RESUMO
Sleep is essential to all animals with a nervous system. Nevertheless, the core cellular function of sleep is unknown, and there is no conserved molecular marker to define sleep across phylogeny. Time-lapse imaging of chromosomal markers in single cells of live zebrafish revealed that sleep increases chromosome dynamics in individual neurons but not in two other cell types. Manipulation of sleep, chromosome dynamics, neuronal activity, and DNA double-strand breaks (DSBs) showed that chromosome dynamics are low and the number of DSBs accumulates during wakefulness. In turn, sleep increases chromosome dynamics, which are necessary to reduce the amount of DSBs. These results establish chromosome dynamics as a potential marker to define single sleeping cells, and propose that the restorative function of sleep is nuclear maintenance.
Assuntos
Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Neurônios/metabolismo , Sono/fisiologia , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos/metabolismo , Feminino , Masculino , Microscopia Confocal , Modelos Animais , Neurônios/citologia , Imagem com Lapso de Tempo , Peixe-ZebraRESUMO
Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.
Assuntos
Cromatina/fisiologia , Lamina Tipo A/metabolismo , Interferência de RNA/fisiologia , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Lamina Tipo A/genética , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , RNA Interferente Pequeno , TelômeroRESUMO
Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , DNA/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Dados de Sequência Molecular , Mapeamento por Restrição , Escleroderma Sistêmico/imunologiaRESUMO
Nucleotide sequences that are cleaved by calf thymus type I topoisomerase have been determined using cloned human Ha-ras and p53 genes. Localization and relative frequency of single-strand cleavages within these sequences were observed to change in the presence of the cytotoxic alkaloid camptothecin.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Eletroforese em Gel de Ágar , Genes ras , Humanos , Dados de Sequência Molecular , Plasmídeos , Timo/enzimologiaRESUMO
The properties and functions of the DNA topoisomerases are reviewed on the basis of the literature and the author's own data. The techniques of isolation and characterization of the covalent complexes of the topoisomerases with DNA are described.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Animais , Sequência de Bases , Ciclo Celular , Células Cultivadas , Cricetinae , Cricetulus , DNA Topoisomerases Tipo I/isolamento & purificação , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Ágar , Feminino , Peixes , Cinética , Novobiocina/farmacologia , Conformação de Ácido Nucleico , Oócitos/enzimologiaRESUMO
Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases.
Assuntos
Clonagem Molecular , DNA Topoisomerases Tipo I/genética , DNA/genética , Expressão Gênica , Placenta/enzimologia , Anticorpos Monoclonais , DNA Topoisomerases Tipo I/isolamento & purificação , Feminino , Vetores Genéticos , Humanos , GravidezRESUMO
Anomalous diffusion, observed in many biological processes, is a generalized description of a wide variety of processes, all obeying the same law of mean-square displacement. Identifying the basic mechanisms of these observations is important for deducing the nature of the biophysical systems measured. We implement a previously suggested method for distinguishing between fractional Langevin dynamics, fractional Brownian motion, and continuous time random walk based on the ergodic nature of the data. We apply the method together with the recently suggested P-variation test and the displacement correlation to the lately measured dynamics of telomeres in the nucleus of mammalian cells and find strong evidence that the telomeres motion obeys fractional dynamics. The ergodic dynamics are observed experimentally to fit fractional Brownian or Langevin dynamics.
Assuntos
Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Modelos Biológicos , Telômero/fisiologia , Telômero/ultraestrutura , Animais , Simulação por Computador , Difusão , Humanos , Modelos EstatísticosAssuntos
Radiação , Televisão , Cor , Concentração Máxima Permitida , Monitoramento de Radiação , Radiometria , U.R.S.S.RESUMO
Recovery of the histomorphometric indices of rat skin at the period from 38 to 150 days following local X-irradiation with doses of 2.5-20 Gy has been studied. It is shown that the rate of restitution of the epidermis and dermis thickness is 0.0021 and 0.0037 days-1, respectively. However, the dermis thickness is less than in the control. The hair follicle density not only fails to restore after irradiation with a dose of above 10 Gy but continues decreasing in time.
Assuntos
Pele/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Masculino , Ratos , RegeneraçãoRESUMO
A new DNAase was isolated from unfertilized loach eggs. The enzyme is activated by bivalent metal ions (Mg2+, Ca2+, Mn2+) and is inhibited by Na+ and N-ethylmaleimide. The maximal activity of DNAase lies within the pH range of 9.0-9.5. The enzyme is thermostable and predominantly hydrolyzes the two-chain DNA by producing one-chain ruptures. The DNAase activity during the purification procedure and enzymatic analysis was determined by a highly sensitive method based on registration of superhelical plasmid or phage DNA conversion into open circular or linear molecules.
Assuntos
Endodesoxirribonucleases/isolamento & purificação , Óvulo/enzimologia , Animais , Cátions Bivalentes , Endodesoxirribonucleases/metabolismo , Feminino , Peixes , Concentração de Íons de Hidrogênio , CinéticaRESUMO
The specificity of splitting of the cloned alpha A-globin gene from chicken erythrocytes induced by three topoisomerases I differing in molecular masses was demonstrated. The localization and relative number of topoisomerase breaks in the alpha A-globin gene vary in different topoisomerase I forms.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Globinas/genética , Animais , Bovinos , Galinhas , Clonagem Molecular , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Eritrócitos/análise , Globinas/metabolismo , Immunoblotting , Peso Molecular , Mapeamento de Nucleotídeos , PlasmídeosRESUMO
The hybridization kinetics of nuclear RNAs of loach embryos labelled with [3H]uridine for 1 hour with DNA excess shows that during embryogenesis (from the blastula to the gastrula stage) the number of newly formed RNA molecules transcribed from repeating DNA sequences in considerably reduced. This occurs both in the RNA fraction extracted from the nuclei with phenol pH 7.7 and having a low sedimentation coefficient and a low degree of polyadenylation, and in the RNA fraction extracted with phenol pH 9.0 having a higher sedimentation coefficient and a higher degree of polyadenylation.
Assuntos
Núcleo Celular/metabolismo , DNA/genética , RNA/genética , Animais , Blastocisto/metabolismo , Embrião não Mamífero , Peixes , Gástrula/metabolismo , Cinética , Hibridização de Ácido NucleicoRESUMO
The interaction of sera from 34 patients with different autoimmune diseases with the expressed fusion protein cloned in lambda gt11 vector (topoisomerase I--beta galactosidase) and monoclonal antibodies against enzyme was studied. It was demonstrated that 100% of Scl cases possessed positive activity against fusion protein. It was shown that this test is not absolutely specific for Scl, i. e. 57.1% of Sle and 84.6% of RA demonstrated positive activity against "topoisomerase test". Autoimmune sera had the positive activity against monoclonal antibodies for topoisomerase I. This activity was shown to be due to the presence of antiidiotypic antibodies against topoisomerase in the autoimmune sera.