Correction/completion of the yeast actin, alanine scan alleles.

IN 1992, the first alanine scanning mutagenesis was performed on the yeast actin gene ACT1 (Wertman et al. 1992). This collection of mutants has proven to be a very useful set of reagents for probing the structure/ function relationships in the actin cytoskeleton. Recently, we realized that several of the most devastating mutants have additional mutations that in several cases lead to complete null alleles. Given how useful these mutants have been, we decided to reconstruct and characterize the seven incorrect alleles. We found that in almost all cases the corrected alleles confer a different phenotype than previously reported. Strains with the corrected alleles, either as heterozygous diploids or as homozygous diploids, and haploids for the viable alleles are now available to the research community. The essential yeast actin gene has proven to be a particularly difficult target for the isolation of conditional mutants by traditional random mutagenesis and screening strategies. Despite numerous attempts, only three conditional alleles were isolated by these methods: act1-1 (P32L), act1-2 (A58T), and act1-4 (E259V). It was expected that actin would have many binding partners, so it was reasoned that many conditional mutations that distribute over the surface of the actin protein could be generated. A strategy was designed to mutate clusters of charged residues in actin with the idea that these would affect clusters of charged residues on the surface of the protein and thereby affect electrostatic interactions with key actin-binding proteins. This procedure yielded 16 new conditional alleles, 11 lethal alleles, 2 dominant lethal alleles, and 7 wildtype alleles (Wertman et al. 1992). These alleles have been very useful to the yeast actin research community and to our lab in particular for mapping actin-binding sites by two-hybrid analysis (Amberg et al. 1995) and for whole-genome-level genetic interaction analysis (Haarer et al. 2007). However, we recently discovered that the original template plasmids for 7 alleles contain additional mutations that, in 5 of the cases, cause frameshifts and in the other 2 cases cause oneand twoamino-acid substitutions. Given the utility of the actin alanine scan alleles, we decided to correct these 7 alleles, characterize their proper mutant phenotypes, and make the correct strains available to the community. As shown in Table 1, 7 of the 36 original ACT1 alanine scan alleles were found to have mutations in addition to the intended one. In five cases, the M13-based mutagenesis procedure introduced frameshift mutations (act1-107, -118, -127, -128, and -130). In one case, a point mutation was introduced (act1-108), and in the case of act1-136, 2 alanine scan alleles were introduced: the expected D2A change plus R37A and R39A (the act1-132 allele). In 1992, the mutations were confirmed by restriction digest because it would have been onerous to determine the complete sequence of ACT1 for all 36 alleles, and therefore the additional mutations were not identified at that time. Five of the alleles in question were reconstructed by overlap fusion PCR (Ho et al. 1989; Dillon and Rosen 1990) and integrated into a BY4741 3 BY4742 diploid background. Two of the alleles (act1-108 and act1-136) were corrected on plasmids by replacing the extra mutations with wild-type DNA by traditional cloning methods, followed by integration into the BY (the knock-out collection) background. We were unable to recover two of the mutants despite numerous efforts (act1-118 and act1-130) and concluded that these are likely dominant lethal mutations. Homozygous diploids were constructed with the viable alleles, and basic phenotype analysis was performed on haploids, homozygous diploids, and heterozygous diploids. The phenotypes of mutants carrying lethal alleles were determined with heterozygous diploids. The goal was to recapitulate the basic phenotypic analysis performed on the original alanine scan mutants (Wertman et al. 1992). Table 2 shows the strains that were constructed. To assess basic growth phenotype and salt sensitivity, all strains were streaked for single colonies on YPD medium and YPD 1 0.9 m NaCl medium and incubated Corresponding author: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210. E-mail: [email protected]