Genetics, Vol 132, 337-350, Copyright © 1992

INVESTIGATIONS

Systematic Mutational Analysis of the Yeast ACT1 Gene

K. F. Wertman, D. G. Drubin and D. Botstein
Genentech, Inc., South San Francisco, CA 94080 Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720.

We report the isolation and characterization of a synoptic set of site-directed mutations distributed throughout the single actin gene of Saccharomyces cerevisiae. Mutations were systematically targeted to the surface of the protein by identifying clusters of 2 or more charged residues in the primary sequence; every charged residue in a cluster was replaced with alanine. Mutations were recovered in high yield (34 of 36 constructed) as heterozygous diploids. Mutant phenotypes were examined in haploid segregants: 11 were recessive lethal, 16 conditional-lethal (including temperature-sensitive and salt-sensitive) and 7 had no discernible phenotype. Genetic analysis suggested that the two mutations constructed but not recovered in yeast may have a dominant defective phenotype. Location of the mutant residues on the three-dimensional structure of the rabbit muscle actin monomer confirmed that most (81%) of the charged residues we altered lie at or near the surface of the protein, confirming a key assumption of the method. Many of the new act1 alleles have properties readily interpreted in light of the actin structure and should prove useful in both genetic and biochemical studies of actin function.

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