Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique 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 variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), 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 without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.
Longtine
M S
MS
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA. mlunc@isis.unc.edu
McKenzie
A
A
Demarini
D J
DJ
Shah
N G
NG
Wach
A
A
Brachat
A
A
Philippsen
P
P
Pringle
J R
JR
D017931:DNA Primers; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins