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1.
Proc Natl Acad Sci U S A ; 114(31): E6361-E6370, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716924

RESUMO

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.


Assuntos
Citoesqueleto/genética , Evolução Molecular , Genoma de Planta/genética , Porphyra/citologia , Porphyra/genética , Actinas/genética , Sinalização do Cálcio/genética , Ciclo Celular/genética , Parede Celular/genética , Parede Celular/metabolismo , Cromatina/genética , Cinesina/genética , Filogenia
2.
Free Radic Biol Med ; 108: 575-584, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28438660

RESUMO

Hypotaurine and taurine are amino acid derivatives and abundant molecules in many eukaryotes. The biological reaction in which hypotaurine is converted to taurine remains poorly understood. Here, hypotaurine and taurine were observed to react with superoxide anion in vitro to form the novel molecule peroxytaurine. In contrast, hypotaurine reacts with hydrogen peroxide to form taurine, but taurine does not react with hydrogen peroxide in vitro. Mass and NMR spectrometry as well as FTIR and Raman spectroscopy support the molecular characterization of peroxytaurine. Gravitometric and spectroscopy experiments suggest a stoichiometry of two superoxide anions reacting with one hypotaurine or two taurines. The newly identified molecule is a semi-stable, organic peroxysulfonic acid that may be an intermediate metabolite in taurine synthesis.


Assuntos
Ácidos Sulfônicos/química , Superóxidos/química , Taurina/análogos & derivados , Taurina/química , Animais , Sistema Livre de Células , Cisteína/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Químicos
3.
Biotechniques ; 57(5): 267-71, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25391916

RESUMO

Microfluidics chip technology is a powerful and convenient alternative to agarose gels and PAGE, but costs can be high due to certain chips being non-reusable. Here we describe a method to regenerate, re-use, and store Agilent DNA, RNA, and protein electrophoresis chips designed for use in the Bioanalyzer 2100. By washing the sample wells and displacing the old gel matrix with new gel-dye mix, we have run samples on the same chip up to ten times with negligible loss of signal quality. Chips whose wells were loaded with buffer or water were stored successfully for one week before re-use.


Assuntos
DNA/análise , Eletroforese/métodos , Microfluídica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise Serial de Proteínas/métodos , RNA/análise , Eletroforese/instrumentação , Humanos , Microfluídica/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise Serial de Proteínas/instrumentação
4.
Genome Biol ; 14(5): R41, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23663246

RESUMO

BACKGROUND: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. RESULTS: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. CONCLUSIONS: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.


Assuntos
Genoma de Planta , Nelumbo/genética , Adaptação Biológica , Substituição de Aminoácidos , Evolução Molecular , Dados de Sequência Molecular , Taxa de Mutação , Nelumbo/classificação , Nelumbo/fisiologia , Filogenia , Vitis/genética
5.
Plant Cell ; 24(10): 3921-48, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23043051

RESUMO

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, Vitamin C 2 (VTC2), monodehydroascorbate reductase 1 (MDAR1), and conserved in the green lineage and diatoms 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Ferro/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/classificação , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/fisiologia , FMN Redutase/genética , FMN Redutase/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Estresse Fisiológico , Transcriptoma
6.
Plant Cell ; 24(6): 2649-65, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22685165

RESUMO

Fe deficiency is one of several abiotic stresses that impacts plant metabolism because of the loss of function of Fe-containing enzymes in chloroplasts and mitochondria, including cytochromes, FeS proteins, and Fe superoxide dismutase (FeSOD). Two pathways increase the capacity of the Chlamydomonas reinhardtii chloroplast to detoxify superoxide during Fe limitation stress. In one pathway, MSD3 is upregulated at the transcriptional level up to 10(3)-fold in response to Fe limitation, leading to synthesis of a previously undiscovered plastid-specific MnSOD whose identity we validated immunochemically. In a second pathway, the plastid FeSOD is preferentially retained over other abundant Fe proteins, heme-containing cytochrome f, diiron magnesium protoporphyrin monomethyl ester cyclase, and Fe2S2-containing ferredoxin, demonstrating prioritized allocation of Fe within the chloroplast. Maintenance of FeSOD occurs, after an initial phase of degradation, by de novo resynthesis in the absence of extracellular Fe, suggesting the operation of salvage mechanisms for intracellular recycling and reallocation.


Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Ferro/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Chlamydomonas reinhardtii/efeitos dos fármacos , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Citocromos f/metabolismo , Ferredoxinas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Estresse Fisiológico , Superóxido Dismutase/genética
7.
J Biol Chem ; 287(19): 15811-25, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22403401

RESUMO

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.


Assuntos
Aciltransferases/genética , Chlamydomonas reinhardtii/genética , Nitrogênio/metabolismo , Proteínas de Plantas/genética , Triglicerídeos/metabolismo , Aciltransferases/metabolismo , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/metabolismo , Reprodutibilidade dos Testes , Genética Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Fatores de Tempo
8.
J Biol Chem ; 286(24): 21427-39, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21515685

RESUMO

The plastid is a defining structure of photosynthetic eukaryotes and houses many plant-specific processes, including the light reactions, carbon fixation, pigment synthesis, and other primary metabolic processes. Identifying proteins associated with catalytic, structural, and regulatory functions that are unique to plastid-containing organisms is necessary to fully define the scope of plant biochemistry. Here, we performed phylogenomics on 20 genomes to compile a new inventory of 597 nucleus-encoded proteins conserved in plants and green algae but not in non-photosynthetic organisms. 286 of these proteins are of known function, whereas 311 are not characterized. This inventory was validated as applicable and relevant to diverse photosynthetic eukaryotes using an additional eight genomes from distantly related plants (including Micromonas, Selaginella, and soybean). Manual curation of the known proteins in the inventory established its importance to plastid biochemistry. To predict functions for the 52% of proteins of unknown function, we used sequence motifs, subcellular localization, co-expression analysis, and RNA abundance data. We demonstrate that 18% of the proteins in the inventory have functions outside the plastid and/or beyond green tissues. Although 32% of proteins in the inventory have homologs in all cyanobacteria, unexpectedly, 30% are eukaryote-specific. Finally, 8% of the proteins of unknown function share no similarity to any characterized protein and are plant lineage-specific. We present this annotated inventory of 597 proteins as a resource for functional analyses of plant-specific biochemistry.


Assuntos
Bases de Dados de Proteínas , Proteínas de Plantas/química , Algoritmos , Arabidopsis/genética , Bioquímica/métodos , Linhagem da Célula , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Cianobactérias/genética , Cianobactérias/metabolismo , Genoma de Planta , Genômica , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , RNA/metabolismo , RNA de Plantas/genética
9.
Plant Cell ; 23(4): 1273-92, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21498682

RESUMO

In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O2-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.


Assuntos
Chlamydomonas/genética , Chlamydomonas/metabolismo , Cobre/metabolismo , Metabolismo/genética , Fenômenos Fisiológicos da Nutrição/genética , Biologia de Sistemas , Processos Autotróficos/genética , Sequência de Bases , Cobre/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Loci Gênicos/genética , Processos Heterotróficos/genética , Dados de Sequência Molecular , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Photosynth Res ; 106(1-2): 3-17, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20490922

RESUMO

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.


Assuntos
Chlamydomonas reinhardtii/genética , Genoma de Planta/genética , Genômica/métodos , Fotossíntese/genética , Filogenia , Proteínas de Plantas/genética , Aclimatação/genética , Sequência de Bases , Genoma de Planta/fisiologia , Mutação/genética , Fenótipo
11.
Science ; 318(5848): 245-50, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17932292

RESUMO

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.


Assuntos
Proteínas de Algas/genética , Proteínas de Algas/fisiologia , Evolução Biológica , Chlamydomonas reinhardtii/genética , Genoma , Animais , Chlamydomonas reinhardtii/fisiologia , Cloroplastos/metabolismo , Biologia Computacional , DNA de Algas/genética , Flagelos/metabolismo , Genes , Genômica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Dados de Sequência Molecular , Família Multigênica , Fotossíntese/genética , Filogenia , Plantas/genética , Proteoma , Análise de Sequência de DNA
12.
Biochemistry ; 45(27): 8193-208, 2006 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-16819818

RESUMO

N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (PurE) catalyzes the reversible interconversion of acid-labile compounds N5-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on its adaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichia coli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable at pH 3.5, with a > or = 20 degrees C higher thermal unfolding temperature at all pHs. His89 is not essential and does not function as part of a proton relay system. The kcat pH-rate profile was consistent with the assignment of pK1 to unproductive protonation of bound nucleotide and pK2 to deprotonation of His59. A 1.85 A resolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonation of CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme and a water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotide decarboxylation in the forward direction (N5-CAIR --> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., and Stubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in the reverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essential role of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO2-azaenolate intermediate.


Assuntos
Acetobacter/enzimologia , Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Bactérias/química , Transferases Intramoleculares/química , Ribonucleotídeos/química , Sequência de Aminoácidos , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Sequência Conservada , Cristalografia por Raios X , Histidina/química , Histidina/genética , Concentração de Íons de Hidrogênio , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Modelos Moleculares , Mutagênese , Mutação , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribonucleotídeos/metabolismo
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