RESUMO
In order to realize superconductivity in cuprates with the T^{'}-type structure, not only chemical substitution (Ce doping) but also postgrowth reduction annealing is necessary. In the case of thin films, however, well-designed reduction annealing alone without Ce doping can induce superconductivity in the T^{'}-type cuprates. In order to unveil the origin of superconductivity in the Ce-undoped T^{'}-type cuprates, we have performed bulk-sensitive hard x-ray photoemission and soft x-ray absorption spectroscopy on superconducting and nonsuperconducting Nd_{2-x}Ce_{x}CuO_{4} (x=0, 0.15, and 0.19) thin films. By postgrowth annealing, core-level spectra exhibited dramatic changes, which we attributed to the enhancement of core-hole screening in the CuO_{2} plane and the shift of chemical potential along with changes in the band filling. The result suggests that the superconducting Nd_{2}CuO_{4} film is doped with electrons despite the absence of the Ce substitution.
RESUMO
The interface between LaAlO(3) and SrTiO(3) hosts a two-dimensional electron system of itinerant carriers, although both oxides are band insulators. Interface ferromagnetism coexisting with superconductivity has been found and attributed to local moments. Experimentally, it has been established that Ti 3d electrons are confined to the interface. Using soft x-ray angle-resolved resonant photoelectron spectroscopy we have directly mapped the interface states in k space. Our data demonstrate a charge dichotomy. A mobile fraction contributes to Fermi surface sheets, whereas a localized portion at higher binding energies is tentatively attributed to electrons trapped by O vacancies in the SrTiO(3). While photovoltage effects in the polar LaAlO(3) layers cannot be excluded, the apparent absence of surface-related Fermi surface sheets could also be fully reconciled in a recently proposed electronic reconstruction picture where the built-in potential in the LaAlO(3) is compensated by surface O vacancies serving also as a charge reservoir.
RESUMO
Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/fisiologia , Luz , Proteínas Nucleares/genética , Células Fotorreceptoras de Invertebrados , Animais , Proteínas de Ciclo Celular , Criptocromos , Retroalimentação , Flavoproteínas/genética , Regulação da Expressão Gênica , Hibridização In Situ , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Proteínas Circadianas Period , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G , Retina/metabolismo , Núcleo Supraquiasmático/metabolismo , Fatores de TranscriçãoRESUMO
We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.
RESUMO
UV damage endonuclease (UVDE) initiates a novel form of excision repair by introducing a nick imme-diately 5" to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts. Here, we report that apurinic/apyrimidinic (AP) sites are also nicked by Neurospora crassa and Schizosaccharomyces pombe UVDE. UVDE introduces a nick immediately 5" to the AP site leaving a 3"-OH and a 5"-phosphate AP. Apyrimidinic sites are more effectively nicked by UVDE than apurinic sites. UVDE also possesses 3"-repair activities for AP sites nicked by AP lyase and for 3"-phosphoglycolate produced by bleomycin. The Uvde gene introduced into Escherichia coli cells lacking two types of AP endonuclease, Exo III and Endo IV, gave the host cells resistance to methylmethane sulfonate and t-butyl hydroperoxide. We identified two AP endonuclease activities in S.pombe cell extracts. Besides cyclobutane pyrimidine dimers and 6-4 photoproducts, N. crassa UVDE also nicks Dewar photoproducts. Thus, UVDE is able to repair both of the major forms of DNA damage in living organisms: UV-induced DNA lesions and AP sites.
Assuntos
Ácido Apurínico/metabolismo , Carbono-Oxigênio Liases/metabolismo , Dano ao DNA/genética , DNA Glicosilases , Reparo do DNA/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Polinucleotídeos/metabolismo , Proteínas de Schizosaccharomyces pombe , Ácido Apurínico/genética , Bleomicina/farmacologia , Carbono-Oxigênio Liases/genética , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Endodesoxirribonucleases/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/genética , Deleção de Genes , Genes Bacterianos/genética , Teste de Complementação Genética , Glicolatos/metabolismo , Metanossulfonato de Metila/farmacologia , N-Glicosil Hidrolases/metabolismo , Neurospora crassa/enzimologia , Neurospora crassa/genética , Oligodesoxirribonucleotídeos/genética , Oxirredução , Polinucleotídeos/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Raios Ultravioleta , Uracila-DNA Glicosidase , terc-Butil Hidroperóxido/farmacologiaRESUMO
The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion. This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO. A candidate gene coding for the human counterpart of MutY has been cloned as hMYH. However, the function and enzyme activities of the gene product have not been identified. We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein. In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1). A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells. Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues. hMYH cDNA suppressed the mutator phenotype of E.coli mutY. In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate. The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis. These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing
Assuntos
Adenina/metabolismo , Pareamento Incorreto de Bases/genética , Núcleo Celular/enzimologia , DNA Glicosilases , Proteínas de Escherichia coli , N-Glicosil Hidrolases/metabolismo , Processamento Alternativo , Animais , Transporte Biológico , Células COS , Catálise , Reparo do DNA/genética , DNA-Formamidopirimidina Glicosilase , Escherichia coli/enzimologia , Escherichia coli/genética , Éxons/genética , Guanina/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Fenótipo , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Supressão Genética/genética , Timina/metabolismoRESUMO
The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C-->C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.
Assuntos
Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/fisiologia , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Guanina/análogos & derivados , Guanina/metabolismo , Pareamento Incorreto de Bases , Citosina/metabolismo , DNA-Formamidopirimidina Glicosilase , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Humanos , N-Glicosil Hidrolases/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Recombinação GenéticaRESUMO
We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair. By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins. Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity. Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.
Assuntos
Adenosina Trifosfatases , Pareamento Incorreto de Bases/fisiologia , Enzimas Reparadoras do DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA , Endonucleases , Proteínas de Saccharomyces cerevisiae , Alquilantes/farmacologia , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Células CHO/efeitos da radiação , Células Clonais , Cricetinae , Dano ao DNA , Resistência a Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Metilnitronitrosoguanidina/farmacologia , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Tolerância a Radiação/fisiologia , Células Tumorais Cultivadas/efeitos da radiação , Raios UltravioletaRESUMO
The drug-sensitive mutant UVS1, isolated from the Chinese hamster cell line CHO9, was previously found to complement the UV sensitivity of the excision repair-defective rodent mutants representative of groups 1 to 8 (Hata et al., Cancer Res., 51: 195-198, 1991; M. Numata et al., personal communication). Recently two new complementation groups of UV-sensitive CHO mutants, e.g., groups 9 and 10, have been identified (Stefanini et al., Cancer Res., 51: 3965-3971, 1991). In this paper we demonstrate that the repair defect in UVS1 cells is genetically different from those present in the mutants CHO7PV and CHO4PV, representing groups 9 and 10, respectively. Therefore, UVS1 represents a new complementation group of UV-sensitive rodent cell lines, the eleventh group.
Assuntos
Células CHO/efeitos da radiação , Reparo do DNA , DNA/efeitos da radiação , Raios Ultravioleta , Animais , Cricetinae , MutaçãoRESUMO
1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.
Assuntos
Dano ao DNA , Reparo do DNA , Nimustina/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Teste de Complementação Genética , Técnicas In Vitro , Mitomicina , Mitomicinas/farmacologia , Nimustina/metabolismo , Raios Ultravioleta , Raios XRESUMO
UVS1 is an intermediately UV-sensitive Chinese hamster ovary mutant originally isolated by its hypersensitivity to an anticancer drug, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride. By cell fusion analysis, UVS1 complemented the UV sensitivity of the mouse lymphoma cell line US31 from the eighth complementation group of UV-sensitive rodent cell lines. By enzyme-linked immunosorbent assay we found that within 3 h after UV irradiation both pyrimidine dimers and (6-4)photoproducts in UVS1 were not removed from chromosomal DNA in UVS1 at all. Twenty-four h after UV irradiation the removal rate of (6-4)photoproducts was intermediate between CHO9, the parental cell line, and 43-3B, a UV-hypersensitive Chinese hamster ovary mutant of the complementation group 1, whereas the pyrimidine dimers in UVS1 were removed less efficiently as 43-3B. Alkaline elution assay showed that the incising activity to damaged DNA after UV irradiation of UVS1 was as low as that of 43-3B. The number of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride-induced DNA interstrand cross-links of UVS1 was almost equal to that of 43-3B and about 1.5 times more than that of CHO9, suggesting that the gene products defective in UVS1 and 43-3B are essential for the excision repair of DNA damages produced by 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride.
Assuntos
Células CHO/efeitos da radiação , Dano ao DNA , Raios Ultravioleta , Animais , Células CHO/fisiologia , Linhagem Celular , Cricetinae , DNA/efeitos dos fármacos , DNA/genética , DNA/efeitos da radiação , Reparo do DNA , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/efeitos da radiação , Genoma , Cinética , Linfoma/genética , Linfoma/fisiopatologia , Camundongos , Mutação , Nimustina/farmacologia , Sensibilidade e Especificidade , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.
Assuntos
Apoptose , Reparo do DNA , Desoxirribodipirimidina Fotoliase/genética , Macropodidae/genética , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos da radiação , DNA/efeitos da radiação , Fragmentação do DNA , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células HeLa , Humanos , Microscopia de Fluorescência , Dímeros de Pirimidina , Fatores de Tempo , TransfecçãoRESUMO
8-Hydroxyguanine (8-OH-G) is one of the major DNA oxidation products implicated in mutagenesis induced by oxygen radical-forming agents, including ionizing radiation. It is also believed to be involved in spontaneous mutation induced by metabolically produced oxygen radicals. A mammalian homologue of 8-OH-G glycosylase/apurinic, apyrimidinic lyase (mutM homologue, MMH) has been identified in the EST database (for expressed sequence tags) through a homology search with yeast OGG1 protein. The human MMH protein (hMMH), 34% identical to the yeast OGG1 protein, is a member of the DNA repair protein superfamily. The hMMH gene was composed of seven exons, with the alternate last exon, exon 8, producing three major alternative splicing isoforms, because splicing of the sixth intron was optional. The hMMH protein expressed in Escherichia coli revealed the glycosylase activity and apurinic, apyrimidinic lyase activity on duplex DNA containing 8-OH-G. The hMMH protein can rescue a spontaneous mutator strain of E. coli lacking mutM and mutY. By the introduction of recombinant hMMH, the rate of mutation, the formation of rifampicin-resistant revertants, was reduced by 4-7 fold. Genomic structure analysis showed that 3' exons of the hMMH gene are transcribed on the antisense strand of the calcium-dependent calmodulin kinase 1 gene.
Assuntos
DNA Glicosilases , Proteínas de Escherichia coli , Liases/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética) , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA-Formamidopirimidina Glicosilase , Desoxirribonuclease IV (Fago T4-Induzido) , Éxons , Regulação Enzimológica da Expressão Gênica , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Liases/metabolismo , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Homologia de Sequência de AminoácidosRESUMO
Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.
Assuntos
Apoptose/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/deficiência , RNA Polimerases Dirigidas por DNA/metabolismo , Células Eucarióticas/enzimologia , Dímeros de Pirimidina/metabolismo , RNA/biossíntese , Animais , Apoptose/efeitos da radiação , Células CHO , Cricetinae , DNA Helicases , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Desoxirribodipirimidina Fotoliase/genética , Relação Dose-Resposta à Radiação , Células Eucarióticas/efeitos da radiação , Mutação/genética , Dímeros de Pirimidina/antagonistas & inibidores , RNA/genética , Raios UltravioletaRESUMO
The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.
Assuntos
Dano ao DNA/efeitos da radiação , Dímeros de Pirimidina/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Álcalis/metabolismo , Dano ao DNA/genética , Análise Mutacional de DNA , Desoxirribonuclease (Dímero de Pirimidina) , Relação Dose-Resposta à Radiação , Endodesoxirribonucleases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Genes p53/genética , Temperatura Alta , Humanos , Mutagênese/genética , Mutagênese/efeitos da radiação , Neurospora crassa , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/efeitos da radiação , Piperidinas/metabolismo , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Dímeros de Pirimidina/química , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.
Assuntos
Cianobactérias/enzimologia , Desoxirribodipirimidina Fotoliase/química , Cristalização , Difração de Raios XRESUMO
Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cmr nei::Kmr double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization.
Assuntos
Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Sequência de Aminoácidos , Animais , Southern Blotting , Carbono-Oxigênio Liases/metabolismo , Clonagem Molecular , DNA Glicosilases , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Endodesoxirribonucleases/química , Endodesoxirribonucleases/isolamento & purificação , Escherichia coli/genética , Éxons/genética , Etiquetas de Sequências Expressas , Humanos , Íntrons/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Fases de Leitura Aberta/genética , Mapeamento Físico do Cromossomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/genética , Homologia de Sequência de Aminoácidos , Timina/análogos & derivados , Timina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor , Ureia/metabolismoAssuntos
Gastroscopia/efeitos adversos , Exame Ginecológico/efeitos adversos , Diálise Peritoneal/efeitos adversos , Peritonite/etiologia , Amicacina/uso terapêutico , Antibacterianos/uso terapêutico , Antibioticoprofilaxia , Cilastatina/uso terapêutico , Combinação Imipenem e Cilastatina , Combinação de Medicamentos , Enterococcus/isolamento & purificação , Feminino , Humanos , Imipenem/uso terapêutico , Pessoa de Meia-Idade , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Prevenção Secundária , Resultado do Tratamento , Vancomicina/uso terapêuticoRESUMO
A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes Virais , Genes , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , Reparo do DNA , Enzimas de Restrição do DNA , Teste de Complementação Genética , Luz , Plasmídeos , Homologia de Sequência do Ácido NucleicoRESUMO
Garrett et al. [Mol. Gen. Genet. 225 (1991) 483-491] recently reported that an Atp2-lacZ fusion protein was transported into mitochondria in yeast, thus identifying the MFT1 (mitochondrial fusion targeting) gene as a genomic fragment which complements a mutation (mft1) that failed in targeting a fusion protein into mitochondria. They mapped this gene to the ORF, which we have independently identified as a gene homologous to the cyc07 gene, which is expressed specifically in the S phase during the plant cell cycle. We have mapped the MFT1 gene precisely and found that this gene should correspond to the neighboring ORF, rather than the ORF they identified.