RESUMEN
A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.
Asunto(s)
Perfilación de la Expresión Génica , Marcadores Genéticos , Neoplasias de Cabeza y Cuello/genética , ARN Mensajero/genética , Neoplasias de la Tiroides/genética , Transcripción Genética , Empalme Alternativo , Etiquetas de Secuencia Expresada , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Laringe/metabolismo , Boca/metabolismo , Faringe/metabolismo , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismoRESUMEN
Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Micelio/citología , Paracoccidioides/genética , Transcripción Genética , Levaduras/citología , 4-Hidroxifenilpiruvato Dioxigenasa/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Diferenciación Celular , Medios de Cultivo , Ciclohexanonas/farmacología , Inhibidores Enzimáticos/farmacología , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Genes Fúngicos , Humanos , Análisis por Micromatrices , Estructura Molecular , Micelio/genética , Micelio/metabolismo , Nitrobenzoatos/farmacología , Paracoccidioides/citología , Paracoccidioides/efectos de los fármacos , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/etiología , Temperatura , Levaduras/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismoRESUMEN
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Here, we describe the microsatellite patterns observed in a collection of P. brasiliensis random sequence tags. We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis). The majority of these microsatellites (87.5%) are found in noncoding sequences. We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P. brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause. We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.
Asunto(s)
Marcadores Genéticos , Repeticiones de Microsatélite/genética , Paracoccidioides/clasificación , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/fisiopatología , Animales , Armadillos/microbiología , Genoma Fúngico , Humanos , Paracoccidioides/genética , Paracoccidioidomicosis/epidemiología , Paracoccidioidomicosis/microbiología , Filogenia , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.