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

1988, Proceedings of the National Academy of Sciences

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 Agtll 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 rindings. Superoxide dismutases (SODs) are abundant enzymes present in most aerobic organisms and many anaerobic ones. Their known activity is catalysis of the disproportionation of the superoxide radical, O°, to give dioxygen and hydrogen peroxide (20 + 2H +-+02 + H202) (1, 2). They are widely assumed to provide protection in vivo against this reactive metabolic by-product, although this has not been conclusively proven (3). SOD exists in several forms in different organisms. Prokaryotes have two forms, one containing iron and one containing manganese. Eukaryotes have a manganese-containing form in the mitochondria and a copper-and zinc-containing form in the cytoplasm (Cu,Zn-SOD; EC 1.15.1.1). The Mn and Fe proteins are related to each other, whereas Cu,Zn-SOD is unrelated to either (2). The Cu,Zn-SOD protein is very well studied. The crystal structure of the bovine enzyme has been solved (4), and it and the yeast enzyme have been the subject of numerous physical and chemical studies in this and other laboratories (5). The in vivo role of Cu,Zn-SOD is less well defined. There is disagreement over whether or not O2 is a direct cause of dioxygen toxicity and, consequently, over whether SOD has an important protective function, since O2 disproportionates