Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes

• Published in: Gene Vol. 79; no. 1; p. 119
• Main Authors: Waring, R B, May, G S, Morris, N R
• Format: Journal Article
• Language: English
• Published: Netherlands 30-06-1989
• Subjects: Alcohol Dehydrogenase - genetics; Aspergillus nidulans - drug effects; Aspergillus nidulans - genetics; Base Sequence; Benomyl - pharmacology; Chimera; Cloning, Molecular; DNA, Fungal - drug effects; DNA, Fungal - genetics; Drug Resistance, Microbial - genetics; Genes, Fungal; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; RNA, Fungal - biosynthesis; RNA, Fungal - genetics; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Transformation, Genetic; Tubulin - biosynthesis; Tubulin - genetics
• ISSN: 0378-1119
• DOI: 10.1016/0378-1119(89)90097-8

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.