Tackling an essential problem in functional proteomics of Saccharomyces cerevisiae

Abstract

Gene inactivation is the cornerstone of functional genetic analysis, but the analysis of essential genes requires conditional inactivation of the gene product. A new study has adapted a simple method for creating conditional alleles to allow large-scale analysis of essential genes in Saccharomyces cerevisiae and has identified a role in DNA replication for a newly identified protein complex.

Figure 1

The Degron cassette enables the construction of potential td alleles for any gene. The cassette contains KanMX for selection (G418 resistance), the CUP1 promoter (P_CUP1) that is expressed in the presence of copper and repressed in its absence, and the gene encoding the temperature-sensitive Degron (td) protein fused to the HA epitope. A gene-specific pair of oligonucleotides (A and B), with 3' ends matching the cassette (shown by vertical dashed lines), is used to amplify the cassette by PCR. Because the 5' end of each oligonucleotide matches sequences of gene X (shown by slanted dashed lines), transformation of the cassette into yeast cells targets the cassette for insertion upstream of the coding sequence by homologous recombination, replacing the native promoter and creating a gene X-td fusion. Materials and details of construction are available at [20].

Figure 2

Schematic outline of the procedure for td-gene inactivation. Horizontal arrows indicate the anticipated results at each step of the process. Procedures in parentheses are optional steps that may be used to characterize the td protein's function within the cell cycle. Alternative treatments may be used before and/or after td-protein inactivation to determine its effect on different processes. See text for additional description of the scheme.