RESUMO
Skin contamination by alpha-emitting actinides is a risk to workers during nuclear fuel production and reactor decommissioning. Also, the list of items for potential use in radiological dispersal devices includes plutonium and americium. The actinide chemical form is important and solvents such as tributyl phosphate, used to extract plutonium, can influence plutonium behavior. This study investigated skin fixation and efficacy of decontamination products for these actinide forms using viable pig skin in the Franz cell diffusion system. Commonly used or recommended decontamination products such as water, cleansing gel, diethylenetriamine pentaacetic acid, or octadentate hydroxypyridinone compound 3,4,3-LI(1,2-HOPO), as well as diethylenetriamine pentaacetic acid hydrogel formulations, were tested after a 2-h contact time with the contaminant. Analysis of skin samples demonstrated that more plutonium nitrate is bound to skin as compared to plutonium-tributyl phosphate, and fixation of americium to skin was also significant. The data show that for plutonium-tributyl phosphate all the products are effective ranging from 80 to 90% removal of this contaminant. This may be associated with damage to the skin by this complex and suggests a mechanical/wash-out action rather than chelation. For removal of americium and plutonium, both Trait Rouge cleansing gel and diethylenetriamine pentaacetic acid are better than water, and diethylenetriamine pentaacetic acid hydrogel is better than Osmogel. The different treatments, however, did not significantly affect the activity in deeper skin layers, which suggests a need for further improvement of decontamination procedures. The new diethylenetriamine pentaacetic acid hydrogel preparation was effective in removing americium, plutonium, and plutonium-tributyl phosphate from skin; such a formulation offers advantages and thus merits further assessment.
Assuntos
Elementos da Série Actinoide/efeitos adversos , Descontaminação/métodos , Géis/administração & dosagem , Ácido Pentético/administração & dosagem , Pele/efeitos dos fármacos , Água/administração & dosagem , Animais , Quelantes/administração & dosagem , Pele/efeitos da radiação , SuínosRESUMO
Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.
Assuntos
Proteínas Nucleares , Proteínas Proto-Oncogênicas c-fos/genética , Estabilidade de RNA/efeitos da radiação , RNA Mensageiro/metabolismo , Raios Ultravioleta , Animais , Morte Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta à Radiação , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Genes Precoces/efeitos da radiação , Genes fos/efeitos da radiação , Genes p53 , Meia-Vida , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
Skin contamination is one of the most probable risks following major nuclear or radiological incidents. However, accidents involving skin contamination with radionuclides may occur in the nuclear industry, in research laboratories and in nuclear medicine departments. This work aims to measure the penetration of the radiological contaminant Americium (241Am) in fresh and frozen skin and to evaluate the distribution of the contamination in the skin. Decontamination tests were performed using water, Fuller's earth and diethylene triamine pentaacetic acid (DTPA), which is the recommended treatment in case of skin contamination with actinides such as plutonium or americium. To assess these parameters, we used the Franz cell diffusion system with full-thickness skin obtained from pigs' ears, representative of human skin. Solutions of 241Am were deposited on the skin samples. The radioactivity content in each compartment and skin layers was measured after 24 h by liquid scintillation counting and alpha spectrophotometry. The Am cutaneous penetration to the receiver compartment is almost negligible in fresh and frozen skin. Multiple washings with water and DTPA recovered about 90% of the initial activity. The rest remains fixed mainly in the stratum corneum. Traces of activity were detected within the epidermis and dermis which is fixed and not accessible to the decontamination.
Assuntos
Amerício/toxicidade , Pele/efeitos dos fármacos , Compostos de Alumínio/química , Amerício/química , Animais , Autorradiografia , Descontaminação , Congelamento , Compostos de Magnésio/química , Ácido Pentético/química , Silicatos/química , Pele/metabolismo , Pele/patologia , SuínosRESUMO
An understanding of the "bioavailability" of disseminated radiocontaminants is a necessary adjunct in order to tailor treatment and to calculate dose. A simple test has been designed to predict the bioavailability of different actinide forms likely to be found after dissemination of radioactive elements by dispersal devices or nuclear reactor incidents. Plutonium (Pu) or Americium (Am) nitrate or MOX (U,PuO2) are immobilized in culture wells using a static gel phase simulating biological compartments (lung, wound, etc.). Gels are incubated in a fluid phase representing physiological media (plasma, sweat, etc.). Transfer of radionuclide from static to fluid phase reflects contaminant bioavailability. After 48 h of incubation in physiological saline, Am transfer from static to fluid phase was greater than for Pu (70% vs. 15% of initial activity). Transfer of Pu or Am was markedly less from the oxide form of the two elements (1% Am and 0.05% Pu transferred). Medium representing intracellular lysosomal fluid (pH 4) increased transfer of Pu and Am, whereas culture medium including serum reduced actinide transfer. Actinide transfer was also reduced by elements of the extracellular matrix present in the static gel phase. Increasing DTPA concentrations (5 to 500 µM) to the fluid phase significantly enhanced transfer of Pu and Am. Although this agarose gel cannot fully represent in vivo complexity, this simple test can be used to investigate and predict the behavior in vivo of radiocontaminants to support medical treatments and medical forensic investigations.
Assuntos
Amerício/análise , Bioensaio/métodos , Líquidos Corporais/química , Plutônio/análise , Radiometria/métodos , Amerício/química , Disponibilidade Biológica , Previsões/métodos , Humanos , Teste de Materiais/métodos , Plutônio/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Transgenic animal technology, and especially the use of germ line manipulation for gene targeting, offers new strategies for in vivo analysis of the mechanisms of DNA repair and cell cycle control underlying their efficiency. In vivo risk assessment of genotoxic agents is already facilitated by using transgenic mice. Knock-out mice provide an opportunity to study the function of the disrupted gene, to model human diseases related to this inactivation, and to determine the consequences of loss of function after a genotoxic exposure. These models might also allow the development of new gene therapy strategies.
Assuntos
Reparo do DNA/genética , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Animais , Ciclo Celular , Previsões , Humanos , Camundongos , Mutagênese/genéticaRESUMO
We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein. Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies. The cDNA fragment is 601 nucleotide long and was called KIN17(601). It contains an open reading frame coding for a 200 amino acid polypeptide. In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein. Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins.
Assuntos
DNA/genética , Recombinases Rec A/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência de Bases , Clonagem Molecular , Epitopos/imunologia , Genes Homeobox , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Recombinases Rec A/genética , Homologia de Sequência do Ácido NucleicoRESUMO
We used antibodies against E coli RecA protein to identify in mouse cells a 45-kDa DNA-binding protein called kin17, which has an active zinc finger and a nuclear localisation signal. Kin17 protein produced in E coli binds preferentially to the curved DNA of a bacterial promoter in vivo and in vitro, suggesting a transcriptional regulation activity. The fact that in rodent cells kin17 protein levels increase after gamma-irradiation suggests its participation in a cellular response to ionising radiation. We raised polyclonal antibodies against the whole kin17 protein and against its derived synthetic peptides. We report the detection of kin17 protein and of truncated forms of the protein by Western blot or by immunocytochemistry after transient overexpression in cultured human cells. Our results indicate that the cross-reactivity with the anti-RecA antibodies is due to an antigenic determinant located in the core of kin17 protein, between residues 129 and 228. The kin17 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm. The RecA homologous region seems to play an essential role in the localisation of kin17 protein since the deletion of this particular region dramatically changes the form and the distribution of the intranuclear foci. We hypothesise that these dot-like structures reflect nuclear metabolism compartmentalization.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas Nucleares , Animais , Western Blotting , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Camundongos , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/genética , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Recombinases Rec A/genética , Deleção de Sequência , Frações Subcelulares/metabolismo , Transfecção , Dedos de Zinco/genéticaRESUMO
We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Recombinases Rec A/imunologia , Animais , Anticorpos/imunologia , Cromossomos , Clonagem Molecular , Sequência Conservada , DNA Complementar , Proteínas de Ligação a DNA/genética , Genes , Mamíferos , Camundongos , Proteínas Nucleares/genética , Proteínas de Plantas/metabolismo , Proteínas/imunologiaRESUMO
Ionizing radiation elicits a genetic response in human cells that allows cell survival. The human KIN (also known as KIN17) gene encodes a 45-kDa nuclear DNA-binding protein that participates in the response to UVC radiation and is immunologically related to the bacterial RecA protein. We report for the first time that ionizing radiation and bleomycin, a radiomimetic drug, which produce single- and double-strand breaks, increased expression of KIN in human cells established from tumors, including MeWo melanoma, MCF7 breast adenocarcinoma, and ATM+ GM3657 lymphoblast cells. KIN expression increased rapidly in a dose-dependent manner after irradiation. Under the same conditions, several genes controlled by TP53 were induced with kinetics similar to that of KIN. Using the CDKN1A gene as a marker of TP53 responsiveness, we analyzed the up-regulation of KIN and showed that is independent of the status of TP53 and ATM. In contrast, the presence of a dominant mutant for activating transcription factor 2 (ATF2) completely abolished the up-regulation of KIN. Our results suggest a role for ATF2 in the TP53-independent increase in KIN expression after gamma irradiation.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Proteínas Nucleares , Proteína Supressora de Tumor p53/fisiologia , Fator 2 Ativador da Transcrição , Bleomicina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Dano ao DNA , Raios gama , Humanos , Proteínas de Ligação a RNA , Fatores de Transcrição/fisiologia , Células Tumorais CultivadasRESUMO
Kin17 is a mammalian nuclear protein sharing a slight sequence homology with the bacterial RecA protein. Kin17 has a zinc-finger motif and binds efficiently to curved DNA, a genomic topology associated with illegitimate recombination junctions. We investigated the relationship between the level of Kin17 protein and genomic alteration due to either impaired wild-type p53 functions or exposure to gamma rays. We used BP cells, a rodent epithelial cell system. The cell lines used were syngeneic and harbored wild-type or mutant p53 alleles and exhibited different sensitivities to gamma irradiation. In radioresistant cells (wild-type p53 genotype), the level of Kin17 protein peaked 30 min after a low dose of radiation (2 Gy), whereas maximum accumulation of p53 protein was observed 3 h postirradiation. Radiosensitive cells carrying the same mutation in both alleles of the p53 gene showed elevated basal levels of both Kin17 and p53 proteins and failed to accumulate Kin17 and p53 proteins after exposure to ionizing radiation. These cells exhibited enhanced cell death by apoptosis after gamma irradiation. Our results indicate that Kin17 protein accumulated immediately after DNA damage in cells carrying a wild-type p53 genotype, and that levels of constitutive Kin17 protein increased in highly proliferating tumorigenic cells when wild-type p53 functions were abrogated.
Assuntos
Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Proteínas de Ligação a DNA/biossíntese , Expressão Gênica/efeitos da radiação , Proteínas Nucleares , Proteína Supressora de Tumor p53/biossíntese , Animais , Benzo(a)pireno/toxicidade , Ciclo Celular/efeitos da radiação , Linhagem Celular , Radioisótopos de Cobalto , Relação Dose-Resposta à Radiação , Epitélio , Raios gama , Genes p53/efeitos da radiação , Cinética , Pulmão , Mutação Puntual , Ratos , Dedos de ZincoRESUMO
The human KIN17 protein is a chromatin-associated protein involved in DNA replication. Certain tumor cell lines overproduce KIN17 protein. Among 16 cell lines, the highest KIN17 protein level was observed in H1299 non-small cell lung cancer cells, whereas the lowest was detected in MeWo melanoma cells. Cells displaying higher KIN17 protein levels exhibited elevated RPA70 protein contents. High KIN17 protein levels may be a consequence of the tumorigenic phenotype or a prerequisite for tumor progression. Twenty-four hours after exposure to ionizing radiation, after the completion of DNA repair, a co-induction of chromatin-bound KIN17 and RPA70 proteins was detected. Etoposide, an inhibitor of topoisomerase II generating double-strand breaks, triggered the concentration of KIN17 into punctuate intranuclear foci. KIN17 may be associated with unrepaired DNA sites. Flow cytometry analysis revealed that 48 h after transfection the uppermost KIN17-positive RKO cells shifted in the cell cycle toward higher DNA content, suggesting that KIN17 protein induced defects in chromatin conformation. Cells displaying reduced levels of KIN17 transcript exhibited a sixfold increased radiosensitivity at 2 Gy. The KIN17 protein may be a component of the DNA replication machinery that participates in the cellular response to unrepaired DSBs, and an impaired KIN17 pathway leads to an increased sensitivity to ionizing radiation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares , Tolerância a Radiação , Animais , Núcleo Celular/metabolismo , Cromatina/química , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/análise , Ensaio de Imunoadsorção Enzimática , Etoposídeo/farmacologia , Raios gama , Humanos , Camundongos , Proteínas de Ligação a RNA , Células Tumorais CultivadasRESUMO
The recA gene was isolated from Streptomyces ambofaciens DSM40697. Its nucleotide sequence predicted a protein of 372 residues. Two recA mutants, NSAR1001 and NSAR57, obtained by gene disruption encoded a RecA protein lacking respectively 30 and at least 62 amino acids from the C-terminal end. NSAR1001 showed a wild-type sensitivity to UV light and oxolinic acid. In contrast, NSAR57 was highly sensitive to these agents and the loss of the inserted DNA restored the wild-type phenotype. Western blot analysis using antiserum to Escherichia coli RecA showed that overproduction of RecA was correlated with overtranscription of recA in an S. ambofaciens amplified mutant derived from genetic instability.
Assuntos
Recombinases Rec A/genética , Streptomyces/genética , Western Blotting , Dano ao DNA/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutagênese/fisiologia , Plasmídeos , RNA Bacteriano/genética , Recombinases Rec A/análise , Streptomyces/química , Transcrição Gênica/genéticaRESUMO
Kin17 and 8-Oxoguanine DNA glycosylase (Ogg1) are proteins, respectively, involved in illegitimate recombination and DNA repair in eukaryotic cells. To characterize the expression of these proteins in cell types of rodent and avian brains, we combined immunocytochemistry for either Kin17 or Ogg1 proteins with glial fibrillary acidic protein (GFAP, an astrocyte marker) immunodetection on the same tissue section. Both Kin17 and Ogg1 proteins were localized in cell nuclei and were extensively distributed in neuronal populations of quail and rodent brains. However, GFAP-immunoreactive cells were never labeled by Kin17 protein. This was observed in nerve fiber tracts, in the cerebral cortex, the hippocampal formation, the hypothalamic region, and the periventricular regions of the brain of both species studied. These results were confirmed by combining in situ hybridization of kin17 mRNA and GFAP immunodetection. On the contrary, GFAP-immunoreactive cells were often labeled by the Ogg1 protein in brain structures such as fiber tracts, the cortical surface, the cerebellum, and the ependymal surface of both quail and mouse brains. Our results suggest that the expression of the Kin17 protein (observed in neurons) and that of the Ogg1 protein (observed in neurons and glial cells) is conserved in brain phylogeny.
Assuntos
Sistema Nervoso Central/enzimologia , Proteínas de Ligação a DNA/metabolismo , N-Glicosil Hidrolases/metabolismo , Neuroglia/enzimologia , Neurônios/enzimologia , Proteínas Nucleares , Codorniz/metabolismo , Roedores/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Axônios/enzimologia , Axônios/ultraestrutura , Sistema Nervoso Central/citologia , Reparo do DNA/fisiologia , DNA-Formamidopirimidina Glicosilase , Epêndima/citologia , Epêndima/enzimologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Camundongos , Neuroglia/citologia , Neurônios/citologia , Codorniz/anatomia & histologia , Ratos , Ratos Sprague-Dawley , Roedores/anatomia & histologiaRESUMO
In eukaryotic cells, various proteins homologous to the E. coli RecA protein are involved in the elimination of DNA damage. These proteins contribute to the repair of double-strand breaks and to genetic recombination. The mouse Kin17 protein is recognised by antibodies directed against the RecA protein. Kin17 has a zinc-finger domain allowing binding to curved DNA stretching over illegitimate recombination junctions. In the present study, we identified the human counterpart of the mouse Kin17 protein (named HsKin17) in skin cells. We employed an in vitro reconstructed skin model composed of an epidermal sheath lying on a dermal matrix with human fibroblasts embedded in rat collagen type I. The maturation programme (proliferation versus differentiation) of keratinocytes was highly dependent on stromal cells. Immunohistochemical staining of frozen sections obtained from skin specimens was monitored by an interactive laser cytometer. In this way we analysed protein levels in both dermal and epidermal compartments. After having characterised the epithelium, we focused our attention on HsKin17 expression. We detected HsKin17 in human keratinocytes. HsKin17 protein levels increased in proliferating epithelial keratinocytes after 7 days of culture. After 2 weeks of culture, epidermal sheaths acquired most of the differentiated features of mature epithelium. At this time, HsKin17 protein dropped below measurable levels in the stratum corneum, and diminished in nucleated cells. This study showed that HsKin17 is expressed in human reconstructed epithelium under conditions of hyperproliferation.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas Nucleares , Pele/química , Adulto , Animais , Membrana Basal/citologia , Diferenciação Celular , Células Cultivadas , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Feminino , Humanos , Queratinócitos/química , Camundongos , Proteínas de Ligação a RNA , Ratos , Pele/citologiaRESUMO
A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.
Assuntos
Proteínas Nucleares/imunologia , Recombinases Rec A/imunologia , Animais , Western Blotting , Células Cultivadas , Reações Cruzadas , Haplorrinos , Humanos , Técnicas Imunológicas , Camundongos , Microscopia Eletrônica , Mitomicina , Mitomicinas/farmacologia , Peso Molecular , Membrana Nuclear/metabolismo , RatosRESUMO
UVC or ionizing radiation of mammalian cells elicits a complex genetic response that allows recovery and cell survival. Kin17 gene, which is highly conserved among mammals, is upregulated during this response. Kin17 gene encodes a 45 kDa protein which binds to DNA and presents a limited similarity with a functional domain of the bacterial RecA protein. Kin17 protein is accumulated in the nucleus of proliferating fibroblasts and forms intranuclear foci. Using expression vectors, we show that overexpression of kin17 protein inhibits cell-cycle progression into S phase. Our results indicate that growth inhibition correlates with disruption of the nuclear morphology which seems to modify the intranuclear network required during the early steps of DNA replication. We report that a mutant encoding a protein deleted from the central domain of kin17 protein enhanced these effects whereas the deletion of the C-terminal domain considerably reduced them. These mutants will be used to elucidate the molecular mechanism by which kin17 protein alters cell growth and DNA replication.
Assuntos
Núcleo Celular/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares , Animais , Antígenos Transformantes de Poliomavirus/genética , Bromodesoxiuridina/análise , Carcinoma Pulmonar de Células não Pequenas , Divisão Celular/genética , Núcleo Celular/química , Cromatina/genética , DNA Complementar/genética , Herança Extracromossômica , Deleção de Genes , Genes Dominantes , Células HeLa , Humanos , Neoplasias Pulmonares , Mamíferos , Mutagênese/fisiologia , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA , Recombinases Rec A/genética , Fase S/genética , Dedos de Zinco/genéticaRESUMO
Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis. UV-irradiation induced the synthesis of various proteins. Among them the analogue of the RecA protein of Escherichia coli (Angulo et al. 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.
Assuntos
Proteínas Fúngicas/biossíntese , Biossíntese Peptídica , Saccharomyces cerevisiae/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Raios UltravioletaRESUMO
In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K 12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD+) strains carrying mitochondrial DNA or not (rho0). This was also true for the following DNA-repair deficient (rad-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-delta, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.
Assuntos
Escherichia coli/genética , Recombinases Rec A/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Raios Ultravioleta , Anticorpos , Relação Dose-Resposta à Radiação , Genótipo , Cinética , Saccharomyces cerevisiae/genética , Especificidade da EspécieRESUMO
PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 plasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn10 out-going promoter located upstream of psiB. When we inserted a Tn10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient. In an F+ x F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasmid replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory phenotype.
Assuntos
Proteínas de Bactérias/biossíntese , Escherichia coli/genética , Fator F , Resposta SOS em Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Elementos de DNA Transponíveis , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Expressão Gênica , Mapeamento por Restrição , ZigotoRESUMO
We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response.