Transcription of Aspergillus nidulans pacC is modulated by alternative RNA splicing of palB

AbstractFungi have evolved elaborate signal transduction networks for remodeling metabolic pathways to scavenge nutrients, including the secretion of nutritional enzymes. This adaptive response involves the conserved PacC/Pal signal transduction pathway, which mediates the transcriptional response to ambient pH. In this study, we show that transcription of the gene for PacC is modulated in response to nutrient changes, phosphate and carbon sources, and pH. In addition, we show that transcription of pacC is modulated in response to alternative RNA splicing of the palB gene. These results reveal novel aspects of the complex network involved in modulation of pacC

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway

AbstractThe filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

<p>Abstract</p> <p>Background</p> <p>Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus <it>Trichophyton rubrum </it>is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of <it>T. rubrum </it>exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries.</p> <p>Results</p> <p>The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 <it>T. rubrum </it>sequences that had not been previously deposited in public databases.</p> <p>Conclusion</p> <p>In this study, we identified novel <it>T. rubrum </it>genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of <it>T. rubrum </it>to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging <it>T. rubrum </it>with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.</p

MLL leukemia-associated rearrangements in peripheral blood lymphocytes from healthy individuals

Chromosomal translocations are characteristic of hematopoietic neoplasias and can lead to unregulated oncogene expression or the fusion of genes to yield novel functions. In recent years, different lymphoma/leukemia-associated rearrangements have been detected in healthy individuals. In this study, we used inverse PCR to screen peripheral lymphocytes from 100 healthy individuals for the presence of MLL (Mixed Lineage Leukemia) translocations. Forty-nine percent of the probands showed MLL rearrangements. Sequence analysis showed that these rearrangements were specific for MLL translocations that corresponded to t(4;11)(q21;q23) (66%) and t(9;11) (20%). However, RT-PCR failed to detect any expression of t(4;11)(q21;q23) in our population. We suggest that 11q23 rearrangements in peripheral lymphocytes from normal individuals may result from exposure to endogenous or exogenous DNA-damaging agents. In practical terms, the high susceptibility of the MLL gene to chemically-induced damage suggests that monitoring the aberrations associated with this gene in peripheral lymphocytes may be a sensitive assay for assessing genomic instability in individuals exposed to genotoxic stress

The nucleation of microtubules in Aspergillus nidulans germlings

Microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. In fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. We used the antimicrotubule drug Benomyl in block/release experiments to depolymerize and repolymerize microtubules in Aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. Twenty seconds after release from Benomyl short microtubules were formed from several bright (immunofluorescent) dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in A. nidulans germlings. Since nuclear movement is dependent on microtubules in A. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants) have some obvious microtubule defect. Cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. However, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. This suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.<br>Microtúbulos são filamentos compostos por dímeros das tubulinas <FONT FACE="Symbol">a</FONT> e <FONT FACE="Symbol">b</FONT> e têm uma variedade de funções nas células vivas. Em fungos, os corpúsculos polares dos fusos são geralmente considerados os centros organizadores dos microtúbulos. Com o objetivo de contribuir para uma melhor compreensão dos processos de nucleação dos microtúbulos no fungo filamentoso A. nidulans, nós utilizamos a droga antimicrotúbulo Benomil em experimentos de bloqueio e liberação para depolimerizar e repolimerizar os microtúbulos. Após 20 segundos de reincubação em meio sem Benomil, pequenos microtúbulos foram formados a partir de pontos distribuídos pela célula, sugerindo que os pontos de nucleação de microtúbulos são aleatoriamente distribuídos pelas hifas de A. nidulans. Como em A. nidulans o movimento nuclear é dependente de microtúbulos foi analisado se mutantes defectivos na distribuição de núcleos ao longo das hifas (mutantes nud) possuíam algum defeito evidente nos microtúbulos. Os microtúbulos citoplasmáticos, dos fusos e astrais estão presentes e aparentam-se normais em todos os mutantes nud, mas foi observada uma pequena distorção na proporção de fusos mitóticos longos e curtos nestes mutantes, comparados com o controle. Isto sugere que alguns núcleos de mutantes nud não alcançam a fase tardia da divisão celular, em temperatura não restritiva

Antifungal Resistance Mechanisms in Dermatophytes

Although fungi do not cause outbreaks or pandemics, the incidence of severe systemic fungal infections has increased significantly, mainly because of the explosive growth in the number of patients with compromised immune system. Thus, drug resistance in pathogenic fungi, including dermatophytes, is gaining importance. The molecular aspects involved in the resistance of dermatophytes to marketed antifungals and other cytotoxic drugs, such as modifications of target enzymes, over-expression of genes encoding ATP-binding cassette (ABC) transporters and stress-response-related proteins are reviewed. Emphasis is placed on the mechanisms used by dermatophytes to overcome the inhibitory action of terbinafine and survival in the host environment. The relevance of identifying new molecular targets, of expanding the understanding about the molecular mechanisms of resistance and of using this information to design new drugs or to modify those that have become ineffective is also discussed.FAPESPCNPqCAPESFAEP

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium altered during cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling