High-Throughput Functional Genomics for Energy Production

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植物功能基因组学研究的有效工具--RNAi技术

综述了RNA干扰的机制以及在生物体内的功能等方面的研究进展,并对在此基础上发展起来的RNA干扰技术在植物功能基因组学中的应用情况作了简要的介绍.     

基因打靶与RNA干扰技术在丝状真菌基因功能组学研究中的应用

随着丝状真茵模式生物基因组测序工作的进行,其提供的大量未知功能的基因序列,为研究丝状真菌的基因功能开辟了新的方向,并已成为生命科学领域研究的热点.对基因功能研究中最新的两种方法--基因打靶和RNAi技术的原理、技术路线和特点以及应用情况进行综述.     

植物功能基因组学的发展现状与发展趋势

本文将评述植物功能基因组学研究领域最新的发展状况及其趋势。     

New insights into the cell cycle profile of Paracoccidioides brasiliensis

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.     

RNAi分子机制及在植物功能基因组研究中的应用

RNA干涉现象自20世纪90年代被发现以来,现在已逐渐成为分子生物学和细胞生物学研究的有用工具之一,已被广泛应用到植物功能基因组研究和植物品质营养改良中。RNA干涉机制的深入研究以及该技术在植物基因功能分析中的应用,建立了新的功能基因组学研究平台。阐述了RNAi的分子作用机制、基因沉默的主要类型以及该技术在植物功能基因组研究和品质营养改良上的应用。     

Role of bats in the ecology of Paracoccidioides brasiliensis: the survival of Paracoccidioides brasiliensis in the intestinal tract of frugivorous bat, Artibeus lituratus

The fruit-eating bat, Artibeus lituratus, was fed known quantities of viable yeast cells and mycelial particles of Paracoccidioides brasiliensis in an attempt to assess the role of this animal in the distribution of this agent in nature. Results of mycosal cultures of the stomach, upper intestine, lower intestine and rectum clearly showed that the fungal cells were unable to survive more than 8 hours in the digestive tract of the bat. The mycelial particles were more susceptible than the yeast and were killed before passing to the rectum. The fungus died rapidly in the voided fecal material. These findings indicate the improbability of isolating P. brasiliensis from the digestive tract of wild captured bats and show that A. lituratus probably plays no role in the distribution of this fungus in nature.     

Ultrastructure of Dimorphic Transformation in Paracoccidioides brasiliensis

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.     

New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen

By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.     

Morphogenesis of the mycelium-to-yeast transformation in Paracoccidioides brasiliensis

The sequential changes observed during the mycelium to yeast transformation in Paracoccidioides brasiliensis were studied microscopically. The mycelial elements produced terminal and intercalary swellings which, later on, became chlamydospore-like structures. These increased in size, acquired a double contour and, finally, gave rise to multiple budding cells. Transformation was asynchronous. During the observation period, multiple budding cells and chlamydospores remained attached to the parent mycelium.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.     

Evaluation of Paracoccidioides brasiliensis Infection in Dairy Goats

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Brazil and other Latin American countries. The participation of domestic and wild animal species in the ecoepidemiology of paracoccidioidomycosis is not well understood. The objective of this study was to evaluate P. brasiliensis infection in dairy goats. The humoral immune response to the gp43 antigen, the main antigen used for paracoccidioidomycosis serodiagnosis and seroepidemiology, was evaluated in two goats immunized with inactivated P. brasiliensis yeast cells. Both animals produced antibodies against the P. brasiliensis gp43 antigen, detected by ELISA, 2 weeks after immunization. A total of 202 goat serum samples were analyzed by ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens. The seropositivity observed by ELISA was 26.2 % although no reactivity was detected by immunodiffusion. The animals over 18 months of age showed significantly higher positivity (40 %) than animals aged 6–18 months (14.8 %) and 0–6 months (2.6 %). Taking into account that cross-reactivity may occur with other pathogens, the serum samples were also analyzed by ELISA using Histoplasma capsulatum exoantigen as antigen and the positivity observed was 14.3 %. The low correlation (0.267) observed between reactivity to P. brasiliensis gp43 and H. capsulatum exoantigen suggests co-infection rather than cross-reactivity. This is the first report showing serological evidence of P. brasiliensis infection in goats and reinforces that domestic animals are useful epidemiological markers of paracoccidioidomycosis.     

Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice‐enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.     

The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved

Some aspects pertaining to the ecology of the dimorphic fungus, Paracoccidioides brasiliensis, are reviewed. The available facts concerning the interactions among the only known host (man), the environment (limited to certain Latin-American countries) and the parasite (with an unknown habitat), are analysed. Efforts are made to detect clue circumstances which may lead to discovery of the fungus micro-niche. An analysis of P. brasiliensis mycelial form reveals that such a form has the required capabilities to be the natural infectious form. Its requirements for a moist environment in vitro as well as the high relative humidity predominating in the heart of the endemic areas point towards the possibility of an aquatic--or at least, an extremely humid--habitat for P. brasiliensis.     

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective anti-fungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.