Mutational Studies on Yeast Calmodulin

Backgroud Information Phenylalanine Mutants
Classical Mutageneic Studies Studies Using Synthetic Calmodulin

Background Information

Classical Mutagenic Studies

Phenylalanine Mutants

Ohya and Bostein used site-directed mutagenesis to systematically study the role of the phenylalanine residues in yeast calmodulin. They reasoned that since phe residues are more conserved than any other amino acid between yeast and vertebrate calmodulin, and since vertebrate calmodulin can functionally substitute for yeast calmodulin, mutating phenylalanine residues to alanine might lead to a conditional-lethal phenotype.

Ohya, Y. and D. Bostein, Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene. (1994) Genetics 138. 1041-1054.

Studies Using a Synthetic Consensus Calmodulin in Yeast

Roberts et al, developed a synthetic "consensus" calmodulin for use in mutagenesis studies. This calmodulin contains the conserved features of plant and animal calmodulins.

Roberts, D.M., R. Crea, M. Malecha, G. Alvarado=Urbina, R.H. Chiarello, and D.M. Watterson, Chemical synthesis and expression of a calmodulin gene for site-specific mutagenesis. (1985) Biochemistry 24. 5090-5098.

Harris, Watterson, and Thorner sunstituted mutants of this gene for wildtype calmodulin in S. Cerevisiae. The "wildtype" synthetic gene (which has 60% sequence identity with yeast calmodulin) was able to substitute for calmodulin, but several of the mutant synthetic calmodulins caused mutant phenotypes in the yeast cells.

Harris, E., D.M. Watterson and J. Thorner, Functional consequences in yeast of single -residue alterations in a consensus calmodulin

Note: all of the synthetic calmodulins ("wildtype" and mutant) are under the control of a GAL promoter, and therefore the proteins are expected to be present at fairly high copy number.

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