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

Xiao Mai Zhou, Mary Jo Cunha, Jonathan Epstein, Robert Levenson, Lewis C. Cantley, Lloyd G. Cantley

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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 α1- and β1-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-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 MOR6.5 and the 5′-flanking regions of α1- and β1-subunit genes are discussed.

Original languageEnglish (US)
Pages (from-to)4126-4133
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number6
StatePublished - Feb 25 1993

Fingerprint

Ouabain
Adenosine Triphosphatases
Up-Regulation
Messenger RNA
DNA
Transfection
Genes
Polyadenylation
5' Flanking Region
COS Cells
DNA sequences
Nucleic Acid Databases
Transcription
sodium-translocating ATPase
Primates
Open Reading Frames
Toxicity
Cell Survival
Nucleotides
Adenosine Triphosphate

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Zhou, Xiao Mai ; Cunha, Mary Jo ; Epstein, Jonathan ; Levenson, Robert ; Cantley, Lewis C. ; Cantley, Lloyd G. / A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase α/β-subunit mRNA up-regulation and confers ouabain resistance. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 6. pp. 4126-4133.
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abstract = "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 α1- and β1-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-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 MOR6.5 and the 5′-flanking regions of α1- and β1-subunit genes are discussed.",
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A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase α/β-subunit mRNA up-regulation and confers ouabain resistance. / Zhou, Xiao Mai; Cunha, Mary Jo; Epstein, Jonathan; Levenson, Robert; Cantley, Lewis C.; Cantley, Lloyd G.

In: Journal of Biological Chemistry, Vol. 268, No. 6, 25.02.1993, p. 4126-4133.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase α/β-subunit mRNA up-regulation and confers ouabain resistance

AU - Zhou, Xiao Mai

AU - Cunha, Mary Jo

AU - Epstein, Jonathan

AU - Levenson, Robert

AU - Cantley, Lewis C.

AU - Cantley, Lloyd G.

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N2 - 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 α1- and β1-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-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 MOR6.5 and the 5′-flanking regions of α1- and β1-subunit genes are discussed.

AB - 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 α1- and β1-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-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 MOR6.5 and the 5′-flanking regions of α1- and β1-subunit genes are discussed.

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