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 Acremon ium chrysogenum using a heterologous gene probe to screen an A. chryso genum 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 s howed a high degree of similarity when compared with amino-acid sequen ces from beta-tubulins of other eukaryotes. The introns are characteri zed by typical consensus sequences found in intervening sequences from other filamentous fungi. In-vitro mutagenesis of codon 167 of the bet a-tubulin gene resulted in the substitution of a phenylalanine by a ty rosine in the corresponding polypeptide sequence. The mutated gene was used successfully in the transformation and co-transformation of A, c hrysogenum to benomyl resistance. The molecular analysis of transforma nts provided evidence that they contain the mutated beta-tubulin gene in addition to the wild-type gene, as was proved by Southern-hybridiza tion analysis and direct sequencing of PCR amplification products.