[Changes of the genome-wide DNA methylation level in various regions of the rat brain with incomplete cerebral ischemia]. / Izmenenie urovnya polnogenomnogo metilirovaniya DNK v razlichnykh oblastyakh golovnogo mozga krys pri nepolnoi tserebral'noi ishemii.

OBJECTIVE: To quantify genome-wide DNA methylation in the olfactory bulbs, fronto-parietal and occipital regions, and cerebellum in normal male Wistar rats and in modeling incomplete cerebral ischemia caused by permanent bilateral occlusion of the common carotid arteries. MATERIAL AND METHODS: The study was performed on 23 male Wistar rats divided into groups: «Sham operation¼ and «Cerebral ischemia¼. The level of genome-wide methylation of CCGG sites was determined by methyl-sensitive restriction using MspI/HpaII endonucleases followed by densitometric analysis of electrophoregrams by ImageJ software. RESULTS: Incomplete cerebral ischemia on the 7th day leads to 56.3% (95% CI: 33.2-76.90) mortality. In the surviving rats of the «Cerebral ischemia¼ group, compared with the animals of the «Sham operation¼ group, a pronounced neurological deficit was observed, which was accompanied by changes in the level of whole-genome DNA methylation in the nervous tissue of brain structures (p<0.05). Incomplete cerebral ischemia in male Wistar rats was characterized by interhemispheric asymmetry in the severity and direction of the epigenomic reaction of the nervous tissue in both ischemic and non-ischemic areas of the brain. CONCLUSION: It is likely that it is precisely this dynamics of changes in the status of genome-wide DNA methylation in the nervous tissue that imparts plasticity to neuronal function during ischemic damage.

[DNA methylation in experimental ischemic brain injury]. / Metilirovanie DNK pri eksperimental'nom ishemicheskom povrezhdenii golovnogo mozga.

The enrichment of angioneurology with fundamental advances leads to the understanding of new important facets in the pathogenesis of cerebral ischemia. The knowledge of epigenetic mechanisms in the development of stroke, in particular, DNA methylation, which makes a significant contribution to the development and formation of cerebral damage, is becoming more and more relevant. This review reflects an analysis of animal studies proving the relationship of DNA methylation with cerebral ischemia. As a result of the search work, 282 articles from the PubMed database were selected for keywords that corresponded to this topic. Of these publications, 8 studies were devoted to genome-wide DNA methylation, and 6 published the results of DNA methylation of candidate genes in experimental cerebral ischemia. The results have demonstrated that brain DNA methylation in animals is associated with the development of ischemic stroke and may play a role in several pathogenetic mechanisms. In two studies, a decrease in the level of DNA methylation in 2 genes in ischemic brain tissues of laboratory animals was found, at the same time, in four studies, 8 genes, in which methylation increased after ischemic stroke, were reported. These data suggest that the assessment of the level of DNA methylation in stroke is a promising biomarker for the search and improvement of pharmacological and non-pharmacological methods for limiting brain damage in ischemic and reperfusion injury at the stages of preclinical and clinical studies.

Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action.

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.

[THE INFLUENCE OF THE PREPARATION PRETREATMENT ON IN SITU DETECTION OF 5-METHYLCYTOSINE IN METAPHASE CHROMOSOMES AND IN INTERPHASE NUCLEI].

Qualitative and quantitate analysis of DNA methylation in situ at the level of cells, chromosomes and chromosomal domains is extremely important for the diagnosis and treatment of various diseases, the study of ageing and the consequences of environmental impacts. An important question arises, whether the revealed in situ methylation pattern reflects DNA methylation per se and (or) availability of the DNA for antibodies, which in turn depends on the peculiarities of chromatin structure and chromosome condensation. These events can lead to an incorrect evaluation of the actual pattern of DNA methylation. To avoid this shortcoming as far as possible, we have modified the most widely used method of revealing 5-methylcytosine in situ with monoclonal antibodies. Here we have shown that the detection of DNA methylation staining of chromosomes including C-heterochromatin, chromosomal arms and sister chromatids is drastically dependent on pretreatment of chromosomal preparations for immunocytochemical study using fluorescent antibodies. Using undifferentiated stem cells of mouse embryonal carcinoma line F9, it has been found that change in preparations storage results in a sharp fluorescence decrease up to complete disappearance of the signal in centromeric heterochromatin. With the help of the method described in the work, we have first revealed the asymmetry of sister chromatids methylation in metaphase chromosomes of F9 cell and lymphocytes of human periphery blood. This may lead to asymmetry of transcriptional signature of daughter cells after division. The proposed here modification of 5-methylcytosine detection in situ provides a more complete characterization of methylation of chromosomes and chromosomal domains, compared to previously published methods.

[[Length polymorphism of minisatellite repeat B2-VNTR of the bradykinin B2 receptor gene in healthy Russians and in patients with coronary heart disease].

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, pain. It is suggested that this receptor may be one of the factors that have cardioprotective and infarct-limiting effects. It is assumed that certain genetic variants in both coding and non-coding regions ofBDKRB2 gene may influence its expression. In the 3'-untranslated region of BDKRB2 exon 3 the minisatellite repeat B2-VNTR is located. B2-VNTR has previously been shown to affect the BDKRB2 mRNA stability. Therefore, it is important to perform the molecular genetic analysis of this minisatellite in patients with different forms of coronary heart disease in order to reveal possible associations between specific B2-VNTR alleles and certain clinical forms of coronary heart disease. In the present study, a comparative analysis of the allele and genotype frequencies of B2-VNTR was carried out in groups of healthy individuals and patients with two clinical forms of coronary heart disease (angina pectoris and myocardial infarction), ethnically Russian. The results of the B2-VNTR length polymorphism analysis indicate that this tandem repeat may be attributed to a class of low polymorphic and non-hypervariable minisatellite. In all analyzed groups we revealed three B2-VNTR alleles, consisting of 43, 38 and 33 repeat units. Alleles of 43 and 33 repeats were major in all investigated groups. No statistically significant differences were found in the B2-VNTR allele and genotype frequencies between men and women in control group, and also between healthy men and men with angina pectoris and myocardial infarction. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of coronary heart disease in Russian men. However, we do not exclude the possibility of association between the B2-VNTR short alleles (38 and 33 repeats) and cardioprotective effects of bradykinin B2 receptor in women with coronary heart disease. This hypothesis requires further investigation.

[Human intra-intronic minisatellite UPS29 associated with neurological diseases regulates reporter gene EGFP expression depending on cell type].

Earlier, it was established that polymorphism of minisatellite UPS29 located in one of introns of human gene CENTB5 (ACAP3) was associated with Parkinson's disease and epilepsy. The main aim of this work was to elucidate if that minisatellite could regulate reporter gene activity, and if such activity was tissue (cell)-specific. To this end there was used transient transfection of HeLa cells, mouse embryonal carcinoma line F9, and rat astrocytes cultures with plasmides which contained reporter gene EGFP under eukaryotic promoter ROSA26 and different allelles of minisatellite UPS29. It was found that UPS29 possessed enhancer-like activity in neuronal type cells.

[CENTB5 gene expression in human and mouse].

Centaurin beta5 with unclear function belongs to protein family of centaurins. Human centaurin beta5 is encoded by gene CENTB5 whose intron 14-15 contains low variable minisatellite UPS29, and mouse homolog CENTB5 in analogous intron contains imperfect microsatellite repeat (CATG)19. Earlier we found the association between an occurrence of short UPS29 alleles with some forms of Parkinson disease and epilepsy. Besides this, both human and mice CENTB5 are localized in the same synteny group with SCNN1D and ACOT7 genes which are known to be expressed predominantly in nervous system. Mutations in these genes are connected with neurodegenerative processes and epilepsy. It is known that intra-intronic sequences can modulate genes of their location and neighbor and even remote genes. Using RT-PCR we carried out simultaneous analysis of CENTB5, SCNN1D and ACOT7 genes expression. Potential possibility of human intra-intronic tandem repeat UPS29 and of mouse intra-intronic tandem repeat (CATG)19 to regulate/modulate CENTB5, SCNN1D and ACOT7 activity was evaluated in silico. It was found that all these genes were expressed in all studied organs and tissues. It is suggested that minisatellite locus UPS29 can regulate an activity of CENTB5, SCNN1D and ACOT7 in nervous system cells.

[Regulatory mechanisms of mammalian imprinting].

Epigenetic modifications, such as monoallelic DNA methylation, covalent histone modifications, nonhistone proteins, chromatin folding, heterochromatinization, spatial nucleus organization are reviewed with regard to establishment and maintenance of imprinting in mammals. Special attention is paid to repeated DNA sequences as intermediates of the above epigenetic modifications. A suggestion is put forward relative to importance of preimplantation development, in particular, to chromosome organization and segregation in the establishment of imprinting. Some futher directions of imprinting mechanisms are also discussed.

[Transgenerational transmission of bovine satellite DNA in transgenic mice].

Genetical, cytogenetical and molecular analysis was made for 5 generations of mice transgenic for bovine satellite DNA (Sat). In all cases transgenic mice were generated by crosses of transgenic males and females with normal (CBA x C57B1) mice. No abnormalities in the founder development were noticed. A normal (near 50 %) ratio of transgenic and nontransgenic offsprings was observed in blastocysts. However, profound differences occurred in the rate of transgene bearing offsprings, depending on the sex of grandparents rather than of parents. The grandfather Sat transmission resulted in the appearance of 0-52.4 % transgenic grandchildren, whereas the grandmother transmission ended in the theoretically expected rate. This means that stabilization of transsatellite took place upon the female germ line transmission (a positive grandmother effect). It is essential that in hemizygous transsatellite mice Sat integration led to the occurrence of mammary tumors, inflammation of uterine horns, and infringement of mother care of transgenic females. Simultaneous FISH and G-banding showed Sat to be localized in the internal region of chromosome 12 near Pax 9 and Brms 11 genes. Commonly, these genes are implicated in tumorigenesis as their expression decreases. Thus, a kind of silencing effect of these genes' expression may be supposed.

[Instability of repetitive units of foreign centromeric satellite DNA in transgenic mice and transfected cells]. / Analiz nestabil'nosti povtoriaiushchikhsia edinits chuzherodnoi tsentromernoi satellitnoi DNK u transgennykh myshei i v transfektnykh kletkakh.

Cytologically detectable instability of centromeric satellite DNA may cause hereditary disorders in human. To study the mechanisms of such instability, two transgenic mouse lines and 11 clones of transfected F9 mouse embryonic teratocarcinoma cells were obtained with the 3.8-kb repetitive unit (Sat) of Bos taurus satellite DNA IV. Intergeneration and somatic instability of exogenous satellite DNA (satDNA) was observed in transgenic mice and transfected cells as a change in nucleotide sequence of an internal Sat region approximately 1000 bp in size. Since Sat was in the hemizygous state in both cases by the experimental protocol, the instability was attributed to intra-allelic processes. Intergeneration instability probably took place in the premeiotic period of gametogenesis or in early embryo development and led to prenatal death of transgenic embryos after at least one generation. No direct or inverse correlation was observed between methylation and instability of Sat. The results testify that submicroscopic changes in highly repetitive noncoding DNA sequences may already affect the genome function in higher eukaryotes.

Bovine satellite DNA induces heterochromatinization of host chromosomal DNA in cells of trassatellite mouse embryonal carcinoma.

Embryonal teratocarcinoma F9 cells were transfected with a fragment (3.8 kb) of bovine satellite DNA IV (Sat), which is not homologous to mouse satellite DNA. FISH analysis revealed various chromosomal integration sites of integrated Sat in different transsatellite clones. After several passages, transsatellite had a tendency to spread along chromosome bearing Sat in one of the studied lines. The integrated transsatellites were enriched with prolonged single-strand DNA regions (SSR) revealed by FISH without previous chromosomal denaturation, and were unmethylated. The observed SSR are presumably supposed to represent intermediates of transsatellite DNA instability via unequal sister chromatid exchanges. DAPI staining demonstrated that the integrated Sat induced the formation of prominent ectopic neoheterochromatin blocks in regions adjacent to integrated Sat. These blocks were located exclusively between integrated Sat and centromeric heterochromatin. Thus, mouse repetitive centromeric DNA (AT-rich, DAPI-positive) "spreads" along the chromosome in response to integration of the bovine satellite GC-rich DNA. The results obtained are discussed in the context of possible position effect variegation mechanisms operating in undifferentiated cells.

[Single-strand DNA breaks in brain cells of different rat strains under normal condition and during exposure to stress]. / Odnonitevye razryvy DNK v otdel'nykh kletkakh mozga razlichnykh linii krys v norme i pri stressornom vozdeistvii.

This study was aimed to characterize pattern of occurrence of spontaneous single-strand breaks in situ in glial and neuronal nuclei of the cortex, middle brain and hyppocapmi (CA3 field) of rats selected for a threshold of nervous system excitability, and to study the influence of stress of various modality on such breaks. The results obtained evidence that: 1) intact animals possess a subpopulation of glial and neuronal cells revealed following gap filling in situ in opposite to other types of terminally differentiated non-proliferating cells; 2) the size of such a subpopulation differs depending on the lines of examined rats, parts of brain, and the type of stress.

[Modeling heterochromatin regions in transgenic mice]. / Modelirovanie geterokhromatinovykh raionov u transgennykh myshei.

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.

[Satellite DNA and disease--possible mechanisms. Minisatellite instability]. / Satellitnye DNK i bolezni--vozmozhnye mekhanizmy. Nestabil'nost' minisatellitov.

The main possible molecular mechanisms of minisatellite DNA instability are reviewed and compared with those of the trinucleotide repeat instability. Evidence indicating that some human diseases are associated with minisatellite DNA instability is presented including data on minisatellite DNA expansion.

[Satellite DNA and disease--possible mechanisms. Trinucleotide repeats]. / Satellitnye DNK i bolezni--vozmozhnye mekhanizmy. Trinukleotidnye povtory.

The new data on the mechanisms underlying trinucleotide repeat expansion are reviewed with special reference to the role of chromatin structure, DNA replication, methylation, and amplification in repeat expansion during ontogeny. A hypothesis is advanced as to the crucial role of processes that occur at the preimplantation developmental stage, such as sister chromatid exchanges, single-strand DNA breaks, and demethylation. The molecular and cellular events responsible for association between trinucleotide expansion and various diseases are discussed.

[Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells]. / Neindutsirovannye odnonitevye razryvy v DNK kletok linii F9 teratokartsinomy myshi.

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.

[The detection of transgenic animals using a polymerase chain reaction in situ]. / Obnaruzhenie transgennykh zhivotnykh pri pomoshchi polimeraznoi tsepnoi reaktsii in situ.

The technique for detecting both foreign and host specific DNA sequences inside nuclei and chromosomes of single cells of transgenic mice with the help of polymerase chain reaction (PCR) in situ is described. The mouse preimplantation and postimplantation embryonic and adult cells were studied. The methodology is described in detail with particular attention to the optimization of composition of reaction mixture, kind of fixation and preliminary denaturation of target DNA. The reaction takes only several hours and needs no sophisticated equipment.

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria.

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.