A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase α/β-subunit mRNA up-regulation and confers ouabain resistance

Transfection of primate cells with a 6.4-kilobase murine genomic DNA fragment (called ouabain resistance gene or MOR^6.5) has been shown previously to confer ouabain resistance (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493). The mechanism by which this sequence can transfer ouabain resistance remains unclear. In order to further investigate this mechanism, we determined the full-length nucleotide sequence of MOR^6.5. Other than mouse repetitive domains, this DNA does not have significant homology with any coding sequence in GenBank. Although potential open reading frames and polyadenylation signals were found, we were unable to detect an MOR^6.5 transcript in CV-1 or COS-1 cells transfected with this DNA, either at early or late times following transfection. We show that in early passages of MOR^6.5 transfectants which were under ouabain-selective pressure and still contained MOR^6.5 DNA sequence, mRNAs for both α₁- and β₁-subunits of the Na,K-ATPase were amplified approximately 10-fold, compared to parental CV-1 cells. These results suggest that MOR^6.5 may rescue the cells from ouabain toxicity by inducing transient up-regulation of the messages for the Na,K-ATP-ase. This might prolong cell survival on ouabain until mutations in the α₁-subunit occur, which permanently reduce ouabain inhibition of the pump (Cantley, L. G., Zhou, X.-M., Cunha, M., Epstein, J., and Cantley, L. C. (1992) J. Biol. Chem. 267, 17271-17278). Possible mechanisms for the up-regulation of transcription based on sequence similarities found between MOR^6.5 and the 5′-flanking regions of α₁- and β₁-subunit genes are discussed.