[Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids].

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

[Nucleosome positioning within neomycinphosphotransferase gene (NPTII) on yeast plasmid in repressed and active state].

Yeast recombinant plasmid containing FRT-sequence flanked by hybrid GAL-CYC promoter and NPTII gene was developed. GAL-CYC promoter contains four UAS sequences and two closely associated TATA-boxes in CYC part. This construct provides galactose-inducible synthesis of neomycinphosphotransferase from NPTII gene, and, thus, resistance of transformed cells to G418 antibiotic. Nucleosome positioning within NPTII gene in repressed and active states was studied. Under repressive conditions (growth on glucose) stable positioning of three nucleosomes was detected. Two nucleosomes are localized in CYC-part. One of them encompasses both TATA-boxes. The third nucleosome overlaps FRT sequence and start of NPTII gene coding sequence. All three nucleosomes show multiple positioning. It suggests possibility of nucleosome sliding along DNA. After induction of NPTII expression by galactose sliding of two nucleosomes is detected. Sliding leads to exposure of TATA-box and long promoter segment. Sliding results in stable repositioning of nucleosomes at new sites. 5'-distal nucleosome moves closer to UAS-sequences. As a results UAS becomes spatially closer to TATA-box. This proximity facilitates assembly of preinitiation complex. Nucleosomes slides independently from each other. The second nucleosome moves towards FRT-sequence and repositions at its nucleosome positioning signal. Galactose-induced expression does not affect nucleosome positioning with coding region of NPTII gene. Unidirectional sliding and repositioning are detected without induction after deacetylase inhibition with trichostatine A. Basal expression of NPTII gene was shown without activation of GAL-CYC promoter and after spatial uncoupling of coding sequence and promoter by gene inversion. In these cases it seems that expression is driven by TATA-like element in FRT-sequence. This element is located in permanently exposed area (in vivo data).

[The role of DNA methylation and histone modifications in structural maintainance of heterochromatin domains (chromocenters)].

The effects of DNA methylation inhibitor 5-azacytidine (5-aza-C) and histone acetylation inhibitor trichostatine A (TSA) on the structure of pericentric heterochromatin in cultured mouse cells (L929) has been studied. After 48 h of 5-aza-C treatment, about 85% of the cells demonstrate transformation of chromocenters from ovoid to elongated structures. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules, well distinguishable by light microscopy. The same globular units can be revealed in hypotonic-treated control cells. 48 h of TSA treatment causes dramatic decrease in HP 1alpha content in the cells. Chromocenters in 25% of treated cells became highly decondenced and can not be reliably detected by light and electron microscopy. 85% of cells demonstrate globular chromocenters with low HP 1alpha content. Hypotonic treatment causes transformation of compact chromocenters into ring-like structures, which can be either single or clustered. Rings are formed by uniform fiber, in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the roles of epigenetic marks in them.

[The dependence of the 3-dimensional organization and length of the nucleolonema in the hepatocyte nucleoli of normal and regenerating rat livers on the nucleosome structure of the intranucleolar chromatin]. / Zavisimost' trekhmernoi organizatsii i dliny nukleolonemy iadryshek gepatotsitov normal'noi i regeneriruiushchei pecheni krysy ot nukleosomnoi struktury vnutriiadryshkovogo khromatina.

A biochemical and ultrastructural stereo-morphological analysis, with special reference to spatial organization and length of nucleonema and Ag-positive zones, was performed for various modifications of nucleolonemal type nucleoli in normal and regenerating (6 and 22 hours after partial hepatectomy) rat hepatocytes. To determine possible disorders on nucleosomal and supranucleosomal levels, chromatin DNA degradation was carried out during micrococcal nuclease hydrolysis, followed by analysis of electrophoretically separated particles. Functional characterization of intranucleolar chromatin was performed by testing the rate of DNA degradation after DNAase I treatment as well as by detection of free G-C pairs during titration with actinomycin D. Transcriptional activity of nucleoli was determined according to the intensity of [14C]-UTP uptake with isolated nucleoli. It is shown that the total chromatin from control nucleoli contains nucleosomal fibrils, although deprived of high compactization level. Nucleosomes themselves are strongly destabilized. In activated nucleoli structural differences of chromatin are more perceptible. In 6 hour preparations the bulk of chromatin fibrils (about 70%) undergo a further relaxation and lose the nucleosomal structure. Therefore at this point of experiment, the maximum length of nucleolonema and Ag-positive zones was registered in addition to the highest quantity of free G-C pairs, and sensibility to DNAase I transcriptional activity of isolated nucleoli. 22 hours after hepatectomy, the transcriptional activity and functional parameters of intranucleolar chromatin markedly decreased compared to the 6 hour period. Simultaneously, the share of chromatin restituting the nucleosomal structure increased, while the length of nucleolonema was shorter than in nucleoli 6 hours after hepatectomy. The main results could be resumed in the following way: the general composition of nucleolonemal type nucleolus variations described in our experimental conditions is in close relation with the with the compactization grade of ribosomal DNP-fibrils.

[The effect of 5-azacytidine on the cell cycle and chromosome structure in cell cultures of swine embryonal kidney tissue]. / Vliianie 5-azatsitidina na kletochnyi na kletochnyi tsikl i strukturkhromosom v kletdu kul'tury tkani pochki émbriona svin'i.

Incubation of pig kidney cells (PK-cells) in the presence of 5-azacytidine (5-azaC), a DNA enzymatic methylation inhibitor, at concentration of 20 microM for 6 or 24 h results in a dramatic decrease in the DNA methylation level (5mC/C + m5.100) - from 3.0 in control to 1.0 in experiment. This is accompanied by a virtually complete arrest of mitosis and a decrease in the ratio of labeled interphase cells upon simultaneous introduction of 3H-deoxycytidine. The incubation with 5-azaC block PK-cells mainly in the G2-period. The inhibitory action is reversible, for the cells enter into mitosis after removal of the inhibitor. Metaphase chromosomes, whose DNA was replicated in the presence of the 5-azaC, exhibit certain ultrastructural differences from normal ones. The results are being discussed in connection with the earlier data on the anomalous structure of interphase chromatin formed in the course undermethylated DNA replication.

[Internucleosome interactions: detecting the dinucleosome fragmentation of chromatin using micrococcal nuclease. Heterogeneity of nucleosomes and localization of histone H1]. / Mezhnukleosomnoe vzaimodeistvie: vyiavlenie dinukleosomnoi fragmentatsii khromatina mikrokokkovoi nukleazoi. Geterogennost' dinukleosom i lokalizatsiia gistona H1.

The content of histone H1 (H1/H4 ratio) in dinucleosomes with the DNA of various length liberated from L-cell nuclear chromatin by micrococcal nuclease was analyzed. It was found that the histone H1 content in the dichromatosome is two times as low as that in the largest dinucleosome and in the complete mononucleosome. The set of chromatin fragments liberated from the Triton X-100 pretreated nuclei differs considerably from that of chromatin sites devoid of histone H1 (the de novo replicating chromatin and the chromatin formed on the undermethylated DNA). A scheme for asymmetric distribution of histone H1 with molecules oriented along the nucleosomal fibril, which reflects the peculiarities of chromatin fragmentation by micrococcal nuclease with predominant liberation of the dichromatosome, is proposed.

[Internucleosome interaction: detection of dinucleosome fragmentation of chromatin by micrococcal nuclease. Analysis of the products of cleavage of chromatin from rat liver nuclei and L cells by micrococcal nuclease]. / Mezhnukleosomnoe vzaimodeistvie: vydelenie dinukleosomnoi fragmentatsii khromatina mikrokokkovoi nukleazoi. Analiz produktov rasshchepleniia khromatina iader pecheni krysy i L-kletok mikrokokkovoi nukleazoi.

In murine L-cell nuclei micrococcal nuclease causes chromatin fragmentation with predominant liberation of dinucleosomes. Analysis of dynamics of rat liver nuclear chromatin cleavage by micrococcal nuclease revealed that the "dinucleosomal" mode of fragmentation is due to the pretreatment of nuclei with the non-ionic detergent Triton X-100 in the course of the isolation procedure. The set of particles detected in nuclease hydrolysates of nuclear chromatin pretreated with Triton X-100 and those isolated by the standard procedure was shown to be significantly different. In Triton X-100 treated nuclei the dichromatosome is the main hydrolysate component under various experimental conditions of nuclease hydrolysis and the sole component under "mild" conditions, whereas sucrose-treated nuclei contain three types of dinucleosomes. In Triton-treated nuclei prolongation of hydrolysis results in the liberation of the chromatosome which is absent in chromatin hydrolysates of sucrose-treated nuclei. Hydrolysis of Triton-treated nuclear chromatin by micrococcal nuclease is unaccompanied by the liberation (up to the stage of "deep" hydrolysis) of the core particle, the major component of the "sucrose" nuclear hydrolysate under the conditions used. The sharp differences in the accessibility of various types of dinucleosomes observed during pretreatment of nuclei with Triton X-100 are interpreted in terms of the localization of histone H1. The non-random type of the histone H1 molecule orientation along the nucleosome fibril is postulated.

[Intracellular distribution of a DNA and carminomycin complex]. / Issledovanie vnutrikletochnogo raspredeleniia kompleksa DNK s karminomitsinom.

The complex of the tritium labeled DNA with carminomycin was injected intravenously to mice. Five h after the complex injection the rats were sacrificed. The liver was isolated and homogenized. The cells were consecutively divided into fractions: intercellular liquid, cytoplasm, nuclear juice and chromatin. The content of high-molecular exogenous DNA and the products of its degradation, as well as the quantity of free and DNA bound carminomycin were determined radiometrically and spectrofluorometrically in every of the above fractions. It was found that the carminomycin complex located in the liver remained unchanged for a long time after injection and penetrated into the cell in the form of the complex. In the cytoplasm, the complex disintegrated to liberate carminomycin which penetrated into the nucleus where it was bound to chromatin DNA.

[Replicative methylation of DNA in L-cells: effect of S-isobutyladenosine and cycloheximide and possible existence of two DNA-methylases]. / Replikativnoe metilirovanie DNK v L-kletkakh: deistvie S-izobutiladenozina i tsiklogeksimida i vosmozhnoe sushchestvovanie dvukh DNK-metilaz.

Methylation of DNA in cultured cells of mouse fibroblasts (L-cells) occurs at least in two steps, i. e. methylation of Okazaki fragments (up to 100 . m5C/C + m5C = 2.8-2.9) and methylation of linkage sites of DNA formed by ligation of the fragments (up to 6.0). The synthesized Okazaki fragments are not subjected to further methylation, since about one half of their methylation sites (CG) remains non-modified. The transmethylation inhibitor S-isobutyladenosine (SiBA) inhibits the methylation of the "linkage" sites of the newly synthesized DNA without affecting that of the Okazaki fragments. The repression of protein synthesis (including that of histones) by cycloheximide in the course of replication reveals some additional methylation sites. The level of methylation of the newly synthesized polymeric DNA reaches thereby 6.0, which corresponds to modification of all its CG-dinucleotides. A model for replicative methylation of DNA based on the existence of two DNA-methylases differing in their specificity is proposed. It is assumed that one of the DNA methylases is highly specific and functions within the complex with DNA-polymerase, while the other possesses a restricted specificity and functions in a free form (i. e. apart from the replicative complex).

[Nucleomeric organization of chromatin]. / Nukleomernaia arganizatsiia khromatina.

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.

[Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization]. / Ogranichennaia dostupnost' saitov metilirovaniia DNK v khromatine raznykh urovnei organizatsii dlia bakterial'nykh metilaz M.Eco RII i M.Eco dam.

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.

[Replication and methylation of DNA in cells of tobacco suspension culture and the effect of auxin]. / Replikatsiia i metilirovanie DNK v kletkakh suspenzionnoi kul'tury tabaka i vliianie auksina.

After 2 min of incubation of tobacco cell culture in a medium with [3H] -- thymidine the bulk of radioactivity of newly synthesized DNA is found in short (about 5S) fragments, whereas after a prolonged incubation of the cells, i. e. 5--60 min--in long replication fragments as well. Hence DNA replication in tobacco cells occurs discretely via formation and cross-linking of Okazaki fragments. At high cell concentrations in the medium the linking of 5S fragments is suppressed. It was shown that the Okazaki fragments and other fragments of DNA replication are subjected to methylation, the DNA methylation occurring immediately after the onset of replication. The level of methylation of the 4--6S fragments is two times less than that of the linked ones; therefore replicative methylation occurs in at least two steps: at first the Okazaki fragments undergo methylation and once they are linked, an additional methylation of DNA takes place. Auxin (2,4-dichlorophenoxyacetic acid) at concentration of 5 mg per 1 of medium does not affect the ratio of the replication fragments and methylation of the Okazaki fragments, but completely inhibits the second step of replicative methylation of DNA, i. e. methylation of the linked fragments. Phytohormones can probably control the transcription of newly synthesized DNA via regulation of methylation.

[Methylation of newly synthesized DNA in mouse fibroblast culture]. / Metilirovanie vnov' sinteziruemoi DNK v kul'ture myshinykh fibroblastov.

After a 10 min- or more prolonged incubation of transformed mouse fibroblasts (L.-cells) with [3H]-thymidine or [3H-methyl]-methionine and a subsequent centrifugation of cell lysates in an alkaline sucrose gradient the DNA radioactivity is detected in long (28, 33 and 45S) and short (5, 13 and 18S) fragments. An increase in cell concentration in the cultural layer results in inhibition of 5S fragments linkage rather than in inhibition of their synthesis. The blocking of the Okazaki fragment linkage may be regarded as one of the inhibitory molecular mechanisms of cell depletion. Both in the case of normal and suppressed (by 99%) replication by arabofuranosylcytosine [3H]-thymidine and [3H-5-methyl] cytosine are detected in the Okazaki fragments (5S) as well as in some discrete lower molecular weight fractions (lesser than 5S) of newly synthesized DNA. Thus, replicative methylation of DNA in the fibroblasts occurs in the replicative fork during DNA synthesis and the functioning DNA methylase is an indispensable component of the replicative complex. The methylation of Okazaki fragments is non-chaotic and has a specificity other than that of total DNA. This may be due to the multiplicity and different specificity of nuclear DNA-methylases. Thus, there exist in animal cells replicative and post-replicative methylation of DNA, which may differ in the nature of substrates and enzymes, in specificity of recognizable sequences and in their functional significanse.

[Ultrastructure of the DNP fibrils and of the interchromatin granules in isolated nuclei of the rat liver]. / Ul'trastruktura fibrill DNP i interkhromatinovykh granul v izolirovannykh iadrakh pecheni krysy.

The structural organization of DNP fibrils and interchromatin granules of isolated rat hepatocyte nuclei has been studied in various conditions of chromatin solubilization. When observed either in nuclei fixed in situ or in a solution containing 20 mM TEA and 1 mM MgCl2, a DNP fibril consists of globular structures 20--25 nm in diameter. In the nuclei fixed in a magnesium-free solution (20 mM TA), nucleosome structures are revealed in DNP. Condensation of chromatin results from interaction between 20 nm globular fibrils, whereas the complete dispersion of chromatin is a consequence of its conversion into the nucleosomal form. In the conditions of both DNP structuralization and dispersion, the nuclei are revealed to contain zones of interchromatin granules connected by thin fibrils. It is assumed that the different compactness of these granular-fibrillar complexes and of the regions of condensed chromatin may be used for their separation and fractionation.

[Localization of (3H)hydrocortisone in rat liver nuclei]. / Lokalizatsiia (3H)gidrokortizona v iadrakh pecheni krys.

The content of hydrocortisone in rat liver nuclei reaches its maximum in 1 hour after its intravenous injection and remains stable for another hour. In three hours after introduction it falls to the initial level (15 min after the injection of [3H]hydrocortisone). The accumulation of the hormone in nuclei completely coincides with kinetics of reversible DNA supermethylation and correlates with the induction of transcription. No free [3H]hydrocortisone was found in nuclei, in isolated nuclei it is found in the fraction of nuclear membranes, nucleoplasma, nucleolus and chromatin. Specific radioactivity of [3H]hydrocortisone (d. p. m. per 1 mg of DNA) in active chromatin is 10--50-fold as high as in condensed chromatin and is 10--15-fold as high as in nucleolus. It is suggested that there are at least two types of binding hormonereceptor complexes with chromatin. The initial binding of these complexes with condensed chromatin may result in its structural rearrangement (euchromatization), in the appearance of more specific secondary sites for binding hormone-receptor complexes with chromatin DNA, and also in the appearance of methylation and transcription initiation sites in DNA.

[Genome structure in higher plants. Reassociation kinetics of DNA from Vicia faba and Vicia sativa]. / Struktura genomov vysshikh rastenii. Kinetika reassotsiatsii DNK Vicia faba i Vicia sativa.

The work been concerned with a study of the kinetics of reassociation of total DNA and that of the fraction of unique sequences in plants from the Vicia family, i. e. Vicia faba and Vicia sativa. The size of the genome was determined by the kinetics of reassociation of the DNA of the fraction of unique sequences and the amount of DNA per nucleus was determined cytophotometrically. It has been shown that the size of the genome expressed in C(0)t units and the size expressed in gramms are not the same which testifies to the absence of true unique genes in the genome of the species studied. The analysis of the possible methodical errors was carried out. On the basis of the data obtained a suggestion was made of a model of chromosomes organization including 12 units of polynemization for Vicia faba and 4 units for Vicia sativa.

[Replication of DNA associated with nuclear envelope of regenerating rat liver]. / Replikatsiia DNK, assotsiirovannoi s iadernoi obolochkoi regeneriruiushchei pecheni krys.

DNA associated with nuclear envelopes was isolated from rat liver nuclear lysate using centrifugation in sucrose gradient. The specific radioactivity of DNA obtained in nuclear envelope was compared with that of overall nuclear DNA at different stages of the S-period after partial hepatectomy and 1-15 min following intravenous injection of 3H-thymidine. It was found that at various steps of the cell cycle (19, 24 and 27 hrs following partial hepatectomy) the specific radioactivities of nuclear membrane-linked and overall nuclear DNAs do not show any significant differences. No noticeable selective synthesis or initiation of DNA replication on the nuelear envelope were observed either.

[DNA from chromatin-nuclear membrane complex: reassociation kinetics at different stages of the cell cycle]. / DNK iz kompleksa khromatina s iadernoi obolochkoi: kinetika reassotsiatsii na raznykh stadiiakh kletochnogo tsikla

Base composition and reassociation kinetics of DNA from chromatin-nuclear membrane complex of rat liver cells at different stages of the cell cycle. The complex isolated in low ionic strength solutions contains about 9% of total nuclear DNA, and its density in sucrose solution is 1.27 g/cm2. DNA, isolated from the complex, is homogenous in its molecular weight (approximately 2-10(6) daltons) and is similar in its base composition to total nuclear DNA. However, DNAs from complexes, isolated at interphase and different S-phases of the cell cycle, differ from total nuclear DNA in reassociation kinetics: DNAs from complexes are enriched by approximately 10% with unique nucleotide sequences. It is concluded from the similarity of reassociation kinetics, that molecular population of nuclear membrane-bound DNA does not change considerably throughout the cell cycle.

[Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]. / Vliianie gidrokortizona na metilirovanie i molekuliarnuiu populiatsiiu DNK v pecheni krys.

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.