FIG. 1.

Construction of sod2Δ mutant and sod2Δ SOD2 reconstituted strains. (A) PCR overlap was performed to create the sod2Δ::URA5 deletion construct, which consisted of two 1-kb 5′ and 3′ regions of genomic DNA flanking the SOD2 open reading frame and the selectable marker URA5. (B) Southern blot analysis of sod2Δ and the sod2Δ SOD2 reconstituted strains confirmed that a single allele-specific homologous integration of the sod2Δ::URA5 deletion construct occurred at the SOD2 locus and that the SOD2-nat reconstitution construct integrated randomly at a single location in the genome. (C) Cell extracts from strains sod2Δ, sod2Δ SOD2, and wild-type (WT) strain H99 were electrophoresed in a 10% polyacrylamide gel, and superoxide dismutase activity was visualized as described by Beauchamp and Fridovich (6). Strain sod2Δ lacked Sod2 activity, whereas the sod2Δ SOD2 strain and wild-type strain H99 exhibited Sod2 activities. One gel representative of more than five experiments performed is shown.