a parallel pattern. The nuclear envelope is formed.

Rice. 2. The main stages of mitosis.
From left to right: chromosome compaction, spindle formation; alignment of chromosomes along the equator of the cell,
attachment of the spindle to the centromeres; movement of chromatids to the poles of the cell.

With careful observation, one can notice that during the process of cell division in each chromosome, the centromere is in a constant position. It maintains a close dynamic connection with the cell center (pole). Centromere division occurs simultaneously in all chromosomes.

Sequencing methods developed in recent years have made it possible to determine the primary DNA structure of extended sections of human and fruit fly centromeres Drosophila and plants Arabidopsis. It turned out that in the chromosomes of both humans and plants, centromeric activity is associated with a block of tandemly organized DNA repeats (monomers) that are similar in size (170-180 nucleotide pairs, bp). Such sections are called satellite DNA. In many species, including those that are evolutionarily distant from each other, the size of the monomers is almost the same: various species of monkeys - 171 np, corn - 180 np, rice - 168 np, chironomus insect - 155 np. This may reflect general requirements for centromeric function.

Despite the fact that the tertiary structure of human and Arabidopsis centromeres is organized similarly, the primary nucleotide sequences (or nucleotide order) in their monomers turned out to be completely different (Fig. 3). This is surprising for a region of the chromosome that performs such an important and universal function. However, when analyzing the molecular organization of centromeres in Drosophila, a certain structural pattern was discovered, namely the presence of sections of monomers of approximately the same size. Thus, in Drosophila, the centromere of the X chromosome consists mainly of two types of very short simple repeats (AATAT and AAGAG), interrupted by retrotransposons (mobile DNA elements) and “islands” of more complex DNA. All these elements were found in the Drosophila genome and outside the centromeres, but DNA sequences characteristic of each centromere were not found in them. This means that centromeric DNA sequences themselves are insufficient and unnecessary for the formation of a centromere.

Rice. 3. DNA structure in human and plant centromeres.

The rectangles correspond to tandemly organized monomers with identical nucleotide sequences inside (primary DNA structure). In different species, the primary structure of DNA monomers varies, and the secondary structure is a helix. The sequence of monomers reflects the higher level structural organization of DNA.

This assumption is also confirmed by the manifestation of centromeric activity outside normal centromeres. Such neocentromeres behave like normal centromeres: they form a cytologically distinguishable constriction and form kinetochores that bind proteins. However, DNA analysis of two human neocentromeres and a conventional centromere did not reveal common sequences, which indicates the possible role of other structural components of the chromosome. They can be histone and non-histone proteins that bind to DNA, forming the nucleosome structure of chromatin.

The functional role of the centromeric chromatin structure is confirmed by the presence of histone H3 variants specific for each biological species in centromeric chromatin: in humans they are called CENP-A, in plants - CENH3. Among the many proteins present in the kinetochore, only two, CENH3 and centromeric protein C (CENP-C), directly bind to DNA. Perhaps it is CENH3, interacting with other histones (H2A, H2B and H4), that forms and determines the type of nucleosomes specific to centromeres. Such nucleosomes can serve as a kind of anchors for kinetochore formation. Variants of histone H3 in centromeres of various species are similar to the canonical histone H3 molecule in areas of interaction with other histone proteins (H2A, H2B, H4). However, the region of centromeric histone H3 that interacts with the DNA molecule appears to be under the influence of driving selection. As discussed, the primary structure of centromeric DNA differs between species, and centromeric histone H3 has been proposed to coevolve with centromeric DNA, particularly in Drosophila and Arabidopsis.

The discovery of the centromeric histone H3 gave rise to the extreme view that centromeric function and its complete independence from the primary DNA structure are determined by the nucleosomal organization and this histone. But are these factors sufficient for full centromere activity? Models that ignore the role of primary DNA structure must assume a random distribution of changes in centromeric DNA structure across populations in the absence of selection. However, analysis of satellite DNA in human centromeres and Arabidopsis identified conserved regions as well as regions with higher than average variability, indicating selection pressure on centromeric DNA. In addition, artificial centromeres were obtained only with human a-satellite repeats amplified from natural centromeres, but not from a-satellites of pericentromeric chromosome regions.

Models in which the decisive factor in determining the position of the centromere (preserved from generation to generation) and its functions is the tertiary (or even higher order) structure of DNA are encountered with fewer fundamental difficulties for explanation. Its conservatism allows for large variations in the nucleotide sequence and does not exclude fine tuning of the primary structure.

Henikoff and colleagues proposed a model that describes the coordinated evolution of DNA and proteins and leads to the appearance of optimally functioning centromeres using the example of female germ cell division. As is known, in the process of meiosis, one parent cell gives rise to four daughter cells through two successive divisions. Subsequently, only one of them turns into a mature female reproductive cell (gamete), transmitting genetic information to the next generation, while the other three cells die. According to this model, in the process of evolution, due to mutations and other mechanisms in chromosomes, centromeres with longer strands of satellite DNA monomers or with a primary nucleotide structure that is more conducive to binding and coordinated work with specific forms of histones CENH3 and CENP-C can arise. Moreover, in some organisms (Arabidopsis, Drosophila), evidence for positive selection pressure was obtained for CENH3, while for other species (cereals, mammals) for CENP-C (Fig. 4a). As a result, such centromeres with improved kinetochores become “stronger” and can attach a larger number of spindle microtubules (Fig. 4b). If there are more such “strong” centromeres in the gametes, then a process of meiotic drive occurs, which increases the number of such centromeres, and a new variant is fixed in the population.

Rice. 4. Model explaining the evolution of centromeres.

Top - centromeres (gray ovals) contain a specialized set of proteins (kinetochores), including histones CENH3 (H) and CENP-C (C), which in turn interact with spindle microtubules (red lines). In different taxa, one of these proteins evolves adaptively and in concert with the divergence of the primary DNA structure of centromeres.

Bottom - Changes in the primary structure or organization of centromeric DNA (dark gray oval) can create stronger centromeres, resulting in more microtubules being attached.

Comparative genomics helps to understand the mechanisms of formation and activity of centromeric regions of chromosomes. A unique example of diverse centromere structure is chromosome 8 in the rice genome. Along with satellite DNA repeats and retrotransposons, actively transcribed genes were found in it; 48 of them had sequences with high homology to known proteins. These findings refute the opinion, based on studies of centromeres in humans, Drosophila and Arabidopsis, that there are no actively working genes in centromeres.

If the molecular structure of centromeres of various eukaryotic species contains some universal characteristics (organization of DNA in the form of tandem, relatively short monomers and chromatin proteins specific to these loci), then it is difficult to identify any patterns in the sizes of these regions. Yes, in yeast Saccharomyces cerevisiae a 125 bp DNA section is taken as the minimum functional centromere, and in yeast Schizosaccharomyces pombe it is much more complex and longer (from 40 to 120 thousand words), has several levels of organization. In humans, the main component of chromosome centromeres - a-satellite DNA - forms long strands of tandemly organized monomers (from 250 thousand to 4 million bp). Among the 12 rice chromosomes, chromosome 8 has the shortest length of the strand with the CentO satellite (~64 thousand bp); the position of the centromere and its approximate size of 2 million bp were determined. It was possible to obtain the complete DNA sequence of this centromeric region and within it to determine the region (~750 thousand bp) where the kinetochore is directly formed. The main CentO cluster is located in this area.

The remarkable plasticity of centromeres, in particular the active genes found in the centromere of rice chromosome 8, suggests that there is no strict boundary between the centromere and the rest of the chromosome and even the possibility of a dispersed structure of centromeric chromatin. However, the existence of several clusters in the region of the chromosomal constriction is contradicted by recently published data on the presence of a chromatin barrier between the centromere itself and pericentromeric heterochromatin in yeast Schizosaccharomyces pombe. The barrier is the alanine tRNA gene. Deletion or modification of the barrier sequence leads to pericentromeric heterochromatin moving beyond its normal boundaries. Moreover, the absence of a barrier causes abnormal chromosome segregation in meiosis. Of course, it should be remembered that these interesting results so far concern only one type of yeast.

They are double-stranded, replicated chromosomes that are formed during division. The main function of the centromere is to serve as an attachment site for spindle fibers. The spindle elongates cells and separates chromosomes to ensure that each new one receives the correct number of chromosomes when completed or.

The DNA in the centromeric region of the chromosome is composed of tightly packed DNA, known as heterochromatin, which is highly compacted and therefore not transcribed. Due to the presence of heterochromatin, the centromere region is stained with dyes darker than other parts of the chromosome.

Location

The centromere is not always located in the central region of the chromosome (see photo above). A chromosome consists of a short arm (p) and a long arm (q), which join at the centromere region. Centromeres can be located either near the middle or in several positions along the chromosome. Metacentric centromeres are located near the center of the chromosomes. Submetacentric centromeres are shifted to one side from the center, so that one arm is longer than the other. Acrocentric centromeres are located near the end of the chromosome, and telocentric centromeres are located at the end or in the telomere region of the chromosome.

The position of the centromere is easily detected in the human karyotype. Chromosome 1 is an example of a metacentric centromere, chromosome 5 is an example of a submetacentric centromere, and chromosome 13 is an example of an acrocentric centromere.

Chromosome segregation in mitosis

Before mitosis begins, the cell enters a stage known as interphase, where it replicates its DNA in preparation for cell division. Sisters are formed, which are connected at their centromeres.

During prophase of mitosis, specialized areas on the centromeres called kinetochores attach chromosomes to spindle fibers. Kinetochores are composed of a series of protein complexes that generate kinetochore fibers that attach to the spindle. These fibers help manipulate and separate chromosomes during cell division.

At the metaphase stage, chromosomes are held on the metaphase plate by equal forces of polar fibers, pressing on the centromeres.

During anaphase, paired centromeres on each individual chromosome begin to diverge from each other as they first center themselves relative to the opposite poles of the cell.

During telophase, the newly formed ones include individual daughter chromosomes. After cytokinesis, two different ones are formed.

Chromosome segregation in meiosis

In meiosis, the cell goes through two stages of the division process (meiosis I and meiosis II). During metaphase I, the centromeres of homologous chromosomes are oriented to opposite poles of the cells. This means that homologous chromosomes will attach at their centromeric regions to spindle fibers extending from only one of the two poles of the cell.

When spindle fibers contract during anaphase I, homologous chromosomes are pulled toward opposite poles of the cells, but sister chromatids remain together. In meiosis II, spindle fibers extending from both cell poles attach to sister chromatids at their centromeres. Sister chromatids separate in anaphase II, when spindle fibers pull them toward opposite poles. Meiosis results in the separation and distribution of chromosomes among four new daughter cells. Each cell contains only half the number of chromosomes of the original cell.

The eukaryotic chromosome is held on the mitotic spindle by the attachment of microtubules to the kinetochore, which is formed in the centromeric region

Typically, centromeres contain chromatin enriched with satellite DNA sequences

At mitosis sister chromatids migrate to opposite poles of the cell. This movement occurs because the chromosomes are attached to microtubules, the opposite ends of which are connected to the poles. (Microtubules are intracellular cylindrical structures that, during mitosis, are organized in such a way that they connect chromosomes to the poles of the cell.)

Sites in two regions Where the ends of microtubules are organized - near the centriole at the poles and on chromosomes - are called microtubule organizing centers (MTOCs).

Picture below schematically illustrates the process of separation of sister chromatids that occurs between metaphase and telophase of mitosis. The region of the chromosome responsible for its segregation in mitosis and meiosis is called the centromere. Through microtubules, the centromere of each sister chromatid is pulled to opposite poles and pulls the chromosome associated with it. The chromosome provides a mechanism for attaching a large number of genes to the division apparatus.

It contains website, which holds sister chromatids together until the individual chromosomes segregate. It looks like a constriction to which all four arms of the chromosome are attached, as shown in the figure below, which shows sister chromatids in the metaphase stage.

Centromere necessary for chromosome segregation. This is confirmed by the properties of the chromosomes, the integrity of which was disrupted. As a result of the break, one fragment of the chromosome retains the centromere, while the other, called acentric, does not contain it. The acentric fragment is unable to attach to the mitotic spindle, as a result of which it does not enter the nucleus of the daughter cell.

Regions chromosomes, flanking the centromere, usually contain rich satellite sequences and a significant amount of heterochromatin. Since the entire mitotic chromosome is condensed, centromeric heterochromatin is invisible in it. However, it can be visualized using a staining technique that reveals C-bands. In the figure below, there is a dark-colored region in the region of all centromeres. This pattern is most often seen, although heterochromatin may not be found at each centromere. This suggests that centromeric heterochromatin is apparently not a necessary component of the division mechanism.

Field of education centromeres in a chromosome is determined by the primary structure of DNA (although the specific sequence is known only for a small number of chromosomes). The DNA of the centromere binds certain proteins that form a structure that ensures the attachment of the chromosome to the microtubules. This structure is called a kinetochore. It is a stainable fibrillar structure with a diameter or length of about 400 nm.

The kinetochore ensures the creation TsOMT on the chromosome. The figure below shows the hierarchical organization of centromere DNA binding to microtubules. Proteins associated with centromere DNA are associated with other proteins, which in turn are associated with microtubules.

When sister chromatid centromeres begin to move towards the poles, the chromatids remain held together by “gluing” proteins called cohesins. First, the chromatids are separated at the centromere, and then, in anaphase, when cohesins are destroyed, they are completely separated from each other.

Formation kinetochore , conjugation homologous chromosomes and is involved in the control of gene expression.

It is in the region of the centromere that sister chromatids are connected in prophase and metaphase mitosis and homologous chromosomes in prophase and metaphase of the first division meiosis. At the centromeres, the formation of kinetochores occurs: proteins that bind to the centromere form an attachment point for microtubules spindles in anaphase and telophase of mitosis and meiosis.

Deviations from the normal functioning of the centromere lead to problems in the relative position of chromosomes in the dividing nucleus, and as a result, to disruptions in the process of chromosome segregation (their distribution between daughter cells). These violations lead to aneuploidy which can have serious consequences (for example, Down syndrome in humans, associated with aneuploidy (trisomy) on chromosome 21).

Centromeric sequence

In most eukaryotes, the centromere does not have a specific centromere corresponding to it. nucleotide sequence. It typically consists of a large number of DNA repeats (eg, satellite DNA) in which the sequence within the individual repeat elements is similar but not identical. In humans, the main repeat sequence is called the α-satellite, but there are several other types of sequences in this region. However, it has been established that α-satellite repeats are not sufficient to form a kinetochore, and that functional centromeres are known that do not contain α-satellite DNA.

Inheritance

In determining the location of the centromere in most organisms, the epigenetic inheritance. Daughter chromosomes form centromeres in the same places as the mother chromosome, regardless of the nature of the sequence located in the centromeric region. It is assumed that there must be some primary way of determining the location of the centromere, even if its location is subsequently determined by epigenetic mechanisms.

Structure

Centromere DNA is usually represented by heterochromatin, which may be essential for its functioning. This chromatin is normal histone H3 is replaced by the centromere-specific histone CENP-A (CENP-A is characteristic of baker's yeast S. cerevisiae, but similar specialized nucleosomes appear to be present in all eukaryotic cells). The presence of CENP-A is thought to be required for kinetochore assembly at the centromere and may play a role in the epigenetic inheritance of centromere location.

In some cases, for example in the nematode Caenorhabditis elegans , y Lepidoptera, as well as in some plants, chromosomes holocentric. This means that the chromosome does not have a characteristic primary constriction- a specific area to which spindle microtubules are predominantly attached. As a result, kinetochores are diffuse in nature, and microtubules can attach along the entire length of the chromosome.

Centromere aberrations

In some cases, a person has noted the formation of additional neocentromere. This is usually combined with inactivation of the old centromere, since dicentric chromosomes (chromosomes with two active centromeres) are usually destroyed during mitosis.

In some unusual cases, spontaneous formation of neocentromeres on fragments of broken chromosomes has been noted. Some of these new positions were originally composed of euchromatin and did not contain alpha satellite DNA at all.