Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly.

Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens.

BACKGROUND: The incidence of fungal endophthalmitis has dramatically increased in recent years and rapid detection of fungi using nucleic acid-based amplification techniques is helpful in management. AIM: To evaluate semi-nested polymerase chain reaction (PCR) targeting internal transcribed spacer (ITS) region for detection of panfungal genome in ocular specimens. STATISTICAL ANALYSIS USED: Z test for two proportion. MATERIALS AND METHODS: Standardization of PCR targeting ITS primers was carried out by determining analytical sensitivity and specificity. The sensitivity and specificity of PCR was determined by serial tenfold dilutions of C. albicans (ATCC 24433) DNA and DNA extracts of laboratory isolates of Aspergillus fumigatus, Fusarium lichenicola (4), other fungal and closely related bacterial strains and also human DNA. Semi-nested PCR was applied onto a total of 168 ocular specimens with clinically suspected fungal etiology during 2003-2005. RESULTS AND CONCLUSIONS: PCR was specific and sensitive to detect 1fg of fungal DNA with ITS primers. PCR detected fungal genome in 90 (53.57%) in comparison with the conventional technique, positive in 34 (20.23%) by smear examination and in 42 (25%) by culture. The increase in clinical sensitivity by 28.57% using PCR was found to be statistically significant { P < 0.001 using Z test for two proportion}. The accuracy of the test was found to be 70.85%. PCR proved to be a rapid diagnostic technique for detection of panfungal genome directly from clinical specimens.

Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa

DNA isolation from some fungal organisms is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. Beginning with a yeast Saccharomyces cerevisiae genomic DNA isolation method, we developed a 30-min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae of Crinipellis perniciosa, which causes witches' broom disease in cacao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from the yeast S. cerevisiae. Genomic DNA was suitable for PCR of specific actin primers of C. perniciosa, allowing it to be differentiated from fungal contaminants, including its natural competitor, T. stromaticum.

The biology and potential for genetic research of transposable elements in filamentous fungi

Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons) that use RNA and reverse transcriptase and class II elements (DNA transposons) that use DNA. The changes (transposition and recombination) caused by transposons can supply wide-ranging genetic variation, especially for species that do not have a sexual phase. The application of transposable elements to gene isolation and population analysis is an important tool for molecular biology and studies of fungal evolution

Bioinformatics of the Paracoccidioides brasiliensis EST Project

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project--PbGenome Project was developed to study the infection process by analyzing expressed sequence tags--ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible.

Paracoccidioides brasiliensis translation and protein fate machineries revealed by functional genome analysis

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.