RESUMO
We have determined the nucleotide sequence of IS1162, a new insertion sequence (IS) isolated from Pseudomonas fluorescens (Pf) strain ST. This IS element is present in two copies on the pEG plasmid harboured by Pf ST and in a single copy on the chromosome, adjacent to the styrene catabolic genes. IS1162 is 2634 bp in length with 12-bp terminal inverted repeats (IR), and could encode four proteins (ORFs), two for each strand. One strand, Pro1 (62,990 Da), showed a helix-turn-helix motif at the N-terminal region, and Pro2 (25,997 Da) was characterized by the presence of the A and B motives of the NTP (ATP/GTP)-binding site. Comparison of IS1162 of Pf with known IS showed a high homology with IS408 of Burkholderia cepacia [Byrne and Lessie, Plasmid 31 (1994) 138-147]. Pro1 and Pro2 were found to be homologous to the corresponding ORFs of IS408, IS21 [Reimmann et al., Mol. Gen. Genet. 215 (1989) 416-424], IS232 [Menou et al., J. Bacteriol. 172 (1990) 6689-6696] and IS5376 [Xu et al., Plasmid 29 (1993) 1-9]. IS1162 transposed at low frequency and no cointegrates were found among the transposition products. The target duplication sites, variable in length, showed the presence of homologous motives, suggesting a certain degree of specificity of the IS1162 insertion site.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , Pseudomonas fluorescens/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , PlasmídeosRESUMO
In the Pseudomonas fluorescens strain ST the ability to utilize styrene as the sole carbon source is due to the presence of a plasmid, pEG. In the present report we show that pEG contains two inverted repeat sequences and we present evidence indicating that the catabolic genes are localized in these repeats. The region separating the inverted repeats can assume alternative orientations. In the chromosome of strain ST, a 3 kbp region is homologous to sequences present at one end of the plasmid repeats. This region is present in one copy in the chromosome and could be a specific site for integration of the plasmid. We suggest that this sequence, which is present twice in the pEG plasmid and once in the chromosome, might be a transposon-like element.
Assuntos
Plasmídeos , Pseudomonas fluorescens/metabolismo , Estirenos/metabolismo , Biodegradação Ambiental , Elementos de DNA Transponíveis , Genes Bacterianos , Família Multigênica , Pseudomonas fluorescens/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , EstirenoRESUMO
The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.
Assuntos
Nitrato Redutases/genética , Pseudomonas aeruginosa/genética , Southern Blotting , Western Blotting , Clonagem Molecular , Teste de Complementação Genética , Mutagênese , Nitrato Redutase , Nitrato Redutases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Mapeamento por RestriçãoRESUMO
Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.
Assuntos
Vetores Genéticos , Bactérias Gram-Negativas/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Replicon/genética , Sondas de DNA , Dados de Sequência Molecular , Origem de Replicação/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Preoperative serum tumour markers are currently classified as positive or negative according to a predetermined cut-off point. In the present study we examined the dynamic variation of marker levels after radical surgery of breast and colorectal cancer. CEA and CA15.3 were measured in 93 patients with breast cancer, CEA and CA19.9 in 97 patients with colorectal carcinoma before and 30 days after radical surgery. Any variation higher than 3-fold the analytical coefficient of variation of the assay was considered significant. In patients with negative preoperative marker levels a significant decrease was noted after surgery in 15.6% of cases for CEA and 27.8% for CA15.3 in breast cancer and in 46.8% for CEA and 25.7% for CA19.9 in colorectal cancer. Using both cut-off-based and dynamic criteria, we found an overall positivity rate of 19.6% for CEA and 33.3% for CA15.3 in breast cancer; 60.0% for CEA and 37.1% for CA19.9 in colorectal cancer. From the present findings we conclude that the dynamic study of perioperative variations of tumour markers is a sensitive method additional to cut-off-based criteria for the assessment of the phenotypic expression of the marker by the tumour.
Assuntos
Antígenos Glicosídicos Associados a Tumores/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Proteínas de Neoplasias/sangue , Neoplasias da Mama/cirurgia , Neoplasias Colorretais/cirurgia , Humanos , Período Pós-Operatório , Sensibilidade e EspecificidadeAssuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Queratinas/sangue , Neoplasias da Mama/sangue , Feminino , Doença da Mama Fibrocística/sangue , Doença da Mama Fibrocística/diagnóstico , Humanos , Ensaio Imunorradiométrico , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Valores de Referência , Antígeno Polipeptídico TecidualRESUMO
The pathogenetic processes underlying the selective motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are complex and still not completely understood even in the cases of inherited disease caused by mutations in the Cu/Zn superoxide dismutase-dependent (SOD1) gene. Recent evidence supports the view that ALS is not a cell-autonomous disease and that glial-neuron cross-talk, throughout cytokines and other toxic factors like the nitric oxide and superoxide, is a crucial determinant for the induction of motor neuron death. This cell-cell interaction may determine the progression of the disease through processes that are likely independent of the initial trigger and that may converge on the activation of intracellular death pathways in the motor neurons. In this review we provide support to the hypothesis that aberrant expression and activity of p38 mitogen protein-activated kinases cascade (p38MAPK) in motor neurons and glial cells may play a role in the development and progression of ALS. Increased activation of p38MAPK may phosphorylate neuron-specific substrates altering their physiological properties and it may turn on responsive genes leading to neurotoxicity.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Comunicação Celular/fisiologia , Neurônios Motores/metabolismo , Neuroglia/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Humanos , Neurônios Motores/patologia , Neuroglia/patologia , Receptor Cross-Talk/fisiologiaRESUMO
We have compared mitochondrial transcripts from yeast Saccharomyces cerevisiae strain D273-10B grown in the presence of 2% galactose (non-repressed cells) or 15% glucose (glucose-repressed cells). The ethidium-bromide-stained electrophoretic pattern of mitochondrial RNAs from glucose-repressed cells shows a clear decrease of tRNAs. In addition, some RNA bands appear to be specific for a single growth condition. To identify these RNA species we have performed hybridization experiments with 32P-labelled mitochondrial DNA from petite mutant cells. The mitochondrial repeat units of the mutants retained only one of the following genes: oxi1, oxi2, oxi3, oli2, cob and oli1. Unchanged amounts of oxi2 and oli2 transcripts and reduced concentrations of oli1 and oxi1 putative mRNAs are present in glucose-repressed cells. In the same growth condition we observe a decreased processing of a precursor RNA species from the split cob gene and reduced amounts of transcripts corresponding to the first, second and fifth intron of the split oxi3 gene. The oxi3 first and second introns, whose transcripts are the most variable, include long open reading frames in their nucleotide sequence, but at present it is not known whether the corresponding RNA species have a functional role. Our results show that their concentrations are related to the growth condition.
Assuntos
Glucose/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , DNA Fúngico/isolamento & purificação , Mutação , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/metabolismoRESUMO
In the "petite negative" yeast Kluyveromyces lactis (strain CBS 2360) growth is inhibited by the same Erythromycin concentrations on fermentable and non-fermentable carbon sources. The minimal inhibitory concentrations of Erythromycin does not affect respiration, but completely inhibits total aminoacid incorporation by the cells in conditions in which permeability effects can be ruled out. The effect of the antibiotic on the "in vitro" protein synthesis by cytoplasmic and mitochondrial systems shows that inhibition is effective only at the mitochondrial level. Results suggest that the inhibition of growth and of total protein synthesis is a consequence of the inhibition of mitochondrial protein synthesis.
Assuntos
Citoplasma/metabolismo , Eritromicina/farmacologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Leveduras/metabolismo , Sistema Livre de Células , Relação Dose-Resposta a Droga , Consumo de OxigênioRESUMO
Mitochondrial transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae. Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes. This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population. Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.
Assuntos
Glucose/farmacologia , Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae/genética , Transcrição Gênica , Autorradiografia , Fenômenos Químicos , Química , RNA/genética , RNA Fúngico/genética , RNA Mitocondrial , RNA de Transferência/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The transcripts of a cluster of eight tRNA genes localized in the Cap-oxiI region of the mitochondrial genome of Saccharomyces cerevisiae were investigated by hybridization of gene-specific probes on Northern blots of mitochondrial RNA and by S1 mapping of the 5' termini of the transcripts. Two rho- mutants that lack mature tRNA species and accumulate precursors have been used to detect transcripts that are not detectable in wild-type (w.t.) mitochondria. The results have shown the existence of polygenic transcripts carrying at least 5-7 tRNA sequences, both in w.t. and in rho- strains. The existence of several alternative processing pathways, which involve cleavage at the 3' and 5' ends of the tRNA sequences and in the long intergenic regions (possibly at GC clusters), is suggested. Cleavage at the 5' ends of tRNA sequences is defective in the mutant strains. The transcripts of the genes for tRNAThrACN and tRNACys (the tRNA genes immediately downstream from the 21S rRNA gene) have been analyzed; the possibility that these species represent primary transcripts is considered, and potential sites for initiation of transcription of the clustered tRNA genes are discussed.
Assuntos
DNA Mitocondrial/genética , Genes Fúngicos , Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Enzimas de Restrição do DNA , Endonucleases , Mutação , Hibridização de Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
A temperature sensitive mutation mapping in the tRNA region of the mitochondrial genome of S. cerevisiae has been found to abolish 3' processing of tRNA(asp). Mutant cells grown for a few generations at the non-permissive temperature were found to specifically lack mature tRNA(asp) and to accumulate 3' unprocessed precursors of this tRNA. The accumulation of precursors of other mitochondrial tRNAs was also observed under the same conditions. After longer incubation times, a generalized decrease of mitochondrial transcripts could be observed. The mutation was genetically mapped in a limited region surrounding the tRNA(asp) gene and found, by sequencing, to consist of a C- greater than T transition at position 61 of the tRNA(asp) gene.
Assuntos
DNA Mitocondrial/genética , Genes Fúngicos , Mitocôndrias/metabolismo , Mutação , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Ácido Aspártico/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mapeamento por Restrição , Saccharomyces cerevisiae/fisiologiaRESUMO
We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA(Asp) gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3' end-processing of the tRNA(Asp). Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3' end-processing. One such suppressor mutation was further characterized: it restores tRNA(Asp) maturation and growth at 36 degrees C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.
Assuntos
RNA Fúngico/genética , RNA de Transferência de Ácido Aspártico/genética , RNA/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Diploide , Genes Fúngicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenótipo , Mutação Puntual , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mitocondrial , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/metabolismo , Saccharomyces cerevisiae/metabolismo , Supressão GenéticaRESUMO
Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).
Assuntos
Nitrato Redutases/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas putida/genética , Azurina/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Concentração de Íons de Hidrogênio , Cinética , Nitrato Redutase , Nitrato Redutases/metabolismo , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.
Assuntos
Álcoois/toxicidade , Cetonas/toxicidade , Phanerochaete/efeitos dos fármacos , Pseudomonas putida/efeitos dos fármacos , Testes de Toxicidade/métodos , Álcoois/química , Biotransformação , Divisão Celular/efeitos dos fármacos , Ácidos Graxos/química , Cetonas/química , Estrutura Molecular , Phanerochaete/fisiologia , Pseudomonas putida/fisiologia , Fatores de TempoRESUMO
The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the beta-galactosidase reporter system. Expression of the promoter of the stySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescens ST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit beta-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and beta-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the beta-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon.
Assuntos
Regulação Bacteriana da Expressão Gênica , Óperon , Pseudomonas fluorescens/genética , Estireno/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Carbono/metabolismo , Genes Bacterianos , Óperon Lac , Família Multigênica , Regiões Promotoras Genéticas , Pseudomonas fluorescens/crescimento & desenvolvimento , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes de FusãoRESUMO
In this paper evidence is given that in a strain of Pseudomonas fluorescens able to grow on styrene as the sole carbon source, the degradation pathway of styrene is inducible and plasmid dependent. The plasmid, which we have called pEG is self-transmissible between Pseudomonas strains and has a size of 37 kb. A restriction map has been constructed and evidence for an inducible transcription of two separate regions of the plasmid has been obtained.
Assuntos
Plasmídeos , Pseudomonas fluorescens/genética , Estirenos/metabolismo , Mapeamento Cromossômico , Genes Bacterianos , Pseudomonas fluorescens/metabolismo , Estireno , Transcrição GênicaRESUMO
Hepatocellular carcinoma (HCC) is the most frequent malignant tumour of the liver. HCC has an incidence that changes with geographic areas (1.2-2.5% in western countries and 13-53% in Asia and Africa) as the risk of tumour bleeding. The patient arrives to the surgeon in emergency with no possibility of radical resection because of the patient's general conditions, the tumour's stage and the cirrhosis. Palliative treatments are: resection, direct suture of the bleeding tumour, artery embolization and selective binding of the hepatic artery. The Authors describe two cases of spontaneous rupture of HCC observed in the surgical department of Venice Hospital. A review of the literature is also reported.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Hemoperitônio/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Ruptura EspontâneaRESUMO
The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.
Assuntos
Genes Bacterianos , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Estirenos/metabolismo , Sequência de Bases , Sequência Consenso , DNA Bacteriano/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , EstirenoRESUMO
A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.