RESUMO
AIM: To evaluate the safety and efficacy of treatment of patients presenting with acute aneurysmal subarachnoid haemorrhage (SAH) with primary flow-diverting stents (FDS; with or without adjuncts), with comparison to the published literature. MATERIALS AND METHODS: A retrospective single-centre review was undertaken of prospectively obtained data on patients treated for SAH over a 60-month period. Of 354 patients treated for SAH during that time period, 24 patients with a total of 25 aneurysms were identified. Baseline patient demographics were recorded and clinical and imaging outcomes assessed. RESULTS: Eighty-eight per cent (22/25) of the aneurysms were completely occluded (Raymond-Roy 1) at mean 12-month follow-up. The minor complication rate was 12.5% (3/24) without permanent morbidity. Mortality rate was 4% (1/25) after one patient died following aneurysmal rebleed on day 7 post-procedure. Forty-two per cent (10/24) of patients had a high-pressure shunt placed prior to endovascular treatment, no haemorrhagic complications of neurosurgical intervention were observed. CONCLUSION: The necessity of dual antiplatelet therapy (DAPT) therapy when deploying FDS will rightly continue to limit their use in the acutely ruptured setting to a case-by-case basis whereby other treatment options are deemed unsafe. Methods employed to minimise subsequent haemorrhagic risks from DAPT in these patients may be worthy of further investigation.
Assuntos
Aneurisma Roto/cirurgia , Procedimentos Endovasculares , Aneurisma Intracraniano/cirurgia , Stents , Hemorragia Subaracnóidea/cirurgia , Adulto , Idoso , Aneurisma Roto/complicações , Aneurisma Roto/diagnóstico por imagem , Feminino , Humanos , Aneurisma Intracraniano/complicações , Aneurisma Intracraniano/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/etiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: alpha(2)-Adrenoceptor agonists are currently used as primary sedative agents in high dependency patients who are at high risk of sepsis. Clinical surveillance of such patients relies in part on their ability to mount appropriate responses to infection, in particular thermal responses. Thermoregulatory responses to infection are well studied in the rat and in this species, and humans, infection can induce febrile, hypothermic, or mixed hypothermic and febrile responses. The involvement of noradrenergic systems in thermal responses to infection prompted the hypothesis that ligands that act on adrenoceptors may interfere with the normal thermal responses to infection. METHODS: In this study on rats, the effect of infusion of the selective alpha(2)-agonist, mivazerol, on hypothermic and plasma corticosterone responses induced by bacterial lipopolysaccharide (LPS) was investigated. RESULTS: Clinically effective doses of mivazerol (4.8 and 10 microg kg(-1) h(-1)) had no effect on body temperature alone. However, mivazerol significantly inhibited the typical thermoregulatory response to bacterial LPS in a dose-dependent manner. This effect was mimicked by the selective alpha(2)-agonist, UK14304-18 (6 microg kg(-1) h(-1)), and antagonized by the alpha(2)-antagonist, RX811059A (7 microg kg(-1) h(-1)). The alpha(2)-ligands had no effect on basal or LPS-induced corticosterone levels. CONCLUSIONS: These data suggest that early thermoregulatory responses to infection can be selectively antagonized by ligands that activate alpha(2)-adrenoreceptors. High dependency patients receiving alpha(2)-adrenoceptor agonists may not be capable of mounting a normal thermal response to infecting organisms and clinical monitoring using core temperature to detect infection may therefore be unreliable in these vulnerable patients.
Assuntos
Agonistas alfa-Adrenérgicos/uso terapêutico , Infecções Bacterianas/complicações , Hipotermia/prevenção & controle , Imidazóis/uso terapêutico , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Infecções Bacterianas/sangue , Regulação da Temperatura Corporal/efeitos dos fármacos , Corticosterona/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Hipotermia/sangue , Hipotermia/microbiologia , Imidazóis/farmacologia , Ligantes , Lipopolissacarídeos , Masculino , Ratos , Ratos WistarRESUMO
One of the most useful tools for investigating the aetiopathology of cancer is the mutation spectrum, which comprises the type and distribution of mutations within a gene sequence. Many studies have generated mutagen-induced spectra using in vitro or in vivo model systems in an attempt to find correlations with those observed in cancer-associated genes such as the TP53 tumour suppressor gene. Consequently, meaningful similarities in the types of mutation found in induced and human spectra have been demonstrated. However, it is more difficult to draw such conclusions about the distribution or sequence context of mutations when they arise in different target sequences. We have developed an analytical approach for base substitution spectra that capture information for both sequence context and mutation type simultaneously. The resulting mutation signature is a fixed set of data points that allows comparison of multiple mutation spectra regardless of sequence. We have applied this method to a mixed set of mutation spectra observed in exons 5, 7 and 8 of TP53 from cancers of brain, breast, skin, colon, oesophagus, liver, head and neck, stomach and lung (smokers and non-smokers) and spectra induced by benzo[a]pyrene diol epoxide, ultraviolet (UV) B, UVC, simulated sunlight and hydroxyl radicals in the cII, supF and yeast p53 model systems. We demonstrate that this approach allows human cancer and mutagen-induced signatures to be grouped together according to similarity. Specifically, the analysis reveals key differences between smoking- and non-smoking-related lung cancer for TP53 mutations and the mutability of CpG sites between exons in skin cancer.
Assuntos
Análise Mutacional de DNA , Mutação , Neoplasias/etiologia , Neoplasias/genética , Animais , Animais Geneticamente Modificados , Carcinógenos , Humanos , Neoplasias Pulmonares/etiologia , Camundongos , Análise Multivariada , Mutagênicos , Neoplasias/patologia , Fumar/efeitos adversosRESUMO
N-Ethyl-N-nitrosourea-induced mutations occurring within a 180-base pair target in the lacI gene of Escherichia coli were characterized by DNA sequencing. In total, 109 mutations were characterized in a wild-type background and 100 in an excision-repair-deficient (UvrB-) background. The majority of mutations induced in the two backgrounds (77 and 85%, respectively) were G:C = greater than A:T transitions, presumably resulting from miscoding O6-ethylguanine lesions. A significant proportion of the mutations (17 and 15%, respectively) were A:T = greater than G:C transitions, which probably result from miscoding O4-ethylthymine lesions. An analysis of the distribution of both types of mutation in the two backgrounds reveals two distinct influences of neighboring base sequence. These effects apply equally to both the G:C = greater than A:T and A:T = greater than G:C transitions. Firstly, miscoding lesions are most likely to occur at 5'-purine-G-3' or 5'-purine-T-3' sites. Secondly, the excision-repair machinery is less efficient at removing both O6-ethylguanine and O4-ethylthymine lesions which are flanked on both sides by G:C base pairs. Thus, in the wild-type spectrum an overabundance of transitions occurs at a 5'-G-G-G/C-3' or 5'-G-T-G/C-3' sequence (where the mutated base is underlined).
Assuntos
Reparo do DNA , Mutação , Sequência de Bases , Escherichia coli/genética , Etilnitrosoureia/toxicidade , Guanina/análogos & derivados , Guanina/metabolismo , Metilnitronitrosoguanidina , Timina/análogos & derivados , Timina/metabolismoRESUMO
Plasmid DNA containing the human T24 H-ras oncogene, with or without viral transcriptional enhancer sequences, was applied to scarified mouse skin, followed by multiple treatments with the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. This resulted in the formation of vasoformative tumours histologically characterized as lymphangiosarcomas. All of the animals treated developed cystic fluid-filled swellings. Polymerase chain reaction analysis revealed the presence of human H-ras sequences within the cystic fluid from 3 out of 4 swellings. An endothelial cell line established from the cystic fluid removed from one of these swellings was found to contain human H-ras sequences and to express the mutant human p21ras. Injection of the cell line into nude mice, or adult syngeneic mice, resulted in the formation of aggressive angiosarcomas. Further experiments showed that 12-O-tetradecanol-phorbol-13-acetate promotion is not required for tumour formation and would appear to reduce the yield of tumours. These results indicate that a single application of the human H-ras oncogene is sufficient to induce endothelial cell transformation in vivo, even in the absence of any further promotional stimulus.
Assuntos
Transformação Celular Neoplásica , Clonagem Molecular/métodos , Genes ras , Neoplasias Experimentais/genética , Neoplasias Cutâneas/genética , Animais , Sequência de Bases , Southern Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Testes de Precipitina , Acetato de TetradecanoilforbolRESUMO
Functional alterations or loss of tumor-suppressor genes are an important feature of neoplastic progression in humans. The employment of suitable animal model systems would greatly facilitate the detection and manipulation of such genes. We describe here an experimental approach to this problem based on the analysis of skin tumors induced in F1 hybrids between Mus musculus and Mus spretus mice. The results show that loss of heterozygosity on chromosome 11 occurred in 4/13 mouse skin carcinomas, but not in premalignant papillomas. Since the murine p53 gene is located on this chromosome, immunoprecipitation and DNA-sequencing studies were carried out on tumorigenic cell lines and primary tumor DNA respectively to determine the status of p53 alleles. These studies revealed the presence of p53 mutations, both frameshifts and missense, some of which are identical to those found in human tumors. Loss of normal p53 function is found in well-differentiated squamous-cell carcinomas and thus does not appear to be directly responsible for further progression to an undifferentiated spindle cell phenotype.
Assuntos
Genes p53 , Heterozigoto , Mutação , Neoplasias Cutâneas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma/genética , Linhagem Celular , Códon/genética , Cruzamentos Genéticos , Mutação da Fase de Leitura , Íntrons , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Muridae , Transplante de Neoplasias , Oligodesoxirribonucleotídeos , Oligonucleotídeos Antissenso , Reação em Cadeia da Polimerase , Polimorfismo Genético , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced. In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations. This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine. The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis. This analysis revealed a strong influence by the 5' flanking base. On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.
Assuntos
Escherichia coli/genética , Genes Bacterianos/efeitos dos fármacos , Metilnitronitrosoguanidina/farmacologia , Mutação , Composição de Bases/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Escherichia coli/efeitos dos fármacosRESUMO
As the result of two mutually compensating frameshift mutations, three successive codons with third-position A were generated in the Neurospora crassa am (NADP-specific glutamate dehydrogenase: GDH) gene. These codons do not occur at all elsewhere in the gene and only infrequently in other highly expressed Neurospora genes. The double-frameshift strain produces only 25 to 35% of the normal level of GDH, whether measured as enzyme activity or as immunoprecipitable protein, but its level of GDH mRNA is normal. Although the modified enzyme is somewhat more heat-sensitive than the wild-type in vitro, its stability in vivo was found to be indistinguishable from that of the wild-type. It is concluded that the introduction of consecutive rare codons reduces the efficiency of translation of the mRNA. The possible mechanisms of such an effect are discussed.
Assuntos
Glutamato Desidrogenase/genética , Neurospora crassa/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Códon , DNA Fúngico , Eletroforese em Gel de Poliacrilamida , Mutação da Fase de Leitura , Glutamato Desidrogenase/metabolismo , Cinética , Dados de Sequência Molecular , Neurospora crassa/enzimologiaRESUMO
The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Mutação , Proteínas Repressoras , Fatores de Transcrição , Sequência de Aminoácidos , Dados de Sequência MolecularRESUMO
EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.
Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Metanossulfonato de Etila/farmacologia , Genes Bacterianos/efeitos dos fármacos , Mutação , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/efeitos da radiação , Especificidade da Espécie , Raios UltravioletaRESUMO
The acridine half-mustard ICR-170-induced reversion of the mutant am15, which has a single base-pair deletion, at a frequency of between 9 and 28 X 10(-6). In each of three classes of revertants, the mutagen had induced the insertion of a -G- -C- base pair at a -G-G- -C-C- site. The mutant am6, which has a single base pair insertion, is known to be revertible, with UV light, by deletion of a -G- -C- base pair at a -G-G-G- -C-C-C- site. This mutant reverted with ICR-170 at a frequency of 0.1 X 10(-6). These results show that ICR-170 is able to induce addition frameshifts in Neurospora crassa within short, monotonous runs of G:C base pairs, but indicate a lack of deletion activity at such sequences.
Assuntos
Aminoacridinas , Genes Fúngicos , Genes , Glutamato Desidrogenase/genética , Mutação , Neurospora crassa/genética , Neurospora/genética , Compostos de Mostarda Nitrogenada/farmacologia , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Neurospora crassa/enzimologiaRESUMO
Thirteen of 28 patients (46%) with grade 2-3 multifocal transitional cell carcinoma (TCC) of the bladder were found to have p53 mutations using DNA sequence analysis. These were subsequently utilized as tumor-specific biomarkers. Analysis of 17 episodes of recurrence from five of the patients revealed that all but one carried the identical mutation to the primary tumor. Thirty urine samples were collected, at initial diagnosis and during follow-up screening, from eight patients with mutations over a period of 24 months. Sequence analysis of PCR products generated from DNA extracted from the urine sediments was carried out. The p53 mutation seen in the primary tumors was detectable in 24 of 30 urine samples. The remaining six cases coincided with a negative cystoscopic examination. Interestingly, 6 of the 24 urine samples in which mutations were detectable also coincided with negative cystoscopy. The results are consistent with: (a) monoclonality of multifocal TCC; (b) the spread of TCC through a seeding mechanism; and (c) the long-term persistence of tumor cell clones (up to 97 months) within the bladder, even in the absence of obvious tumor growth.
Assuntos
Carcinoma de Células de Transição/genética , Genes p53/genética , Neoplasias da Bexiga Urinária/genética , Biomarcadores/urina , Carcinoma de Células de Transição/patologia , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , DNA de Neoplasias/urina , Humanos , Mutação , Recidiva Local de Neoplasia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Twenty-eight transitional cell carcinomas of the bladder, grade 2 or 3, were analyzed for the presence of p53 mutations. Thirteen tumors were found to contain 14 mutations. These were all base substitution mutations, of which nine were GC-->AT transitions (three at CpG sites). The remaining five mutations were transversions (three GC-->CG, one GC-->TA, and one AT-->TA). Four of the mutations were found at codon 280. A comparison with other studies of bladder tumors reveals that a region encompassing codons 280 and 285 represents a hot spot for p53 mutation in bladder cancer. The 280/285 hot spot lies within two purine-rich sequences that may provide some clues to the identity of potential bladder carcinogens. A comparison of mutations from bladder tumors of smokers and nonsmokers reveals no significant differences.
Assuntos
Carcinoma de Células de Transição/genética , Mutagênese Sítio-Dirigida/genética , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Carcinoma de Células de Transição/epidemiologia , Carcinoma de Células de Transição/patologia , Códon/genética , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Epidemiologia Molecular , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Fumar/efeitos adversos , Fumar/epidemiologia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/epidemiologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
This paper describes specific genetic changes involving chromosome 7 in mouse skin tumors, the most important consequence of which appears to be an alteration in the allelic balance of normal and mutant H-ras genes. The use of restriction-fragment-length polymorphisms in F1 hybrid mice demonstrates that trisomy of chromosome 7 is an early event preceding papilloma formation, and further events, such as mitotic recombination, seem to occur during progression to malignant carcinomas. There is some evidence of a tumor-suppressor locus situated on chromosome 7.
Assuntos
Carcinoma/genética , Genes Supressores de Tumor , Genes ras , Papiloma/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Cutâneas/genética , Alelos , Animais , Carcinoma/induzido quimicamente , Cromossomos/efeitos dos fármacos , Cromossomos/ultraestrutura , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hibridização Genética , Metilnitronitrosoguanidina , Camundongos , Papiloma/induzido quimicamente , Polimorfismo de Fragmento de Restrição , Recombinação Genética , Neoplasias Cutâneas/induzido quimicamente , TrissomiaRESUMO
The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E. coli by a series of alkylating agents has been analyzed. In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted. This preference was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species is the alkyl diazonium ion. In contrast, this preference was not seen following treatment with ethylmethanesulfonate. The observed preference for 5'PuG-3' site over 5'-PyG-3' sites corresponds well with alterations observed in the Ha-ras oncogene recovered after treatment with NMU. This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E. coli also appear to occur in mammalian cells.
Assuntos
Alquilantes/farmacologia , Dano ao DNA , Mutação , Composição de Bases , Sequência de Bases , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos/efeitos dos fármacos , Dados de Sequência Molecular , Proto-Oncogenes/efeitos dos fármacosRESUMO
Alkylating treatments predominantly induce G: C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G: C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.
Assuntos
Alquilantes/farmacologia , Reparo do DNA , DNA/efeitos dos fármacos , Mutação , Composição de Bases , Sequência de Bases , DNA/genética , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Dados de Sequência Molecular , Relação Estrutura-Atividade , Raios UltravioletaRESUMO
A two-probe technique has been developed to measure 125I in the thyroid. In this technique simultaneous emissions from a source are detected as coincidence events between two probes rather than as a sum peak in a single probe (the sum peak method). The background count rate when counting coincidence events between probes is substantially lower than the background in the sum peak region of a single probe. Theoretical equations which enable the activity of a source to be calculated are given. The applicability of these equations to thyroid measurements has been verified by measuring sources of various sizes, so that the geometric distribution and self-absorption of the sources were varied beyond the limits likely to be encountered in thyroid monitoring. Measurements on a simulated thyroid showed a variation in measured activity from +2% at a 5 cm source-to-detector distance to -4% at 10 cm. Measurements on a simulated thyroid containing 310 Bq (8.4 nCi) for a 500 s counting time gave a standard deviation of 21%. When the activity of the source was increased to 5500 Bq (149 nCi) the standard deviation was 6.2%.
Assuntos
Radioisótopos do Iodo/análise , Radiometria/métodos , Glândula Tireoide/análise , HumanosRESUMO
The use of animal models for human cancer has proved effective in the elucidation of those molecular events which are responsible for the various stages of tumour development. Chemical carcinogenesis in mouse skin has been studied as a model for human squamous cancer for several decades, and analysis of this model has led to the identification of a number of the changes which are involved in the evolution of malignancy. The use of transgenic and knockout mice offers a further avenue of advancement, allowing refinement of the model, and the ability to examine the consequences of individual events in vivo in greater detail. Additionally, crossing different transgenic or knockout animals represents a powerful tool to study the cumulative effects of several genetic alterations acting in concert.
Assuntos
Neoplasias Cutâneas/induzido quimicamente , Animais , Genes p53 , Genes ras , Humanos , Camundongos , Camundongos Transgênicos , Fator de Crescimento Transformador alfa/fisiologiaRESUMO
274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.
Assuntos
Escherichia coli/genética , Metilnitronitrosoguanidina/farmacologia , Mutação , Recombinases Rec A/genética , Composição de Bases , Sequência de Bases , Códon , DNA Bacteriano/biossíntese , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Glicina/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas Repressoras/genéticaRESUMO
Our approach to the study of how the molecular nature of DNA modulates the behavior of mutational sites involves the characterisation of distributions of mutations. The Escherichia coli lacI genetic/M13 cloning system allows the comparison of base substitution frequencies at a large number of sites. The observed distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced G:C----A:T transition (the predominant event), and A:T----G:C transition (a relatively rare event), is strikingly non-random. Some sites of G:C----A:T mutation are almost 100 times more often mutated by MNNG than the least susceptible sites. Sites of mutation, however, do not display a continuum of mutability, but rather can be strictly demarcated by their 5' flanking base. Sites with a high frequency of occurrence share a common sequence motif, namely 5'-R-G-N-3', which is the sole apparent feature that distinguishes them from sites less commonly mutated (i.e. 5'-Y-G-N-3'). A corollary of this defined site specificity is the absence of a strand bias in MNNG-induced lacI-d mutation. The availability of specific or non-specific alkylation-repair systems does not appear to alter the distribution of mutation, which suggests that the observed mutational distribution is a direct reflection of the initial damage distribution. MNNG does not belong to that class of compounds typified by ultraviolet light or 4-nitroquinoline-N-oxide which exhibit both random and non-random components of mutagenesis.