Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin |
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Authors: | Masaaki Takahashi Seiji Matsumoto Shigeo Iwasaki and Ichiro Yahara |
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Institution: | (1) Institute of Applied Microbiology, The University of Tokyo, 113 Bunkyo-ku, Tokyo, Japan;(2) Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 113 Bunkyo-ku, Tokyo, Japan |
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Abstract: | Summary Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to -tubulin. ThebenA gene of three independently isolated rhizoxin-resistant (Rhir) mutants ofAspergillus 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 thebenA -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 -tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeastSchizosaccharomyces pombe and the budding yeastSaccharomyces cerevisiae are naturally occurring Rhir organisms whose -tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 ofS. pombe and the Val-100 ofS. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing -tubulin (Asn-100) instead of -tubulin (Ile-100 or Val-100) were found to be Rhis. Haploid yeast expressing -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 -tubulin. |
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Keywords: | Aspergillus nidulans Schizosaccharomyces pombe Saccharomyces cerevisiae Drug resistance -tubulin mutation" target="_blank">gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin mutation |
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