A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities.
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Glucan 1,4-alpha-Glucosidase↗
Biomedical subjects
Reginald Storms
Publications and source records attributed to Reginald Storms.
Generation, annotation, and analysis of an extensive Aspergillus niger EST collection.
BACKGROUND: Aspergillus niger, a saprophyte commonly found on decaying vegetation, is widely used and studied for industrial purposes. Despite its place as one of the most important organisms for commercial applications, the lack of available information about its genetic makeup limits research with this filamentous fungus. RESULTS: We present here the analysis of 12,820 expressed sequence tags (ESTs) generated from A. niger cultured under seven different growth conditions. These ESTs identify about 5,108 genes of which 44.5% code for proteins sharing similarity (E < or = 1e(-5)) with GenBank entries of known function, 38% code for proteins that only share similarity with GenBank entries of unknown function and 17.5% encode proteins that do not have a GenBank homolog. Using the Gene Ontology hierarchy, we present a first classification of the A. niger proteins encoded by these genes and compare its protein repertoire with other well-studied fungal species. We have established a searchable web-based database that includes the EST and derived contig sequences and their annotation. Details about this project and access to the annotated A. niger database are available. CONCLUSION: This EST collection and its annotation provide a significant resource for fundamental and applied research with A. niger. The gene set identified in this manuscript will be highly useful in the annotation of the genome sequence of A. niger, the genes described in the manuscript, especially those encoding hydrolytic enzymes will provide a valuable source for researchers interested in enzyme properties and applications.
Aspergillus niger↗
OrfPredictor: predicting protein-coding regions in EST-derived sequences.
OrfPredictor is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with a hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments, otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences. The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where the coding region begins and ends. OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects. OrfPredictor is available at https://fungalgenome.concordia.ca/tools/OrfPredictor.html.
Algorithms↗
TargetIdentifier: a webserver for identifying full-length cDNAs from EST sequences.
TargetIdentifier is a webserver that identifies full-length cDNA sequences from the expressed sequence tag (EST)-derived contig and singleton data. To accomplish this TargetIdentifier uses BLASTX alignments as a guide to locate protein coding regions and potential start and stop codons. This information is then used to determine whether the EST-derived sequences include their translation start codons. The algorithm also uses the BLASTX output to assign putative functions to the query sequences. The server is available at https://fungalgenome.concordia.ca/tools/TargetIdentifier.html.
Algorithms↗
Microplate-based carboxymethylcellulose assay for endoglucanase activity.
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Carboxymethylcellulose Sodium↗
Microplate-based filter paper assay to measure total cellulase activity.
The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.
Cellulase↗
Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger.
We constructed three sets of plasmids for use in Aspergillus niger. These plasmids were assembled using various combinations of a series of modular DNA cassettes that included a selectable marker, pyrG, derived from Aspergillus nidulans; two promoter regions for directing protein expression; a cassette derived from the AMA1 replicator sequence to support autonomous replication; and a reporter gene based on the A. niger lacA gene. One set included integrating and autonomously replicating plasmids for the expression of homologous and heterologous proteins. The second was a set of autonomously replicating plasmids, with a secreted beta-galactosidase encoding reporter gene, for studying gene regulation events. The third set included pyrG-derived gene-blaster cassettes suitable for genome manipulation by targeted gene replacement.
Aspergillus nidulans↗
Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery.
Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.
Alleles↗