RESUMO
Germline DNA alterations affecting homologous recombination pathway genes have been associated with pancreatic cancer (PC) risk. BRCA2 is the most studied gene and affects the management of PC patients and their families. Even though recent reports have suggested a similar role of germline ATM pathogenic variants (PV) in familial PC, there is still a disagreement between experts on how it could affect patient management given the lack of proper PC risk estimates. We retrospectively analyzed the germline data of 257 PC patients among whom nearly 50% were sporadic cases. We showed similar frequencies of BRCA2 (4.9%) and ATM (4.4%) PV or likely pathogenic variants, which were not related to familial history. Based on our findings and that of the literature, we suggest including ATM gene among the panel of genes analyzed in PC patients pending the publication of prospective studies.
Assuntos
Predisposição Genética para Doença , Neoplasias Pancreáticas , Humanos , Estudos Retrospectivos , Estudos Prospectivos , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologiaRESUMO
Vegetation cover can be used in the phytomanagement of polluted areas by adding value to abandoned sites and reducing the dispersion of pollutants by erosion. Appropriate amendments, that allow both efficient plant growth and the immobilization of contaminants in the soil must be chosen in order to optimize the efficiency of this process. We used a mining technosol mainly contaminated by arsenic (1068â¯mgâ¯kg-1) and lead (23387â¯mgâ¯kg-1) to study the effect of three amendments (biochar, compost and iron grit) on (i) physico-chemical properties of the soil and soil pore water, (ii) metal(loid) mobility, bioavailability and bioaccessibility (CaCl2 and Simple Bioaccessibility Extraction Test (SBET)), and (iii) the capability of Trifolium repens to germinate and grow. All the amendments used increased the pH and electrical conductivity of the SPW, resulting in a 90% decrease in the concentration of lead in the soil pore water (SPW). We also demonstrated a decrease in Pb phytoavailability. The amendments allowed the establishment of a plant cover, although the addition of iron grit alone did not allow any clover germination. For the Pontgibaud technosol, the combination of the three amendments resulted in a significant decrease in As and Pb concentrations in clover tissues, mainly in the aerial organs. The amendments also made it possible for some of them to halve the phytoavailable fraction of arsenic. However, for compost, both the As concentrations in the SPW, and the bioavailable fraction of As increased. All the amendments used had contrasting effects on the bioaccessible fractions of metal(loid)s. The most efficient amendment combination was the addition of 5% biochar and 5% compost.
Assuntos
Arsênio/química , Carvão Vegetal/química , Recuperação e Remediação Ambiental/métodos , Chumbo/química , Poluentes do Solo/química , Trifolium/química , Compostagem , Ferro/química , Mineração , Solo/química , Poluentes do Solo/análiseRESUMO
The threat of bacterial resistance to antibiotics has created an urgent need to develop new antimicrobials. The aim of this study was to characterize the chemical diversity of Litsea cubeba leaf essential oil (EO) and its impacts on the antibacterial activity against pathogenic bacteria. Essential oils collected from seven provinces in North Vietnam (n = 25) were characterized by their high content in either 1,8-cineole or linalool. Linalool-type EOs were more effective against the eight bacterial strains tested than 1,8-cineole-type. Oil samples, LC19 (50% 1,8-cineole) and BV27 (94% linalool), were selected to investigate their antibacterial mechanisms against Escherichia coli. A strong bactericidal effect was observed after 4 and 2 h of exposure respectively. Microscopic analysis of treated E. coli cultures clearly showed that EOs caused changes in cell morphology, loss of integrity and permeability of the cell membrane, as well as DNA loss. However, the effects of both EOs were distinct. LC19 mostly affected cell membrane, led to a significant cell filamentation rate and altered cell width, whereas BV27 damaged cell membrane integrity leading to cell permeabilization and altered nucleoid morphology with the appearance of spot and visibly altered compaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study aimed to characterize the chemical diversity of Litsea cubeba leaf essential oil (EO) and its impacts on its antibacterial activity. Two major chemotypes (1,8-cineole or linalool rich) were identified in North Vietnam and both were bactericidal against several pathogenic bacteria. A distinct inhibitory effect of EO samples on Escherichia coli was observed. 1,8-cineole-rich sample (LC19) affected cell membrane, led to cell filamentation and perturbation of cell width, while the linalool-rich one (BV27) induced damages in the cell membrane and changes in the nucleoid morphology. The study demonstrates the importance of considering chemotype variations in terms of chemical composition as well as the mode of action.
Assuntos
Antibacterianos/farmacologia , Cicloexanóis/farmacologia , Escherichia coli/efeitos dos fármacos , Litsea/química , Monoterpenos/farmacologia , Óleos Voláteis/farmacologia , Monoterpenos Acíclicos , Membrana Celular/efeitos dos fármacos , Eucaliptol , Testes de Sensibilidade Microbiana , Folhas de Planta/química , VietnãRESUMO
Species of Pyricularia (magnaporthe-like sexual morphs) are responsible for major diseases on grasses. Pyricularia oryzae (sexual morph Magnaporthe oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, while Pyricularia grisea (sexual morph Magnaporthe grisea) is responsible for foliar diseases of Digitaria. Magnaporthe salvinii, M. poae and M. rhizophila produce asexual spores that differ from those of Pyricularia sensu stricto that has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation. Magnaporthe salvinii was recently allocated to Nakataea, while M. poae and M. rhizophila were placed in Magnaporthiopsis. To clarify the taxonomic relationships among species that are magnaporthe- or pyricularia-like in morphology, we analysed phylogenetic relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as LSU, ITS, RPB1, actin and calmodulin. Species of Pyricularia s. str. belong to a monophyletic clade that includes all P. oryzae/P. grisea isolates tested, defining the Pyriculariaceae, which is sister to the Ophioceraceae, representing two novel families. These clades are clearly distinct from species belonging to the Gaeumannomyces pro parte/Magnaporthiopsis/Nakataea generic complex that are monophyletic and define the Magnaporthaceae. A few magnaporthe- and pyricularia-like species are unrelated to Magnaporthaceae and Pyriculariaceae. Pyricularia oryzae/P. grisea isolates cluster into two related clades. Host plants such as Eleusine, Oryza, Setaria or Triticum were exclusively infected by isolates from P. oryzae, while some host plant such as Cenchrus, Echinochloa, Lolium, Pennisetum or Zingiber were infected by different Pyricularia species. This demonstrates that host range cannot be used as taxonomic criterion without extensive pathotyping. Our results also show that the typical pyriform, 2-septate conidium morphology of P. grisea/P. oryzae is restricted to Pyricularia and Neopyricularia, while most other genera have obclavate to more ellipsoid 2-septate conidia. Some related genera (Deightoniella, Macgarvieomyces) have evolved 1-septate conidia. Therefore, conidium morphology cannot be used as taxonomic criterion at generic level without phylogenetic data. We also identified 10 novel genera, and seven novel species. A re-evaluation of generic and species concepts within Pyriculariaceae is presented, and novelties are proposed based on morphological and phylogenetic data.
RESUMO
Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.
Assuntos
Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Membranas Intracelulares/metabolismo , Lipoilação , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Western Blotting , Células Cultivadas , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Citosol/metabolismo , Fibroblastos/citologia , Imunofluorescência , Proteínas de Choque Térmico HSP20/genética , Humanos , Imunoprecipitação , Mutação/genética , Transporte Proteico , Transdução de Sinais , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/metabolismoRESUMO
CONTEXT: Heterozygous GNAS inactivating mutations are known to induce pseudohypoparathyroidism type 1a when maternally inherited and pseudopseudohypoparathyroidism when paternally inherited. Progressive osseous heteroplasia (POH) is a rare disease of ectopic bone formation, and studies in different families have shown that POH is also caused by paternally inherited GNAS mutations. OBJECTIVE: Our purpose was to characterize parental origin of the mutated allele in de novo cases of POH and to draw phenotype/genotype correlations according to maternal or paternal transmission of a same GNAS mutation. DESIGN AND SETTING: We conducted a retrospective study on patients addressed to our referral center for the rare diseases of calcium and phosphorus metabolism. PATIENTS AND METHODS: We matched 10 cases of POH with cases of pseudohypoparathyroidism type 1a carrying the same GNAS mutations. MAIN OUTCOME MEASURES: The parental origin of the mutated allele was studied using informative intragenic polymorphisms and subcloning of PCR products. RESULTS: Paternal origin of GNAS mutations was clearly demonstrated in eight POH cases including one patient with mutation in exon 1. Genotype/phenotype analyses suggest that there is no direct correlation between the ossifying process and the position of the inactivating GNAS mutation. It is, however, more severe in patients in whom origin of the mutation is paternal. Severe intrauterine growth retardation was clearly evidenced in paternally inherited mutations. CONCLUSIONS: Clinical heterogeneity makes genetic counseling a delicate matter, especially in which paternal inheritance is concerned because it can lead to either a mild expression of pseudopseudohypoparathyroidism or a severe expression of POH.
Assuntos
Osso e Ossos , Coristoma/genética , Coristoma/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Mutação/genética , Mutação/fisiologia , Criança , Pré-Escolar , Cromograninas , Metilação de DNA , Bases de Dados Genéticas , Feminino , Impressão Genômica , Genótipo , Humanos , Lactente , Masculino , Hormônio Paratireóideo/fisiologia , Linhagem , Polimorfismo de Nucleotídeo Único , Pseudo-Hipoparatireoidismo/genética , Pseudopseudo-Hipoparatireoidismo/genética , RNA/genéticaAssuntos
Abscesso/diagnóstico por imagem , Valva Aórtica , Bioprótese/efeitos adversos , Endocardite Bacteriana/diagnóstico por imagem , Próteses Valvulares Cardíacas/efeitos adversos , Infecções Relacionadas à Prótese/diagnóstico por imagem , Abscesso/tratamento farmacológico , Abscesso/cirurgia , Anti-Infecciosos/uso terapêutico , Insuficiência da Valva Aórtica/diagnóstico por imagem , Insuficiência da Valva Aórtica/tratamento farmacológico , Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/cirurgia , Ecocardiografia Transesofagiana , Endocardite Bacteriana/tratamento farmacológico , Endocardite Bacteriana/cirurgia , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-Idade , Falha de Prótese , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/cirurgia , ReoperaçãoRESUMO
Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.
Assuntos
Proteínas Fúngicas/química , Fungos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Sequência de Aminoácidos , Southern Blotting , Botrytis/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Éxons , Etiquetas de Sequências Expressas , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Íntrons , Magnaporthe/metabolismo , Dados de Sequência Molecular , Neurospora crassa/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , SoftwareRESUMO
Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.
Assuntos
Actomiosina/fisiologia , Proteínas de Protozoários/fisiologia , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Actomiosina/química , Sequência de Aminoácidos , Animais , Apicomplexa/fisiologia , Apicomplexa/ultraestrutura , Adesão Celular/fisiologia , Citoesqueleto , Interações Hospedeiro-Parasita , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Movimento , Organelas/química , Fagocitose , Dobramento de Proteína , Infecções por Protozoários/parasitologia , Proteínas de Protozoários/química , Alinhamento de Sequência , Toxoplasma/ultraestruturaRESUMO
Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.
Assuntos
Cromatina/fisiologia , Proteína HMGA1a , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Níquel/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/fisiologia , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Resistência a Medicamentos , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas de Grupo de Alta Mobilidade/química , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/fisiologiaRESUMO
Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms. Plant protection against metal toxicity occurs, at least in part, through control of root metal uptake and of long distance metal transport. Inside cells, proteins such as ferritins and metallothioneins, and glutathion-derived peptides named phytochelatins, participate in excess metal storage and detoxification. Low molecular weight organic molecules, mainly organic acids and amino acids and their derivatives, also play an important role in plant metal homeostasis. When these systems are overloaded, oxidative stress defense mechanisms are activated. Molecular and cellular knowledge of these processes will be necessary to improve plant metal resistance. Occurrence of naturally tolerant plants which hyperaccumulate metals provides helpful tools for this research.
Assuntos
Metais/toxicidade , Fenômenos Fisiológicos Vegetais , Plantas/efeitos dos fármacos , Metais/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas , Plantas/genéticaRESUMO
A complementation approach of the yeast fet3fet4 mutant strain, defective in both low- and high-affinity iron transport, was initiated as an attempt to characterize the Fe(III)-mugineic acid (MA) transporter from grasses. A maize cDNA encoding a novel MYC transcription factor, named 7E, was cloned by screening an iron-deficient maize root cDNA expression library on a minimum media containing Fe(III)-deoxyMA as a unique iron source. 7E expression restored growth specifically to the fet3 fet4 mutant strain. It did not affect growth rate of a trk1trk2 potassium transport defective yeast strain or parental W303 strain growth rate. No 55Fe uptake increase was observed in 7E transformed fet3 fet4 yeast during short-term kinetics. However, the iron accumulation in these cells was 1.3-fold higher than in untransformed cells after a 24-h period. The 7E protein contained 694 amino acids and had a predicted molecular mass of 74.2kDa. It had 44% identity with the RAP-1 protein, a 67.9-kDa MYC-like protein from Arabidopsis thaliana which binds the G-box sequence via a basic region helix-loop-helix (bHLH), without requiring heterodimerization with MYB proteins. Phylogenic comparisons revealed that the maize 7E protein was related to the Arabidopsis thaliana RAP-1 protein and to the Phaseolus vulgaris PG1. This similarity was particularly evident for the bHLH domain, which was 95% identical between maize 7E and Arabidopsis thaliana RAP-1. 7E, RAP-1 and PG-1 proteins revealed a plant MYC-like sub-family that was more related to the maize repressor-like IN1 than to maize R proteins. 7E mRNA was detected in both roots and leaves by the Northern analysis. The amount of 7E mRNA increased, in response to iron starvation, by 20 and 40% in roots and leaves, respectively. The relationship between iron metabolism and myc expression in animal cells is discussed.
Assuntos
DNA Complementar/genética , Proteínas Proto-Oncogênicas c-myc/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Zea mays/genética , Sequência de Aminoácidos , Arabidopsis/genética , Clonagem Molecular , DNA Complementar/química , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Ferro/metabolismo , Ferro/farmacologia , Dados de Sequência Molecular , Mutação , Folhas de Planta/química , Folhas de Planta/genética , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/químicaRESUMO
Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a 'domain-swapping' strategy-replacement of the cell wall anchor of IniA by the membrane anchor of ActA- we show that the reduced ability to adhere and enter cells of strains expressing IniA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.
Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Sequência de Aminoácidos , Células CACO-2 , Membrana Celular/metabolismo , Células Epiteliais , Humanos , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/metabolismo , Dados de Sequência MolecularRESUMO
In plants, synthesis of the iron-storage protein ferritin in response to iron is not regulated at the translational level; this is in contrast to ferritin synthesis in animals. Part of the response is mediated through a transduction pathway which involves the plant hormone abscisic acid. In this work, we report the cloning and sequencing of two maize ferritin genes (ZmFer1 and ZmFer2) coding for members of the two ferritin mRNA subclasses, FM1 and FM2, respectively. Although plant and animal ferritins are closely related proteins, a major difference is observed between the organisation of the genes. Both maize ferritin genes are organised as eight exons and seven introns, the positions of which are identical within the two genes, while animal ferritin genes are interrupted by three introns, at positions different from those found in maize genes. Sequence divergence between the 3' untranslated regions of these genes has allowed the use of specific probes to study the accumulation of FM1 and FM2 transcripts in response to various environmental cues. Such probes have shown that FM1 and FM2 transcripts accumulate with differential kinetics in response to iron; FM1 mRNA accumulate earlier than FM2 mRNA and only FM2 transcripts accumulate in response to exogenous abscisic acid or water stress. Mapping of the transcriptional initiation region of these two genes defined their 5' upstream regions and allowed a sequence comparison of their promoters, which appeared highly divergent. This raises the possibility that the differential accumulation of FM1 and FM2 mRNAs in response to iron, abscisic acid and drought could be due to differential transcription of ZmFer1 and ZmFer2.
Assuntos
Ácido Abscísico/fisiologia , Ferritinas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Ferro/fisiologia , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA de Plantas , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido NucleicoRESUMO
The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14% of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of M(r) 34,330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.
Assuntos
Acetiltransferases/metabolismo , Arabidopsis/enzimologia , Isoenzimas/metabolismo , Pisum sativum/enzimologia , Frações Subcelulares/enzimologia , Acetiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Clonagem Molecular , Citosol/enzimologia , DNA Complementar , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Plasmídeos , Homologia de Sequência de Aminoácidos , Serina O-AcetiltransferaseRESUMO
pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.
Assuntos
Adenosina Trifosfatases/genética , Cádmio/farmacologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Listeria monocytogenes/genética , Plasmídeos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico Ativo/genética , Cádmio/metabolismo , Resistência Microbiana a Medicamentos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Especificidade por Substrato , Zinco/farmacologiaRESUMO
The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.
Assuntos
Cádmio/farmacologia , Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Listeria monocytogenes/genética , Plasmídeos/genética , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Resistência Microbiana a Medicamentos/genética , Regulação da Expressão Gênica , Rearranjo Gênico , Dados de Sequência Molecular , Nucleotidiltransferases/classificação , Nucleotidiltransferases/genética , Plasmídeos/classificação , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransposasesRESUMO
The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.