In Vitro Mutagenesis and Plasmid Shuffling: From Cloned Gene to Mutant Yeast

Robert S. Sikorski, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

This chapter describes the In Vitro mutagenesis and plasmid shuffling in yeast genes. method for generating mutant alleles uses replicating yeast episomes as a means of exchanging the wild-type gene for mutant copies. The basic scheme for the exchange, known as plasmid shuffling In the first step, one copy of the gene of interest is inactivated in a diploid, and a wild-type copy is propagated in the cell on an episome. This allows the generation of a haploid strain with a chromosomal null allele. Mutagenized copies of the gene are then introduced into this cell on a second episome and exchanged (or shuffled) with the wild-type version. Removal of the wild-type gene, YFG in our example, is the key step in any plasmid shuflting scheme. This can be accomplished by taking advantage of two factors. First, even relatively stable YCp episomes are lost from a cell by missegregation or misrepfication at a rate of 10–2 per generation. Second, compounds are available that prevent the growth of cells carrying specific yeast genes, and in the presence of such compounds these genes act as counterselectable markers, allowing one to directly select for cells which have lost this marker. By including one of these counterselectable markers on the same plasmid that contains the wild-type YFG gene, an investigator can select for cells that have lost the entire plasmid.

Original languageEnglish (US)
Pages (from-to)302-318
Number of pages17
JournalMethods in enzymology
Volume194
Issue numberC
DOIs
StatePublished - Jan 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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