The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity

Abstract

The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity. The gene was isolated from a lambda gt11 library by using a long, unique deoxyoligonucleotide probe. The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table. The sequence of the coding and flanking regions is reported. The cloned gene was expressed and shown to be active in vivo. A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity. The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain. Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant. The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.