SYSTEMATIC MUTATIONAL ANALYSIS OF THE YEAST BETA-TUBULIN GENE

A systematic strategy was used to create a synoptic set of mutations t hat are distributed throughout the single beta-tubulin gene of Sacchar omyces cerevisiae. Clusters of charged amino acids were targeted for m utagenesis and converted to alanine to maximize alterations on the pro tein's surface and minimize alterations that affect protein folding. O f the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers h eat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that beta- tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yea st at 30 degrees C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most crit ical for beta-tubulin function. Interestingly, most of the conditional -lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaff ected. The exceptions are two mutants that contain abnormally long cyt oplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of beta-tubulin mutants and should be valuable in dissecting spindle function.