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1.
Reprod Toxicol ; 74: 1-9, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28843703

RESUMO

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.


Assuntos
Compostos Benzidrílicos/toxicidade , Cromátides/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Estrogênios não Esteroides/toxicidade , Fenóis/toxicidade , Animais , Linhagem Celular Tumoral , Cromátides/genética , Embrião de Mamíferos/metabolismo , Feminino , Células HEK293 , Humanos , Metáfase , Camundongos
3.
Tsitologiia ; 57(1): 39-46, 2015.
Artigo em Russo | MEDLINE | ID: mdl-25872374

RESUMO

Transmission of foreign mtDNA along the paternal lineage founded by male mice (F0), and distribution of that mtDNA in their progeny at early stages of prenatal development were studied. Transmitochondrial males of F0 obtained after injection of human mitochondria into mouse zygotes has been shown to transmit foreign mtDNA to subsequent generations. Individual peculiarities among the males studied, concerning transmission of foreign mtDNA to the progeny, are likely to exist. Besides, the distribution of human mtDNA among blastomeres of transmitochondrial embryos under study differed from that observed in previous investogation of its inheritance along the maternal lineage.


Assuntos
DNA Mitocondrial/genética , Embrião de Mamíferos/metabolismo , Padrões de Herança , Mitocôndrias/genética , Zigoto/metabolismo , Animais , Quimera , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitocôndrias/química , Fatores Sexuais , Zigoto/crescimento & desenvolvimento
4.
Tsitologiia ; 50(11): 983-7, 2008.
Artigo em Russo | MEDLINE | ID: mdl-19140345

RESUMO

Distribution of human mitochondrial DNA (mtDNA) among separate murine blastomeres was analyzed during the splitting of embryos in which the suspension of human mitochondria had been injected at the one- or two-cell stage. Human mtDNA was detected by PCR with species specific primers. The total amount of the two- and four-cell murine embryos analyzed in the study was 339. In all embryos examined the copies of human mitochondrial genome were revealed along with murine mtDNA, which indicated the phenomenon of an artificially modeled heteroplasmy. The foreign mtDNA was not ubiquitous among the blastomeres of transmitochondrial embryos. Mathematical analysis of the results showed that in the period between the injection of human mitochondria and the subsequent splitting no equal distribution of the human mtDNA occurred in the cytoplasm. These results also point at the presence of more than 2-3 segregation units of mtDNA in the entire pool of mitochondria (about 5 x 10(2)) introduced into an embryo by microinjection.


Assuntos
Blastômeros/metabolismo , DNA Mitocondrial/metabolismo , Embrião de Mamíferos/metabolismo , Animais , Blastômeros/química , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Frequência do Gene , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias Hepáticas/química
5.
Genetika ; 40(8): 1034-45, 2004 Aug.
Artigo em Russo | MEDLINE | ID: mdl-15523841

RESUMO

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.


Assuntos
Centrômero , DNA Satélite/genética , Animais , Metilação de DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Reação em Cadeia da Polimerase
6.
Tsitologiia ; 46(1): 53-61, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15112432

RESUMO

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.


Assuntos
Cromossomos/metabolismo , DNA Satélite , Heterocromatina/metabolismo , Animais , Carcinoma Embrionário , Bovinos , Linhagem Celular Tumoral , Cromossomos/genética , Células Clonais , Heterocromatina/genética , Hibridização in Situ Fluorescente , Camundongos , Transfecção
7.
Tsitologiia ; 43(3): 269-73, 2001.
Artigo em Russo | MEDLINE | ID: mdl-11387756

RESUMO

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.


Assuntos
Encéfalo/ultraestrutura , Dano ao DNA , DNA de Cadeia Simples/ultraestrutura , Neurônios/ultraestrutura , Estresse Psicológico/genética , Animais , Masculino , Ratos , Ratos Wistar , Especificidade da Espécie
8.
Tsitologiia ; 42(11): 1060-8, 2000.
Artigo em Russo | MEDLINE | ID: mdl-11204650

RESUMO

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.


Assuntos
Dano ao DNA , DNA de Cadeia Simples/genética , Teratocarcinoma/genética , Células 3T3 , Animais , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Replicação do DNA , Camundongos , Troca de Cromátide Irmã , Teratocarcinoma/patologia , Tretinoína/farmacologia , Células Tumorais Cultivadas
9.
Zygote ; 6(4): 351-8, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9921646

RESUMO

The effects of two demethylating drugs with a different mechanism of action (5-azacytidine (Aza) and L-ethionine (Eth)) on mouse preimplantation development were investigated. Preimplantation embryos were cultured for 24 h in the presence of the drug and for an additional 24 or 48 h (depending on the cleavage stage) in medium supplemented with bromodeoxyuridine to reveal sister chromatid exchanges (SCEs) and the number of cell cycles performed before harvesting. Striking differences between the two drugs were observed in their influence on proliferation of blastomeres, primary differentiation and sister chromatid differentiation (SCD), and in the pattern of DNA methylation and the frequency of SCEs per cell. At a final concentration of 1 microM Aza had no effects, whereas higher concentrations stopped development of all stages except the zygote. In contrast Eth treatments (5 mM) resulted in a severe reduction of the mean cell number per embryo in comparison with controls. Moreover both the absence of blastocyst formation and no effects on mitotic activity were detected. The most prominent effect of Eth was detected at the zygote and 4-cell stages. An unexpected decrease in SCE frequency in Eth-treated morulae and 4-cell embryos has been observed. Data are explained taking into account the different mechanisms of action of the agents.


Assuntos
Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Etionina/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Feminino , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Troca de Cromátide Irmã/efeitos dos fármacos , Zigoto/efeitos dos fármacos
10.
Genome ; 38(2): 381-4, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7774804

RESUMO

The nick translation and gap filling procedures, without external addition of nicking enzymes, were performed in situ on fixed chromosomes of mouse preimplantation and postimplantation embryos and of bone marrow in order to detect possible DNA single-strand breaks (nicks and (or) gaps). All chromosome preparations were made using the same technique. Nick translation of chromosomal DNA with DNA polymerase I (Pol I) or gap filling with the Klenow fragment of Pol I in the presence of biotinylated-dUTP, demonstrated a regular absence of label on chromosomes of postimplantation embryos and bone marrow. No difference in sensitivity was found between the holoenzyme and the Klenow fragment. In preimplantation embryos, the chromosome reactivity in nick translation was highest at the blastocyst stage and varied according to cleavage divisions of the zygote.


Assuntos
Aberrações Cromossômicas , Dano ao DNA , DNA de Cadeia Simples/análise , Embrião de Mamíferos/ultraestrutura , Animais , Blastocisto/ultraestrutura , Medula Óssea/ultraestrutura , DNA Polimerase I , Idade Gestacional , Camundongos , Sensibilidade e Especificidade
11.
Tsitologiia ; 37(5-6): 458-64, 1995.
Artigo em Russo | MEDLINE | ID: mdl-8658975

RESUMO

The occurrence and distribution pattern of spontaneous single-strand breaks (nicks) and/or gaps of mouse chromosomal DNA were studied with the help of nick-translation procedure omitting exogenous nucleases. The holoenzyme and a Klenow's fragment were used at a concentration of 0.I. U/20 microl of reaction mixture, resp. Bio-dUTP and streptavidin-alkaline phosphatase were used for labeling and detection. Chromosomes of postimplantation embryos and bone marrow were not stained. Chromosomes of all preimplantation stages of development were homogeneously stained with prominent dots of various size and intensities of grayish. DNA Pol I and the Klenow enzyme demonstrated a similar pattern of labelling. The centromeric heterochromatin was not labeled. The label was localized asymmetrically exclusive of NOR and telomeric regions.


Assuntos
Cromossomos/genética , Dano ao DNA/genética , DNA de Cadeia Simples/genética , Embrião de Mamíferos/ultraestrutura , Animais , Medula Óssea/ultraestrutura , Cruzamentos Genéticos , Desenvolvimento Embrionário/genética , Feminino , Técnicas Genéticas , Metáfase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Gravidez
12.
Cytobios ; 79(319): 235-40, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7748262

RESUMO

The nick translation procedure without external addition of nicking enzymes was performed in situ on fixed nuclei of mouse preimplantation, and postimplantation embryos, as well as bone marrow in order to detect possible DNA single-strand breaks. All preparations of nuclei were made using the same technique. Nick translation of nuclear DNA with DNA polymerase I in the presence of biotinylated-dUTP demonstrated a characteristic absence of label on nuclei of postimplantation embryos and bone marrow. The nuclear reactivity varied according to the cleavage divisions of the zygote, being highest at the four-cell stage.


Assuntos
Blastômeros/metabolismo , Dano ao DNA , DNA de Cadeia Simples/análise , Zigoto/metabolismo , Animais , Medula Óssea/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA de Cadeia Simples/metabolismo , Desenvolvimento Embrionário e Fetal , Feminino , Técnicas Genéticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Gravidez
13.
Cytogenet Cell Genet ; 66(1): 31-2, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8275704

RESUMO

The phenomenon of spontaneous differentiation (without bromodeoxyuridine in the culture medium) of sister chromatids of mouse chromosomes at the blastocyst stage of embryogenesis is described. The frequency of sister chromatid exchanges in such differentiated chromosomes was calculated.


Assuntos
Blastocisto/citologia , Cromátides/ultraestrutura , Troca de Cromátide Irmã , Animais , Cruzamentos Genéticos , Feminino , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Gravidez
14.
Tsitologiia ; 35(9): 12-7, 1993.
Artigo em Russo | MEDLINE | ID: mdl-8266578

RESUMO

The frequency of sister-chromatid exchanges (SCE) was analysed in spontaneously and superovulated morulae and blastocyst of CBA, C57Bl, F1 CBA x C57Bl mice in culture with bromodeoxyuridine (BrdU). The background level for SCE was found to be 4-5 times higher in early embryos than in fetal or adult tissues of the mouse. The phenomenon of spontaneous sister-chromatid differentiation (SCD) of blastocyst chromosomes without any treatment with BrdU was observed. The SCE frequency was calculated on such chromosomes and was the same as with BrdU. A possible mechanism of this phenomenon is suggested.


Assuntos
Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário , Troca de Cromátide Irmã , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Bromodesoxiuridina/farmacologia , Bandeamento Cromossômico , Cruzamentos Genéticos , Técnicas de Cultura , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mórula/efeitos dos fármacos , Mórula/ultraestrutura , Gravidez , Troca de Cromátide Irmã/efeitos dos fármacos , Superovulação/efeitos dos fármacos
15.
Ontogenez ; 23(6): 632-7, 1992.
Artigo em Russo | MEDLINE | ID: mdl-1294927

RESUMO

Transcriptionally active NORs of chromosomes visualized by AgNO3 staining were studied in bone marrow and embryos (day 10 of gestation) of CBA and C57BL mice, as well as of (CBA x C57BL)F1 hybrids. These mouse strains were shown to differ by the average number of Ag-positive NORs in marrow cells; in hybrids, the number of NORs is greater than in the parent strains. During embryogenesis, the number of chromosomes carrying silver-stained NORs increases; however, no significant differences by this parameter was detected between hybrid and C57BL embryos. The average number of silver-stained NORs was the smallest in embryos of CBA mice.


Assuntos
Cromossomos/ultraestrutura , Cruzamentos Genéticos , Camundongos Endogâmicos C57BL/embriologia , Camundongos Endogâmicos CBA/embriologia , Região Organizadora do Nucléolo/ultraestrutura , Animais , Medula Óssea/ultraestrutura , Feminino , Masculino , Metáfase , Camundongos , Coloração pela Prata/métodos , Especificidade da Espécie
17.
Tsitologiia ; 34(6): 55-8, 1992.
Artigo em Russo | MEDLINE | ID: mdl-1455559

RESUMO

The picture of differential staining of early mouse embryogenesis metaphasic chromosomes, from the first cleavage up to 10 days of gestation, after digestion by restriction endonuclease AluI was studied. It was shown that depending on the degree of digestion by endonuclease differential bandings of G+C- or C-type were observed. After the least digestion only the first cleavage chromosomes were differently stained. A slight difference in intensity of staining between paternal and maternal chromosomes of the zygote was observed. All the mouse chromosomes were identified after AluI digestion and staining after Giemsa.


Assuntos
Bandeamento Cromossômico/métodos , Cromossomos/efeitos dos fármacos , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Animais , Cromossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fatores de Tempo , Zigoto/efeitos dos fármacos , Zigoto/ultraestrutura
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