ISOLATION AND CHARACTERIZATION OF MUTATIONS IN THE ß-TUBULIN GENE OF SACCHAROMYCES CEREVISIAE

James H. Thomas ¹, Norma F. Neff ¹, and David Botstein ¹

¹ Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (Ben^R), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the ben^R mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding ß-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the Ben^R and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.

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