ADDITIONAL MODULES FOR VERSATILE AND ECONOMICAL PCR-BASED GENE DELETION AND MODIFICATION IN SACCHAROMYCES-CEREVISIAE

An important recent advance in the functional analysis of Saccharomyce s cerevisiae genes is the development of the one-step PCR-mediated tec hnique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a var iety of gene modifications. Using as selectable marker the S. cerevisi ae TRP1 gene or modules containing the heterologous Schizosaccharomyce s pombe his5(+) or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promote r), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or wit hout concomitant protein tagging). Because of the modular nature of th e plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these p lasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. (C) 1998 John Wiley & Sons, Ltd.