RESUMEN
Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.(AU)
A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu sub sequentesequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).(AU)
Asunto(s)
ADN de Archaea/genética , ARN Ribosómico 16S/análisis , Reacción en Cadena de la Polimerasa , FilogeniaRESUMEN
Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.
Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).
Asunto(s)
Archaea/genética , Filogenia , ARN Ribosómico 16S/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Cartilla de ADN/genética , Genes de ARNrRESUMEN
Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.
A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu sub sequentesequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).
Asunto(s)
ADN de Archaea/genética , Filogenia , /análisis , Reacción en Cadena de la PolimerasaRESUMEN
Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.
Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).
RESUMEN
Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.
Asunto(s)
Archaea , Archaea/genética , Cartilla de ADN/genética , Genes de ARNr , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The Archaea domain was recognized as a separate phylogenetic lineage in the tree of life nearly 3 decades ago. It is now known as part of the human microbiome; however, given that its roles in oral sites are still poorly understood, this review aimed to establish the current level of evidence regarding archaea in the oral cavity to guide future research, providing insights on the present knowledge about the human oral archaeome. A scoping review was conducted with the PRISMA Extension for Scoping Reviews checklist. Five electronic databases were searched, as well as gray literature. Two independent reviewers performed the selection and characterization of the studies. Clinical studies were included when the target population consisted of humans of any age who were donors of samples from the oral cavity. A qualitative analysis was performed, based on the type of oral site and by considering the methods employed for archaeal identification and taxonomy, including the DNA extraction protocols, primers, and probes used. Fifty articles were included in the final scoping review, published from 1987 to 2019. Most studies sampled periodontal sites. Methanogens were the most abundant archaea in those sites, and their presence could be associated with other periodontal pathogens. No consistent relationship with different disease conditions was observed in studies that evaluated the microbiota surviving in endodontic sites. Few articles analyzed the presence of archaea in dental caries, saliva, or tongue microbiota, as well as in archaeologic samples, also showing a relationship with healthy microbiota. Archaea have been detected in different oral niches of individuals from diverse geographic locations and clinical conditions, suggesting potential roles in oral diseases. Methodological limitations may hamper our current knowledge about archaeal diversity and prevalence in oral samples, and future research with diversified methodological approaches may lead to a better comprehension of the human oral archaeome.
Asunto(s)
Caries Dental , Microbiota , Archaea/genética , Humanos , Boca , FilogeniaRESUMEN
Although the richness of Bacteria and Fungi in Cerrado' soils has been reported, here we report, for the first time, the archaeal community in Cerrado's soils. DNA extracted from soil of two distinct vegetation types, a dense subtype of sensu strict (cerrado denso) and riverbank forest (mata de galeria), was used to amplify Archaea-specific 16S rRNA gene. All of the fragments sequenced were classified as Archaea into the phylum Thaumarchaeota, predominantly affiliated to groups I.1b and I.1c. Sequences affiliated to the group I.1a were found only in the soil from riverbank forest. Soils from 'cerrado denso' had greater Archaea richness than those from 'mata de galeria' based on the richness indexes and on the rarefaction curve. ß-Diversity analysis showed significant differences between the sequences from the two soil areas studied because of their different thaumarchaeal group composition. These results provide information about the third domain of life from Cerrado soils.
Asunto(s)
Archaea/clasificación , Archaea/genética , Microbiología del Suelo , Archaea/aislamiento & purificación , Brasil , Genes Arqueales , Genes de ARNr , Metagenoma , Filogenia , ARN Ribosómico 16S/genética , Suelo/química , ÁrbolesRESUMEN
AIMS: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe-egl1) encoding a putative endoglucanase. METHODS AND RESULTS: Pe-egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5.0-9.0), and an optimal temperature of 60 degrees C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre-incubation at 70 degrees C). Calcium exerted a strong stimulatory effect over EGL1 activity. CONCLUSIONS: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro-organism.
Asunto(s)
Celulasa/genética , Celulasa/metabolismo , Regulación Fúngica de la Expresión Génica , Penicillium/enzimología , Penicillium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cationes/farmacología , Celulasa/química , Celulosa/metabolismo , Clonación Molecular , ADN Complementario/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Fúngicos/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , TemperaturaRESUMEN
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.
Asunto(s)
Humanos , Animales , Etiquetas de Secuencia Expresada/metabolismo , Paracoccidioides/patogenicidad , Transcripción Genética/genética , ADN Complementario , ADN de Hongos , Datos de Secuencia Molecular , Paracoccidioides/enzimología , Paracoccidioides/genética , Paracoccidioidomicosis/virología , Regulación Fúngica de la Expresión Génica , Secuencia de Bases , Transcripción Genética/fisiología , Virulencia/genéticaRESUMEN
The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.
Asunto(s)
Humanos , Origen de la Vida , Etiquetas de Secuencia Expresada , Factores de Transcripción/genética , Paracoccidioides/genética , Factores de Transcripción/fisiología , Genoma Fúngico , Paracoccidioides/fisiología , ARN de Hongos/genética , ARN Polimerasa II/genética , ARN Polimerasa II/fisiología , Reproducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Transcripción Genética/fisiologíaRESUMEN
The translational and post-translational modification machineries of Paracoccidioides brasiliensis were assessed by means of comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags) with sequences deposited on different databases. Of the 79 sequences corresponding to cytosolic ribosomal proteins, we were able to find 78 in the P. brasiliensis transcriptome. Nineteen of the 27 Saccharomyces cerevisiae genes related to translation initiation were also found. All eukaryotic elongation factors were detected in P. brasiliensis transcriptome, with eEF1A as one of the most expressed genes. Translation termination is performed, in eukaryotes, by factors 1 and 3 (eRF1, eRF3). In P. brasiliensis transcriptome it was possible to identify eRF3, but not eRF1. Sixteen PbAESTs showing aminoacyl-tRNA synthetase-predicted activities were found in our analyses, but no cysteinyl-, leucyl-, asparagyl- and arginyl-tRNA synthetases were detected. Among the mitochondrial ribosomal proteins, we have found 20 and 18 orthologs to S. cerevisiae large and small ribosomal subunit proteins, respectively. We have also found three PbAESTs similar to Neurospora crassa mitochondrial ribosomal genes, with no similarity with S. cerevisiae genes. Although orthologs to S. cerevisiae mitochondrial EF-Tu, EF-G and RF1 have been found in P. brasiliensis transcriptome, no sequences corresponding to functional EF-Ts were detected. In addition, 64 and 28 PbAESTs associated to protein modification and degradation, respectively, were found. These results suggest that these machineries are well conserved in P. brasiliensis, when compared to other organisms.
Asunto(s)
Genoma Fúngico/genética , Modificación Traduccional de las Proteínas/genética , Paracoccidioides/metabolismo , Proteínas Ribosómicas/metabolismo , Etiquetas de Secuencia Expresada/metabolismo , Paracoccidioides/genética , Proteínas Ribosómicas/genética , Regulación de la Expresión Génica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 degrees C and as yeasts (Y) at 37 degrees C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Micelio/crecimiento & desarrollo , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Secuencia de Bases , Cisteína/genética , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , Proteínas Fúngicas/química , Intrones , Modelos Moleculares , Datos de Secuencia Molecular , Micelio/genética , Paracoccidioides/citología , Filogenia , ARN de Hongos/análisis , ARN Mensajero/análisis , Homología de Secuencia de AminoácidoRESUMEN
Diffusely adhering Escherichia coli (E. coli) strains (DAEC) represent a potential cause of diarrhoea in infants, and the detection of type three secretion system (TTSS) genes in DAEC would substantiate their pathogenic nature. In this work, four isolates of DAEC, recovered from stools of diarrhoeic children, were analysed by PCR, in order to detect the presence of TTSS genes. Primers targeted to the escC, escJ, escN and escV, some of the most conserved TTSS genes in enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), were used in order to verify the occurrence of homologous genes in our DAEC isolates. By this approach, we were able to characterise DNA fragments corresponding to putative escJ and escN genes in all DAEC isolates. Furthermore, DNA fragments homologous to the escC and escV genes were also amplified from all isolates. Besides the similarity found among the DAEC esc homologues with EPEC and EHEC esc genes, the nucleotide sequence analysis of the flanking regions of the amplified DNA fragments suggests that the putative DAEC esc genes are organised in the same manner as observed in EPEC and in EHEC strains. The results described here provide strong evidence for the presence of a TTSS in the DAEC strains analysed, implicating a pathogenic nature of these isolates.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Niño , Secuencia Conservada , ADN Bacteriano , Diarrea/microbiología , Escherichia coli/clasificación , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Filogenia , Homología de Secuencia de Ácido Nucleico , SerotipificaciónRESUMEN
Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.
Asunto(s)
Etiquetas de Secuencia Expresada , Genoma Fúngico , Paracoccidioides/genética , Secuencia de Bases , Brasil , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
Paracoccidioides brasiliensis is a dimorphic human pathogenic fungus that is the causal agent of paracoccidioidomycosis, a systemic disease that predominantly affects rural communities in South and Central America. Dimorphism is a common characteristic of systemic human pathogenic fungi. Here we describe the use of differential display (DD) to isolate and identify differentially expressed genes of P. brasiliensis, in the two cell types, yeast (Y) and mycelium (M), as well as at different time intervals during temperature-induced M to Y transition. Using two oligo-deoxythymidine-anchored primers combined with 10 arbitrary ones, we were able to detect the presence of at least 20 differentially transcribed cDNA fragments. Some of these fragments were further analysed by reverse-northern blot and northern blot in order to confirm their differential expression. The M32, M51 and M73 cDNA fragments were specific for the mycelial form of P. brasiliensis. Furthermore, we found two cDNA fragments (M-Y1 and M-Y2) that were upregulated during M-Y transition. This method was efficient and useful in the detection of differentially expressed genes in P. brasiliensis.