RESUMO
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.
Assuntos
Basidiomycota/genética , Genômica , Lignina/metabolismo , Basidiomycota/classificação , Hidrólise , Dados de Sequência Molecular , Oxirredução , Filogenia , Especificidade da EspécieRESUMO
Ceriporiopsis subvermispora is a white-rot fungus with a high specificity towards lignin mineralization when colonizing dead wood or lignocellulosic compounds. Its lignocellulose degrading system is formed by cellulose hydrolytic enzymes, manganese peroxidases, and laccases that catalyze the efficient depolymerization and mineralization of lignocellulose. To determine if this metabolic specialization has modified codon usage of the lignocellulolytic system, improving its adaptation to the fungal translational machine, we analyzed the adaptation to host codon usage (CAI), tRNA pool (tAI, and AAtAI), codon pair bias (CPB), and the number of effective codons (Nc). These indexes were correlated with gene expression of C. subvermispora, in the presence of glucose and Aspen wood. General gene expression was not correlated with the index values. However, in media containing Aspen wood, the induction of expression of lignocellulose-degrading genes, showed significantly (p < 0.001) higher values of CAI, AAtAI, CPB, tAI, and lower values of Nc than non-induced genes. Cellulose-binding proteins and manganese peroxidases presented the highest adaptation values. We also identified an expansion of genes encoding glycine and glutamic acid tRNAs. Our results suggest that the metabolic specialization to use wood as the sole carbon source has introduced a bias in the codon usage of genes involved in lignocellulose degradation. This bias reduces codon diversity and increases codon usage adaptation to the tRNA pool available in C. subvermispora. To our knowledge, this is the first study showing that codon usage is modified to improve the translation efficiency of a group of genes involved in a particular metabolic process.
Assuntos
Uso do Códon , Lacase/metabolismo , Lignina/metabolismo , Peroxidases/metabolismo , Polyporales/metabolismo , RNA de Transferência/genética , Catálise , Hidrólise , Lacase/genética , Peroxidases/genética , Filogenia , Polyporales/genética , Polyporales/crescimento & desenvolvimentoRESUMO
We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivariate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Biópsia , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genéticaRESUMO
BACKGROUND: Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. METHODOLOGY: We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. CONCLUSIONS: Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.
Assuntos
DNA/isolamento & purificação , Morus/química , Papel , DNA/química , DNA/genética , Humanos , Repetições de Microssatélites/genética , Morus/genética , Museus , Casca de Planta/química , Casca de Planta/genéticaRESUMO
In this work, we explore the use of the unbiased cDNA-AFLP strategy to identify genes involved in Mn(2+) homeostasis in Ceriporiopsis subvermispora. In this ligninolytic white-rot fungus, whose genome has not yet been sequenced, three Mn peroxidase genes responding to Mn(2+) have been characterized. Using cDNA-AFLP to identify transcript-derived fragments (TDFs), a total of 37 differentially expressed cDNA fragments were identified by comparing band intensities among cDNA-AFLP patterns obtained from mycelia from cultures supplemented with different concentrations of Mn(2+). Of 21 differentially expressed TDFs, nine were classified as upregulated, five as downregulated and seven as unregulated. Of these, six upregulated and two downregulated TDFs were selected for further characterization. The expected TDFs for the known Mn peroxidases were not isolated, but several genes encoding proteins related to protein sorting, storage and excretion of excess Mn(2+) were identified. Transcripts induced under Mn(2+) supplementation exhibited homologies to the elongation factor eEF3, a HDEL sequence binding protein and the ARD1 subunit of the N-acetyltransferase complex, among others. Overall, the results obtained in this study suggest a complex picture of Mn(2+) homeostasis and provide the possibility to search for common regulatory elements in the promoters of the novel putatively identified genes.
Assuntos
Coriolaceae/genética , Regulação Fúngica da Expressão Gênica , Manganês/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Técnicas Genéticas , Genoma Fúngico , Glicosilação , Proteínas Ferro-Enxofre/química , Manganês/química , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Oligonucleotídeos/química , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.
Assuntos
Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/genética , Proteínas de Membrana/genética , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
In this report we describe the isolation and characterization of a gene encoding the transcription factor Ace1 (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-ace1 encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Ace1, Mac1 and Haa1 from Saccharomyces cerevisiae. The Pc-ace1 gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae ace1 null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-ace1. Moreover, Northern blot hybridization studies indicated that Pc-ace1 cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Ace1 transcription factor in basidiomycetes.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Phanerochaete/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Northern Blotting , Clonagem Molecular , Cobre/farmacologia , DNA Complementar , Regulação Fúngica da Expressão Gênica , Modelos Genéticos , Phanerochaete/efeitos dos fármacos , RNA Mensageiro/análiseRESUMO
We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivaríate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diabetes Mellitus Tipo 1/complicações , /complicações , Nefropatias Diabéticas/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Biópsia , Estudos de Casos e Controles , Genótipo , Íntrons/genética , Análise Multivariada , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Splicing de RNA/genética , Transcrição Gênica/genéticaRESUMO
Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , /genética , Nefropatias Diabéticas/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Polimorfismo Genético/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Genótipo , Marcadores Genéticos/genética , Projetos PilotoRESUMO
In this report we describe the isolation and characterization of a gene encoding the transcription factor Acel (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-acel encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Acel, Macl and Haal from Saccharomyces cerevisiae. The Pc-acel gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae acel null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-acel. Moreover, Northern blot hybridization studies indicated that Pc-acel cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Acel transcription factor in basidiomycetes.