Methods for generating hypermutable yeast
AssigneesThe Johns Hopkins University, Morphotek, Inc.
InventorsNicholas C. Nicolaides, Philip M. Sass, Luigi Grasso, Bert Vogelstein, Kenneth W. Kinzler
Life of this patent
Estimating the term…
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 be rendered genetically stable by restoring the mismatch repair system to proper functionality.
Claims42
A method for making a hypermutable yeast, comprising the step of:
- introducing into a yeast a polynucleotide comprising a dominant negative allele of a heterologous mismatch repair gene, wherein said heterologous mismatch repair gene is a mammalian or plant MutL homolog, whereby the cell becomes hypermutable.
A homogeneous composition of cultured, hypermutable yeast comprising a dominant negative allele of a mismatch repair gene, wherein said mismatch repair gene is a mammalian or plant MutL homolog.
A method of generating a hypermutable cell comprising introducing into a cell a polynucleotide encoding PMSR2, wherein said cell becomes hypermutable.
A method for generating a mutation in a gene of interest comprising the steps of
- growing a cell culture comprising the gene of interest and a polynucleotide encoding a PMSR2 operably linked to expression control sequences;
- testing said cell to determine whether the gene of interest harbors a mutation.
An isolated hypermutable cell comprising an expression vector comprising a PMSR2 coding sequence operably linked to expression control sequences.
A method of generating a hypermutable cell comprising introducing into a cell a polynucleotide encoding PMSR3.
A method for generating a mutation in a gene of interest comprising the steps of:
- growing a cell culture comprising the gene of interest and a polynucleotide encoding a PMSR3 operably linked to expression control sequences;
- testing said cell to determine whether the gene of interest harbors a mutation.
An isolated hypermutable cell comprising an expression vector comprising a PMSR3 coding sequence operably linked to expression control sequences.
Dependencies are inferred from the claim text.