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Updated: Aug 4, 2026
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Mak mutants of yeast: mapping and characterization
Abstract:
Killer strains of Saccharomyces cerevisiae are those carrying a 1.5 x 10(6)-dalton double-stranded (ds) ribonucleic acid (RNA) (M) in virus-like particles and secreting a protein toxin. Most yeast (koller or not) also carry a 3 x 10(6)-dalton dsRNA (L). We have mapped mutations in eight of the chromosomal genes needed for maintaining M (mak genes). The mak genes are widely distributed on the yeast map, with no multigene complexes. We show that mutants defective in these and other mak genes lose M dsRNA, but not L dsRNA. The mak3-1 mutation results in markedly decreased cellular levels of L dsRNA, but mak3-1 stains do not lose L dsRNA completely. Mutation of mak16 results in temperature-sensitive growth, whereas mutations in mak13, mak15, mak17, mak20, mak22, and mak27 result in slow growth at any temperature. No effect of mak mutations on mating, meiosis, sporulation, germination, homothallism, or ultraviolet sensitivity has been found. The specificity of mak mutations is discussed.
Insights
Killer yeast Saccharomyces cerevisiae strains require specific M double-stranded (ds) RNA. Researchers mapped mutations in eight chromosomal maintenance (mak) genes, finding they cause M dsRNA loss but not L dsRNA loss, impacting yeast growth.
Area of Science:
- Molecular biology
- Yeast genetics
- Virology
Background:
- Killer strains of Saccharomyces cerevisiae harbor M double-stranded (ds) RNA within virus-like particles, enabling toxin secretion.
- Most yeast strains also possess L dsRNA, a larger dsRNA molecule.
- Understanding the genetic basis for dsRNA maintenance is crucial for yeast biology.
Purpose of the Study:
- To identify and map chromosomal genes (mak genes) essential for maintaining the M dsRNA in killer yeast.
- To investigate the effects of mutations in these mak genes on dsRNA stability and yeast physiology.
Main Methods:
- Genetic mapping of mutations in eight chromosomal mak genes.
- Analysis of dsRNA content (M and L) in wild-type and mutant yeast strains.
- Phenotypic characterization of mak mutants, including growth rates and temperature sensitivity.
Main Results:
- Eight chromosomal mak genes were mapped across the yeast genome, showing no complex formation.
- Mutations in these mak genes led to the loss of M dsRNA but not L dsRNA.
- Specific mutations (e.g., mak3-1) affected L dsRNA levels, while others (e.g., mak16) caused temperature-sensitive growth, and several resulted in slow growth.
Conclusions:
- The identified mak genes are specifically required for the maintenance of M dsRNA in killer yeast.
- These genes are essential for yeast viability and growth, with distinct mutations conferring different physiological defects.
- The study provides a genetic framework for understanding M dsRNA maintenance and its impact on killer yeast phenotypes.
