Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Resultados da Pesquisa
Item Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi(Genetics and Molecular Biology, 2002) Gomes, Eliane A.; Kasuya, Maria Catarina M.; Barros, Everaldo G. de; Borges, Arnaldo C.; Araújo, Elza F.Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp.), and Laccaria (L. bicolor and L. amethystea). Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.Item The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao(Genetics and Molecular Biology, 2009) Lima, Juliana O.; Pereira, Jorge F.; Rincones, Johana; Barau, Joan G.; Araújo, Elza F.; Pereira, Gonçalo A. G.; Queiroz, Marisa V.This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.Item PCR amplification and sequence analyses of Reverse Transcriptase-like genes in Crinipellis perniciosa isolates(Fitopatologia Brasileira, 2007-09) Pereira, Jorge F.; Ignacchiti, Mariana D. C.; Araújo, Elza F.; Brommonschenkel, Sérgio H.; Cascardo, Júlio C. M.; Pereira, Gonçalo A. G.; Queiroz, Marisa V.Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from the gypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.Item Analysis of Tc1-Mariner elements in Sclerotinia sclerotiorum suggests recent activity and flexible transposases(BMC Microbiology, 2014) Santana, Mateus F.; Silva, José C. F.; Mizubuti, Eduardo S. G.; Araújo, Elza F.; Queiroz, Marisa V.Sclerotinia sclerotiorum is a necrotrophic fungus that is pathogenic to many plants. Genomic analysis of its revealed transposable element expansion that has strongly influenced the evolutionary trajectory of several species. Transposons from the Tc1-Mariner superfamily are thought to be ubiquitous components of fungal genomes and are generally found in low copy numbers with large numbers of deleterious mutations in their transposase coding sequence.This study shows that the genome of S. sclerotiorum has a large number of copies of Tc1-Mariner transposons, and in silico analysis shows evidence that they were recently active. This finding was confirmed by expressed sequence tag (EST) analysis. Fourteen new Tc1-Mariner transposon families that were distributed throughout the genome were identified, and in some cases, due to the excision/retention of introns, different transcripts were observed for the same family, which might be the result of an efficient strategy to circumvent mutations that generate premature stop codons in the RNA sequence. In addition, the presence of these introns shows that the transposase protein has a flexible coding sequence and, consequently, conformation. No evidence for RIP-like gene silencing mechanisms, which are commonly found in fungi, was found in the identified Tc1-Mariner elements, and analysis of the genomic insertion sites of these elements showed that they were widely distributed throughout the genome with some copies located near the 3′ regions of genes. In particular, EST analysis demonstrated that one of these copies was co-expressed with a gene, which showed the potential for these elements to undergo exaptation.Fourteen novel Tc1-Mariner families were characterized. Some families had evidence of introns, which might or might not be excised depending on the family or element in question, and this finding demonstrates a possible strategy for overcoming possible mutations that generate premature stop codons in a RNA sequence. Tc1-Mariner elements likely play an important role in the structure and evolution of the S. sclerotiorum genome.Item Fermentation of maltose and starch by Klebsiella oxytoca P2(Biotechnology Letters, 1998-12) Santos, Vera L. dos; Guimarães, Walter V.; Barros, Everaldo G. de; Araújo, Elza F.Klebsiella oxytoca P2, which has genes from Zymomonas mobilis encoding the alcohol dehydrogenase and pyruvate decarboxylase integrated in its chromosome, fermented 50 g maltose/l to 25.4 g of ethanol/l. It also fermented 10, 20 and 40 g starch/l yielding 4, 8.4, and 17.7 g ethanol/l, respectively, representing 72, 75 and 78% of the theoretical yield.Item MpSaci is a widespread gypsy-Ty3 retrotransposon highly represented by non-autonomous copies in the Moniliophthora perniciosa genome(Current Genetics, 2015-05) Araújo, Elza F.; Queiroz, Casley B.; Queiroz, Marisa V.; Pereira, Jorge F.; Brommonschenkel, Sérgio H.; Costa, Gustavo G. L.; Carazzolle, Marcelo F.; Pereira, Gonçalo A. G.Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15 kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag gene is constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.Item Small heat shock proteins in the development of thermotolerance in Pisolithus sp.(Journal of Thermal Biology, 2005-12) Ferreira, Adão S.; Kasuya, Maria C. M.; Araújo, Elza F.; Borges, Arnaldo C.; Tótola, Marco. R.Small heat shock proteins (HSPs) have been shown to confer thermotolerance in many organisms. Here, we demonstrate that small HSPs (sHSPs) can also be involved in development of thermotolerance in Pisolithus sp. In heat shock response, Pisolithus isolate RV82 synthesized proteins of molecular mass 28, 26 and 15–18 kDa. These group of proteins are synthesized when mycelial mass are exposed to heat shock temperature (42 °C) for short period (30 min) and incubated back at 28 °C, the optimal temperature for growth. Our results show sHSPs are an important biochemical alteration in ectomycorrhizal fungi under thermal stress.Item The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao(Genetics and Molecular Biology, 2008-12-05) Lima, Juliana O.; Pereira, Jorge F.; Rincones, Johana; Barau, Joan G.; Araújo, Elza F.; Pereira, Gonçalo A.G.; Queiroz, Marisa V.This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.