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A 6.5-kilobase fragment of genomic DNA from mutant mouse cells under ouabain selection pressure conferred ouabain resistance when transfected into ouabain-sensitive CV1 green monkey fibroblasts. Ouabain resistance was induced in the presence of 10 microM ouabain. Amiloride (500 microM) completely blocked ouabain-insensitive 86Rb+ uptake into these cells. Plasma membranes from these cells demonstrated little sodium-dependent adenosine triphosphatase (ATPase) activity but had potassium-dependent and ouabain-resistant p-nitrophenylphosphatase activity. Like Na+,K+-ATPase this activity was vanadate- and sodium-inhibitable. Also, like the Na+,K+-ATPase, sodium inhibition of the p-nitrophenylphosphatase was reversed by 10 microM adenosine 5'-triphosphate.

The structural gene, PHO13, for the specific p-nitrophenyl phosphatase of Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The deduced PHO13 protein consists of 312 amino acids and its molecular weight is 34635. The disruption of the PHO13 gene produced no effect on cell growth, sporulation, or viability of ascospores. The PHO13 locus was mapped at 1.9 centimorgans from the HO locus on the left arm of chromosome IV. By chromosome fragmentation, the PHO13 locus was found to be located about 72 kb from the left-hand telomere of chromosome IV and distal to the HO locus.