RESUMO
Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx-1 and nlg-1, respectively. Here we show that nrx-1-deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx-1 mutants was markedly different to nlg-1-deficient mutants; thus, while the former had a 'hyper-reversal' phenotype increasing the number of changes of direction with respect to the wild-type strain, the nlg-1 mutants presented a 'hypo-reversal' phenotype. On the other hand, the nrx-1- and nlg-1-defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma-aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx-1-deficient mutants were rescued in transgenic strains expressing either human alpha- or beta-NRXN-1 isoforms under the worm nrx-1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Comportamento Exploratório , Mutação , Proteínas do Tecido Nervoso/metabolismo , Aldicarb/farmacologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/deficiência , Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Antagonistas GABAérgicos/farmacologia , Humanos , Levamisol/farmacologia , Movimento , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
No disponible
Assuntos
Humanos , Transtorno Autístico/genética , Polimorfismo de Nucleotídeo Único/genética , Amplificação de Genes/genética , Reação em Cadeia da PolimeraseRESUMO
The steroidal glycoalkaloid alpha-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and purified to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, beta-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by alpha-tomatine.
Assuntos
Fusarium/genética , Regulação Fúngica da Expressão Gênica , Peptídeo Sintases/genética , Sequência de Aminoácidos , Antifúngicos/farmacologia , Sequência de Bases , Clonagem Molecular , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Alinhamento de Sequência , Tomatina/farmacologiaRESUMO
Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and gamma-irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.
Assuntos
Arabidopsis/genética , DNA Complementar/genética , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli , Sequência de Aminoácidos , Arabidopsis/enzimologia , Northern Blotting , Carbono-Oxigênio Liases/metabolismo , Clonagem Molecular , DNA Glicosilases , Reparo do DNA , DNA Complementar/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Oxirredução , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Distribuição TecidualRESUMO
Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.
RESUMO
Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.
Assuntos
Fusarium/enzimologia , Fusarium/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Solanum lycopersicum/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Sequência de Carboidratos , Biblioteca Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tomatina/análogos & derivados , Tomatina/química , Tomatina/metabolismoRESUMO
The antifungal glycoalkaloid alpha-tomatine of the tomato plant (Lycopersicon esculentum) is proposed to protect the plant against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a vascular pathogen of tomato, produces a tomatinase enzyme which hydrolyses the glycoalkaloid into non-fungitoxic compounds. Detoxification of alpha-tomatine may be how this fungus avoids the plant glycoalkaloid barrier. As an initial step to evaluate this possibility we have studied the induction of tomatinase; (i) in fungal cultures containing extracts from leaf, stem or root of tomato plants; and (ii) in stem and root of tomato plants infected with the pathogen at different infection stages. The kinetics of tomatinase induction with leaf extract (0.6% dry weight) was similar to that observed with 20 micrograms ml-1 of alpha-tomatine. In the presence of stem extract, tomatinase activity was less than 50% of that induced with leaf extract, whereas in the presence of root extract tomatinase activity was very low. In the stem of infected tomato plants tomatinase activity was higher at the wilt stage than in previous infections stages and in root, tomatinase activity appeared with the first symptoms and was maintained until wilting. TLC analysis showed that the tomatinase induced in culture medium with plant extracts and in infected tomato plants had the same mode of action as the enzyme induced with pure alpha-tomatine, hydrolysing the glycoalkaloid into its non-fungitoxic forms, tomatidine and beta-lycotetraose. The antisera raised against purified tomatinase recognized in extracts of root and stem of infected tomato plants a protein of 50000 (45000 when proteins were deglycosylated), corresponding to the tomatinase enzyme. Therefore, it is concluded that F. oxysporum f. sp. lycopersici express tomatinase in vivo as a result of the infection of tomato plant.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Glicosídeo Hidrolases/metabolismo , Micoses/enzimologia , Doenças das Plantas/microbiologia , Solanum lycopersicum/microbiologia , Western Blotting , Sequência de Carboidratos , Cromatografia em Camada Fina , Indução Enzimática , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/biossíntese , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/biossíntese , Dados de Sequência Molecular , Micoses/microbiologiaRESUMO
The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.
Assuntos
Enzimas/isolamento & purificação , Fusarium/enzimologia , Glicosídeo Hidrolases/isolamento & purificação , Enzimas/metabolismo , Glicosídeo Hidrolases/metabolismo , Tomatina/metabolismoRESUMO
This work attempted to derive a quantitative relationship between mutagenicity and carcinogenicity by examining the association between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance. Lethality was measured at equivalent experimental conditions to those of mutant yield by using a mixed population of a pair of isogenic strains distinguished by their differential nutritional requirements. The study was carried out with a group of 11 direct-acting monofunctional alkylating agents, which failed to show any quantitative correlation in the histidine reverse-mutation test. Our data suggest that the mutagenic efficiency of the compounds is directly proportional to the magnitude of the maximum yield of L-arabinose resistance mutants and inversely proportional to the dose and the number of lethal hits at which the maximum yield occurs. A highly significant correlation (r10 = 0.86, P less than 0.01) was found between the mutagenic efficiencies of the compounds in the Ara test and their carcinogenic potencies in rodents, expressed as TD50 ('tumor dose' 50) values. The result suggests that the Ara forward-mutation test of S. typhimurium might be capable of reflecting the relative potency of animal carcinogens, at least when confined to particular chemical classes. A more generic and definitive conclusion about the predictive value of the Ara test would require this analysis to be extended to other types of genotoxic carcinogens.
Assuntos
Alquilantes/toxicidade , Testes de Carcinogenicidade , Testes de Mutagenicidade , Alquilantes/farmacologia , Animais , Arabinose/metabolismo , Marcadores Genéticos/análise , Mutação , Neoplasias Experimentais/patologia , Salmonella typhimurium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
S9 mix produces an effect similar to that of D-glucose in the L-arabinose resistance test of Salmonella typhimurium, releasing the growth inhibition exerted by L-arabinose. Two elements are responsible for this effect: the glucose-6-phosphate present in the cofactors of the S9 mix and the S9 fraction itself. UV light was used as a mutagen to compare the efficiency of S9 mix and D-glucose in allowing phenotypic expression of mutants in selective plates with L-arabinose; 0.5 ml of S9 mix per plate showed and efficacy similar to that of 0.5 mg of D-glucose per plate. To verify that the S9 mix is equivalent to D-glucose traces in selective plates with respect to the number of induced mutants in compounds requiring metabolic activation, we utilized 2 direct-acting nitrofurans. Our conclusion is that activation of agents could be erroneously attributed to the S9 mix, when plates with 0.5 mg of D-glucose are compared to plates with 0.5 ml of S9 mix plus 0.5 mg of D-glucose. Our results suggest that D-glucose traces be omitted in experiments requiring the presence of the S9 mixture.
Assuntos
Arabinose/farmacologia , Meios de Cultura/farmacologia , Expressão Gênica , Mutagênicos/metabolismo , Mutação , Salmonella typhimurium/genética , Animais , Resistência Microbiana a Medicamentos/genética , Glucose/farmacologia , Fígado/enzimologia , Testes de Mutagenicidade , Ratos , Salmonella typhimurium/efeitos dos fármacos , Raios UltravioletaRESUMO
A simple method is described for the determination of the lethal effects of chemicals assayed with the L-arabinose resistance test of Salmonella typhimurium. The method uses a mixed culture of 2 isogenic bacterial strains which are distinguished on the basis of their different nutritional requirements: sensitivity or resistance to L-arabinose, auxotrophy or prototrophy to histidine and leucine. BA13 (the mutation indicator strain) is the strain for routine screening of mutagens and allows the selection of forward mutation from L-arabinose sensitivity to L-arabinose resistance. BAL13 (the survival indicator strain) is a derivative of BA13. Both bacterial strains are found to be equally sensitive to the lethal effects of mutagens. The method described permits the measurement of cell survival at the same high cell concentration as used in the measurement of the mutant yield and in the same type of minimal medium with L-arabinose and glycerol, except for the histidine supplement in the mutant plates or the leucine in the survival plates.
Assuntos
Arabinose , Testes de Mutagenicidade , Mutagênicos , Salmonella typhimurium/genética , 2-Naftilamina/farmacocinética , 2-Naftilamina/toxicidade , Biotransformação , Resistência Microbiana a Medicamentos , Metanossulfonato de Etila/farmacocinética , Metanossulfonato de Etila/toxicidade , Metanossulfonato de Metila/farmacocinética , Metanossulfonato de Metila/toxicidade , Metilnitronitrosoguanidina/toxicidade , Mutagênicos/farmacocinéticaRESUMO
While delayed photoreversal (PR) mutagenesis has been interpreted as a measure of misincorporation step in targeted mutagenesis, the specificity to produce glutamine tRNA suppressor mutations (C to T transitions) at sites in DNA where a thymine-cytosine dimer (T = C) may target mutation suggests a deamination model: deamination T = C to T = U and trans-U DNA replication after PR. We describe here two enquires that did not support the latter model: (a) Uracil DNA glycosylase activity as estimated from the restricted plating efficiency of phage T5 containing uracil-substituted DNA showed no variation that might allow an exceptional opportunity for mutation at U in DNA, and (b). The kinetics of delayed PR mutagenesis were unaltered if UV-irradiated cells were held in buffer suspension for 2 h at 41 degrees C (a procedure known to allow deamination T = C to T = U) and then assayed. Other results with cells containing both umuC and ung (uracil DNA glycosylase) defects showed the magnitude of T = C deamination sufficient to provide T = U at the critical site of mutation to an extent greater than the mutation frequencies produced by delayed PR mutagenesis, and considerations of the kinetics led to the suggestion that the deamination model could apply if there were an optimum period 30-130 min post-UV for efficient recovery of DNA replication after PR. The results underscored the feasibility of delayed PR mutagenesis by deamination and trans-U replication, but a selection between the two models could not be determined.
Assuntos
Citosina , DNA Glicosilases , Reparo do DNA , N-Glicosil Hidrolases/metabolismo , Dímeros de Pirimidina , Sequência de Bases , Desaminação , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Mutação/efeitos da radiação , Supressão Genética , Fatores de Tempo , Uracila-DNA GlicosidaseRESUMO
UV mutation frequency responses for two types of Escherichia coli prototrophic mutant were measured. Only the response associated with a mutation targeted by a thymine-cytosine pyrimidine dimer was reduced in the dark in cells with amplified DNA photolyase. This specific reduction is attributed to the interruption of mutational DNA synthesis by a photolyase complex at the targeting dimer.
Assuntos
Desoxirribodipirimidina Fotoliase/metabolismo , Escherichia coli/efeitos da radiação , Liases/metabolismo , Mutação , Dímeros de Pirimidina , Raios Ultravioleta , Relação Dose-Resposta à Radiação , Escherichia coli/enzimologia , Escherichia coli/genética , Transcrição GênicaRESUMO
The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered. The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form. We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations. With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation. As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein). The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized.
Assuntos
Escherichia coli/efeitos da radiação , Genes Bacterianos/efeitos da radiação , Mutação , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Tirosina/genética , Supressão Genética/efeitos da radiação , Raios Ultravioleta , Escherichia coli/genética , RNA de Transferência de Tirosina/efeitos da radiaçãoRESUMO
UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photoreversal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective alpha subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective epsilon proofreading subunit) had any effect on the misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of epsilon proofreading activity by recA protein are possible. In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3'-5' proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.
Assuntos
DNA Polimerase III/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Mutação/efeitos da radiação , Escherichia coli/efeitos da radiação , Raios UltravioletaRESUMO
We introduced the dnaE486 and polC74 mutations (which are associated with decreased DNA polymerase III replication fidelity) into excision defective Escherichia coli strains with varying SOS responses. These mutations increased the UV-induced frequency of base pair substitution mutations in all strains tested, except recA430 and umuC122 derivatives. This UV mutator effect therefore requires expression of the SOS error-prone repair system. In recA441 lexA51 strains where the SOS system is constitutively expressed, the UV mutator effect of the dnaE alleles was similar in relative terms (though greater in absolute terms). Since these dnaE alleles decrease rather than increase survival after UV it is argued that they promote a burst of untargeted mutations close to UV photoproducts ("hitch-hiking" mutations) rather than increase the number of translesion synthesis events. The fact that there was no UV mutagenesis in dnaE486 umuC122 or polC74 umuC122 strains indicates that infidelity associated with these dnaE alleles did not of itself enable translesion synthesis to occur. The spontaneous mutator effect conferred by dnaE486 and polC74 was not affected by umuC122 or recA430 indicating that it is not dependent upon error-prone repair ability. In recA441 lexA51 bacteria, where SOS error-prone repair is constitutively induced, the mutator effect of dnaE486 was greater and was largely blocked by umuC122. It is suggested that spontaneously occurring cryptic lesions that are themselves unable to induce the SOS system are subject to translesion synthesis under these conditions and trigger a burst of hitch-hiking mutations that are therefore effectively umuC dependent.
Assuntos
Alelos , DNA Polimerase III/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Mutação , Raios Ultravioleta , Relação Dose-Resposta à Radiação , Escherichia coli/efeitos da radiação , Genes/efeitos da radiação , Genes Bacterianos/efeitos da radiação , Genótipo , CinéticaRESUMO
Prototrophic mutants produced by UV light in Escherichia coli K-12 strains with argE3(Oc) and hisG4(Oc) defects are distinguished as backmutations and specific nonsense suppressor mutations. In strains carrying a umuC defect, mutants are not produced unless irradiated cells are incubated and then exposed to photoreversing light (delayed photoreversal mutagenesis). The mutants thus produced are found to be specifically suppressor mutations and not backmutations. The suppressor mutations are primarily glutamine tRNA ochre suppressor mutations, which have been attributed previously to mutation targeted at T = C pyrimidine dimers. In a lexA51 recA441 strain, where the SOS mutagenesis functions are constitutive, targeting at dimers is confirmed by demonstrating that the induction of glutamine tRNA suppressor mutations is susceptible to photoreversal. In the same strain induction of backmutations is not susceptible to photoreversal. Thus delayed photoreversal mutagenesis produces suppressor mutations that can be targeted at pyrimidine dimers and does not produce backmutations that are not targeted at pyrimidine dimers. This correlation supports the idea that delayed photoreversal mutagenesis in umuC defective cells reflects a mutation process arrested at a targeting pyrimidine dimer photoproduct, which is the immediate cause of both the alteration in DNA sequence and the obstruction (unless repaired) to mutation fixation and ultimate expression.
Assuntos
Escherichia coli/genética , Mutação , Dímeros de Pirimidina/genética , Supressão Genética , Arginina/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Histidina/metabolismo , Luz , Aminoacil-RNA de Transferência/genética , Raios UltravioletaRESUMO
UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions. Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.
Assuntos
Escherichia coli/genética , Mutação , Pirimidinas/metabolismo , Reparo do DNA , DNA Bacteriano/efeitos da radiação , Luz , Polímeros , Recombinases Rec A/metabolismo , Proteínas Repressoras/metabolismo , Raios UltravioletaRESUMO
The mutagenicity of pyrazole and seven pyrazole derivatives (4-nitropyrazole, 4-bromopyrazole, 1-methyl-4-nitropyrazole, 3,5-dimethyl-4-nitropyrazole, 1-methyl-4-bromopyrazole, 4,4'-dinitro-1, 1'-methylene-dipyrazole and 4,4'-dibromo-1,1'-methylene-dipyrazole) has been investigated with the L-arabinose forward mutation assay of Salmonella typhimurium. Two nitroimidazoles (1-methyl-5-nitroimidazole and metronidazole) were included as reference drugs. The mutagenicity of each chemical was determined by both preincubation and liquid tests, in the presence or absence of S9 microsomal fraction. The mutagenic response was expressed as the absolute number of L-arabinose resistant mutants growing in selective plates, supplemented with traces of D-glucose. Strain BA13 with a wildtype lipopolysaccharide barrier was used as a comparison to the deep rough derivative BA9. No mutagenic effect was detected with pyrazole and two of its derivatives, 1-methyl-4-bromopyrazole and 4,4'-dibromo-1,1'-methylene-dipyrazole. The other five pyrazole derivatives were mutagenic to different degrees, although their mutagenic potencies were always considerably lower than those of the two nitroimidazoles. The results suggest that 4-nitropyrazoles, as well as 4,4'-dinitro-1, 1'-methylene-dipyrazoles, should be investigated further as alternatives to, or even substitutes for, the currently used nitroimidazoles.
Assuntos
Arabinose/farmacologia , Mutagênicos , Nitroimidazóis/toxicidade , Pirazóis/toxicidade , Antitricômonas/toxicidade , Resistência Microbiana a Medicamentos , Testes de Mutagenicidade , Salmonella typhimurium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Strain TA102 of S. typhimurium is a new histidine-requiring mutant, particularly suited to the detection of oxidative mutagens acting at A.T base pairs. 10 oxidizing chemicals, previously tested in strain TA102, were used to evaluate the mutagenic sensitivity of the L-arabinose forward mutation assay of S. typhimurium with respect to those types of mutagens. The mutagenicity of each compound was determined by liquid test, measuring both the frequency of mutants among the survivors and the absolute number of mutants growing in selective plates with traces of D-glucose. Strain BA13 with a wild-type lipopolysaccharide barrier was used as compared to the deep rough derivative strain BA9. The chemicals studied were: bleomycin, t-butyl hydroperoxide, chromium trioxide, cumene hydroperoxide, formaldehyde, glyoxal, glutaraldehyde, hydrogen peroxide, paraquat, and phenylhydrazine. Additionally, ultrasonic oscillation was used as a presumable non-mutagenic lethal control treatment. The L-arabinose forward mutation assay detected the mutagenic activity of all the chemicals under study with a high degree of sensitivity, including paraquat which is unable to revert strain TA102. Positive responses were obtained at doses equivalent to or 10 times lower than the doses detected by strain TA102. The results support the idea that the L-arabinose forward mutation assay could replace the set of specific tester strains used by the histidine reverse mutation assay in general screening for genetic toxins.