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ABSTRACT: We have generated a collection of yeast strains, each of which has an essential yeast gene under the control of the tetracycline-responsive, tetO, promoter. Screens using first-generation promoter-swap strains uncovered the non-specific responsiveness of the tetO7 promoter to a known human transcription factor (hIRF-1). Non-specific regulation was not observed with the tetO2 promoter. Reporter assays have been used to demonstrate this phenomenon. Subsequent efforts to generate a collection of tetracycline-regulatable strains have focused on the tetO2 promoter. These strains are available to the yeast community and can be used for functional genomics studies.
Yeast 04/2006; 23(4):325-31. · 1.89 Impact Factor
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ABSTRACT: Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.
Applied and Environmental Microbiology 06/2005; 71(5):2737-47. · 3.83 Impact Factor
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ABSTRACT: Whole genome sequencing of several filamentous ascomycetes is complete or in progress; these species, such as Aspergillus nidulans, are relatives of Saccharomyces cerevisiae. However, their genomes are much larger and their gene structure more complex, with genes often containing multiple introns. Automated annotation programs can quickly identify open reading frames for hypothetical genes, many of which will be conserved across large evolutionary distances, but further information is required to confirm functional assignments. We describe a comparative and functional genomics approach using sequence alignments and gene expression data to predict the function of Aspergillus nidulans genes. By highlighting examples of discrepancies between the automated genome annotation and cDNA or EST sequencing, we demonstrate that the greater complexity of gene structure in filamentous fungi demands independent data on gene expression and the gene sequence be used to make confident functional assignments.
Mycological Research 09/2004; 108(Pt 8):853-7. · 2.81 Impact Factor
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ABSTRACT: The science of taxonomy is constantly improving as new techniques are developed. Current practice is to construct phylogenetic trees based on the analysis of the DNA sequence of single genes, or parts of single genes. However, this approach has recently been brought into question as several tree topologies may be produced for the same clade when the sequences for various different genes are used. The availability of complete genome sequences for several organisms has seen the adoption of microarray technology to construct molecular phylogenies of bacteria, based on all of the genes. Similar techniques have been used to reveal the relationships between different strains of the yeast Saccharomyces cerevisiae. We have exploited microarray technology to construct a molecular phylogeny for the Saccharomyces sensu stricto complex of yeast species, which is based on all of the protein-encoding genes revealed by the complete genome sequence of the paradigmatic species, S. cerevisiae. We also analyze different strains of S. cerevisiae itself, as well as the putative species S. boulardii. We show that in addition to the phylogeny produced, we can identify and analyze individual ORF traits and interpret the results to give a detailed explanation of evolutionary events underlying the phylogeny.
Genome Research 07/2004; 14(6):1043-51. · 13.61 Impact Factor
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ABSTRACT: The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.
Fungal Genetics and Biology 03/2004; 41(2):199-212. · 3.74 Impact Factor
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Yeast 01/1994; 10(2):247-255. · 1.89 Impact Factor
