Methods for Generating Hypermutable Yeast
AssigneesThe Johns Hopkins University, Morphotek, Inc.
InventorsNicholas C. Nicolaides, Philip M. Sass, Luigi Grasso, Bert Vogelstein, Kenneth W. Kinzler
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Abstract
Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be rendered genetically stable by restoring the mismatch repair system to proper functionality.
Claims24
A method for generating a population of mismatch repair-proficient yeast with new output traits, comprising the steps of:
- growing a yeast cell comprising a gene of interest and a polynucleotide encoding a PMS2, wherein said PMS2 comprises an ATPase domain and has the ability to inhibit mismatch repair, to create a population of mutated, hypermutable yeast cells;
- selecting yeast cells having a mutation in the gene of interest;
- and restoring normal mismatch repair activity to the selected yeast cells.
A method for generating a genetically altered clonal yeast cell population comprising:
- introducing a polynucleotide encoding a PMS2, wherein said PMS2 comprises an ATPase domain and has the ability to inhibit mismatch repair, said polynucleotide operably linked to a promoter, in a population of yeast cells in culture, thereby inhibiting mismatch repair in said cells,
- separating said population into individual members of the population,
- identifying members of the population comprising a mutation in a gene of interest,
- restoring mismatch repair activity to said members of the population comprising a mutation in the gene of interest, and expanding said members comprising a mutation in the gene of interest,
- thereby generating a genetically altered clonal yeast cell population.
Dependencies are inferred from the claim text.