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

Gene. 1989 Jun 30;79(1):119-30. doi: 10.1016/0378-1119(89)90097-8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • DNA, Fungal
  • RNA, Fungal
  • RNA, Messenger
  • Tubulin
  • Alcohol Dehydrogenase
  • Benomyl