causes sensitivity to metal stresses. (A) Diagram of the DNA organization around the wild type and mutant allele of PCS1. The three exons of PCS1 are the large blue boxes in the wild type, with the region replaced by the URA5 marker illustrated with the white box. The positions of primers (AS###) are indicated by arrows. AS008-AS002 and AS003-AS009 were used to generate 5′ and 3′ fragments for targeted homologous replacement. Primers AS020-AS004 were used to amplify the probe used in the Southern blot, and primers AS005-AS006 used for the PCR in panel B. Restriction enzyme sites relevant to the Southern blot are provided along with the relative positions in parentheses. (B) PCR analysis using primers AS005-AS006 on the wild type and pcs1 deletion strains. (C) Southern blot analysis of gene replacement of PCS1 with the URA5 gene. Genomic DNA was digested with BamHI, EcoRI or HindIII restriction enzymes and resolved on a 1% agarose gel. 32P-labelled DNA amplified with primers AS020-AS004 was hybridized to the blot. Note that the amplification of the probe yielded a second non-specific product, which hybridizes to the bands marked with *. (D) Phenotypes of the gene replacement strain compared to wild type and complemented (Δ + PCS1) strain. Ten-fold serial dilutions were plated onto YPD medium with or without supplements, and cultured for 3 d at 22°C.