Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Mycol Med ; 28(2): 257-268, 2018 Jun.
Artigo em Francês | MEDLINE | ID: mdl-29545121

RESUMO

Among the subdivision of Saccharomycotina (ascomycetes budding yeasts), the CTG clade (formerly the Candida clade) includes species that display a particular genetic code. In these yeasts, the CTG codon is predominantly translated as a serine instead of a leucine residue. It is now well-known that some CTG clade species have a major impact on human and its activities. Some of them are recognized as opportunistic agents of fungal infections termed candidiasis. In addition, another series of species belonging to the CTG clade draws the attention of some research groups because they exhibit a strong potential in various areas of biotechnology such as biological control, bioremediation, but also in the production of valuable biocompounds (biofuel, vitamins, sweeteners, industrial enzymes). Here we provide an overview of recent advances concerning the biology, clinical relevance, and currently tested biotechnological applications of species of the CTG clade. Future directions for scientific research on these particular yeasts are also discussed.


Assuntos
Candida , Candidíase/microbiologia , Microbiologia Industrial , Códon , Humanos
2.
Methods Enzymol ; 576: 167-206, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27480687

RESUMO

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.


Assuntos
Catharanthus/enzimologia , Catharanthus/genética , Genes de Plantas , Genômica/métodos , Biologia Sintética/métodos , Transcriptoma , Leveduras/genética , Vias Biossintéticas , Catharanthus/citologia , Catharanthus/metabolismo , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Transformação Genética , Leveduras/citologia , Leveduras/enzimologia , Leveduras/metabolismo
3.
Plant Biol (Stuttg) ; 13(1): 36-41, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21143723

RESUMO

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.


Assuntos
Calmodulina/metabolismo , Catharanthus/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Calmodulina/química , Calmodulina/genética , Catharanthus/química , Catharanthus/genética , Linhagem Celular , Clonagem Molecular , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência
4.
Plant Mol Biol ; 45(4): 477-88, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11352466

RESUMO

The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step in the biosynthesis of therapeutically valuable terpenoid indole alkaloids. In Catharanthus roseus the Str gene was shown to be regulated by a wide variety of signals including auxin, methyl jasmonate and fungal elicitors in cell suspension cultures and by tissue-specific control in plant organs. The Str promoter contains a functional G-box (CACGTG) cis-regulatory sequence. In order to understand better the mechanisms involved in the regulation of Str gene expression, we isolated the C. roseus cDNAs encoding G-box binding factors Crgbf1 and Crgbf2. The binding specificity of their protein products CrGBF1 and CrGBF2 was analysed by competitive electrophoresis mobility and saturation binding assays. CrGBF1 had a high binding specificity for class I G-boxes including the Str G-box. CrGBF1 showed a lower affinity for class II G-boxes and for the G-box-like element (AACGTG) found in the tryptophan decarboxylase (Tdc) gene which encodes another enzyme involved in TIA biosynthesis. CrGBF2 showed a high affinity for all types of G-boxes tested and to a lesser extent for the Tdc G-box-like element. Transient bombardment experiments demonstrated that both CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str promoter via direct interaction with the G-box. These data indicate that GBFs may play functional role in the regulation of expression of the terpenoid indole alkaloid biosynthetic gene Str.


Assuntos
Carbono-Nitrogênio Liases/genética , Proteínas de Ligação a DNA/genética , Plantas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva , Northern Blotting , Southern Blotting , DNA/genética , DNA/metabolismo , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação G-Box , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Células Vegetais , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Fatores de Transcrição/metabolismo
5.
Plant J ; 23(4): 497-506, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10972876

RESUMO

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.


Assuntos
Fosfatidilinositóis/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Diester Fosfórico Hidrolases/isolamento & purificação , Poaceae/enzimologia , Fosfolipases Tipo C/metabolismo , Animais , Anticorpos Monoclonais , Western Blotting , Encéfalo/enzimologia , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Iluminação , Microssomos/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Folhas de Planta/enzimologia , Poaceae/metabolismo , Testes de Precipitina , Protoplastos/enzimologia , Ratos , Extratos de Tecidos/metabolismo , Fosfolipases Tipo C/imunologia , Fosfolipases Tipo C/isolamento & purificação
6.
Plant Cell ; 8(4): 573-586, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12239393

RESUMO

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.

7.
Cytometry ; 23(3): 241-9, 1996 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8974869

RESUMO

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is a key enzyme of photosynthesis in C4 plants; it is specifically localized in the cytosol of mesophyll cells and is regulated by a phosphorylation/dephosphorylation process. The light-dependent phosphorylation of PEPC is triggered by an increase in the cytosolic pH (pHc) of mesophyll cell protoplasts. An epifluorescence and confocal microscopy analysis showed that the specific pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM), when used at low concentration, was essentially localized in the protoplast cytosol. By the nigericin null-point method and flow cytometry, the pHc of freshly isolated protoplasts was estimated to be 6.4. To observe the full activity of PEPC kinase and maximal phosphorylation of PEPC in vivo, such protoplast suspensions must first be treated with a permeant weak base. The present report shows that 20 mM NH4Cl raised the final pHc to 7.4. This method can be useful for estimating rapid changes of pHc in plant cells.


Assuntos
Citometria de Fluxo/métodos , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Poaceae/metabolismo , Cloreto de Amônio/farmacologia , Citosol , Nigericina/farmacologia , Protoplastos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...