A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase alpha/beta-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 MOR6.5) 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 MOR6.5. 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 MOR6.5 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 MOR6.5 transfectants which were under ouabain-selective pressure and still contained MOR6.5 DNA sequence, mRNAs for both alpha 1- and beta 1-subunits of the Na,K-ATPase were amplified approximately 10-fold, compared to parental CV-1 cells. These results suggest that MOR6.5 may rescue the cells from ouabain toxicity by inducing transient up-regulation of the messages for the Na,K-ATPase. This might prolong cell survival on ouabain until mutations in the alpha 1-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 MOR6.5 and the 5'-flanking regions of alpha 1- and beta 1-subunit genes are discussed.