Development of an homologous transformation system for Acremonium chrysogenum based on the beta-tubulin gene.
The beta-tubulin gene was isolated from the filamentous fungus Acremonium chrysogenum using a heterologous gene probe to screen an A. chrysogenum lambda library. Sequencing of the A. chrysogenum gene revealed a mosaic gene which contains five exons and four intervening sequences. The exons encode for a polypeptide of 447 amino-acid residues which showed a high degree of similarity when compared with amino-acid sequences from beta-tubulins of other eukaryotes. The introns are characterized by typical consensus sequences found in intervening sequences from other filamentous fungi. In-vitro mutagenesis of codon 167 of the beta-tubulin gene resulted in the substitution of a phenylalanine by a tyrosine in the corresponding polypeptide sequence. The mutated gene was used successfully in the transformation and co-transformation of A. chrysogenum to benomyl resistance. The molecular analysis of transformants provided evidence that they contain the mutated beta-tubulin gene in addition to the wild-type gene, as was proved by Southern-hybridization analysis and direct sequencing of PCR amplification products.
Nowak
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Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum, Germany.
Kück
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D000164:Acremonium / Q000187:drug effects / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001542:Benomyl / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005799:Genes, Dominant; D005800:Genes, Fungal; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012326:RNA Splicing / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D005819:Genetic Markers; D014404:Tubulin; D001542:Benomyl