Deletion of the Trichodiene Synthase Gene of Fusarium venenatum: Two Systems for Repeated Gene Deletions

The trichodiene synthase (tri5) gene of Fusarium venenatum was cloned from a genomic library. Vectors were created in which the tri5 coding sequence was replaced with the Neurospora crassa nitrate reductase (nit3) gene and with the Aspergillus nidulans acetamidase (amdS) gene flanked by direct repea...

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Bibliographic Details
Published in:Fungal genetics and biology 1999-10, Vol.28 (1), p.68-78
Main Authors: Royer, John C, Christianson, Lynne M, Yoder, Wendy T, Gambetta, Greg A, Klotz, Alan V, Morris, Carin L, Brody, Howard, Otani, Suzie
Format: Article
Language:English
Subjects:
acetamidase
acetamide
amds gene
amide hydrolases
Amidohydrolases - genetics
Aspergillus nidulans
Aspergillus nidulans - enzymology
Aspergillus nidulans - genetics
biosynthesis
Blotting, Southern
Carbon-Carbon Lyases - genetics
cloning
deletion
deletions
diacetoxyscirpenol
enzymes
Fungal Proteins - genetics
Fusarium
Fusarium - enzymology
Fusarium - genetics
Fusarium venenatum
Gene Deletion
gene transfer
Gene Transfer Techniques
genes
Genes, Fungal
genetic markers
genetic transformation
Genetic Vectors
in vitro selection
mutagenesis
Neurospora crassa
nit3 gene
Nitrate Reductase
Nitrate Reductases - genetics
nitrates
nitrogen metabolism
phenotype
plasmid vectors
Quorn
replacement
Transformation, Genetic
tri5
tri5 gene
Trichothecenes - metabolism
tricothecene
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Summary:The trichodiene synthase (tri5) gene of Fusarium venenatum was cloned from a genomic library. Vectors were created in which the tri5 coding sequence was replaced with the Neurospora crassa nitrate reductase (nit3) gene and with the Aspergillus nidulans acetamidase (amdS) gene flanked by direct repeats. The first vector was utilized to transform a nitrate reductase (niaD) mutant of F. venenatum to prototrophy, and the second vector was utilized to confer acetamide utilization to the wild-type strain. Several of the transformants lost the capacity to produce the trichothecene diacetoxyscirpenol and were shown by hybridization analysis to have gene replacements at the tri5 locus. The nit3 gene was removed by retransformation with a tri5 deletion fragment and selection on chlorate. The amdS gene was shown to excise spontaneously via the flanking direct repeats when spores were plated onto fluoroacetamide.
ISSN:1087-1845
1096-0937
DOI:10.1006/fgbi.1999.1162