Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin

Mol Gen Genet. 1990 Jul;222(2-3):169-75. doi: 10.1007/BF00633814.

Abstract

Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to beta-tubulin. The benA gene of three independently isolated rhizoxin-resistant (Rhir) mutants of Aspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhis) strain. In all three Rhir mutants, the AAC codon for Asn-100 of the benA beta-tubulin gene was altered to ATC, coding for Ile. Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism. The amino acid sequences of beta-tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are naturally occurring Rhir organisms whose beta-tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 of S. pombe and the Val-100 of S. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing beta-tubulin (Asn-100) instead of beta-tubulin (Ile-100 or Val-100) were found to be Rhis. Haploid yeast expressing beta-tubulin (Asn-100) is normal except for its sensitivity to rhizoxin. These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in beta-tubulin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antifungal Agents / pharmacology*
  • Aspergillus nidulans / drug effects*
  • Aspergillus nidulans / genetics
  • Base Sequence
  • Blotting, Southern
  • Cloning, Molecular
  • Codon
  • DNA, Fungal
  • Drug Resistance, Microbial / genetics
  • Genes, Fungal*
  • Lactones / pharmacology
  • Macrolides
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Mutation
  • Restriction Mapping
  • Saccharomyces cerevisiae / genetics
  • Schizosaccharomyces / genetics
  • Sequence Homology, Nucleic Acid
  • Transformation, Genetic
  • Tubulin / genetics*

Substances

  • Antifungal Agents
  • Codon
  • DNA, Fungal
  • Lactones
  • Macrolides
  • Tubulin
  • rhizoxin