Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.
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.
Waring
R B
RB
Department of Pharmacology, UMDNJ, R.W. Johnson Medical School, Piscataway 08854.
May
G S
GS
Morris
N R
NR
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D001233:Aspergillus nidulans / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D001542:Benomyl / Q000494:pharmacology; D002678:Chimera; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000187:drug effects / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics
D004271:DNA, Fungal; D012331:RNA, Fungal; D012333:RNA, Messenger; D014404:Tubulin; D000426:Alcohol Dehydrogenase; D001542:Benomyl