Use of <Emphasis Type="Italic">MET3</Emphasis> promoters for regulated gene expression in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> |
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Authors: | Alexander Dünkler Jürgen Wendland |
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Institution: | Department of Microbiology, Friedrich-Schiller-University, Jena, Germany. |
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Abstract: | A central tool for gene function analysis is the construction mutant strains. This can be done conveniently in A. gossypii using PCR-based tools. The deletion of essential genes can be performed since initial transformants are sheltered in a heterokaryotic
mycelium, which contains nuclei with both wild type and mutant alleles. The analysis of mutant phenotypes in A. gossypii is regularly started by germinating spores, which contain only one nucleus. Thus, selection can be used to identify mutant
germ cells and germlings. However, such an analysis yields only mutant mycelia if the deleted gene is not essential. We describe
the use of the regulatable Saccharomyces cerevisiae and A. gossypii
MET3 promoters as novel tools to regulate gene expression in A. gossypii. Conditional expression was tested using GFP and lacZ-reporter genes. Regulation of MET3 promoters was found to be dependent on methionine but not on cysteine and down-regulation to about 1/10 of the initial expression
levels was achieved. We used the A. gossypii
WAL1 and CYK1 genes as models to demonstrate that MET3 promoters could regulate the expression of these genes and reveal their mutant phenotypes depending on the presence or absence
of methionine. Finally, we show that the AgMET3 promoter contains two Cpf1-binding sites and that AgCPF1 can complement the S. cerevisiae
cpf1 methionine auxotrophy. |
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Keywords: | Filamentous fungus Gene expression Regulatable promoter Morphogenesis |
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