Molecular cloning of rat brain Na,K-ATPase α-subunit cDNA

J. W. Schneider, R. W. Mercer, M. Caplan, J. R. Emanuel, K. J. Sweadner, E. J. Benz, R. Levenson

Research output: Contribution to journalArticle

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Abstract

We have isolated a cDNA clone for the rat brain Na,K-ATPase α subunit. A λgt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, λrb5, containing a 1200-base-pair cDNA insert expressed a β-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the α subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of λrb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney > brain > liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to λrb5 cDNA were amplified ≃40-fold. Analysis of the λrb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase α-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the λrb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase α subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.

Original languageEnglish (US)
Pages (from-to)6357-6361
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume82
Issue number18
DOIs
StatePublished - Dec 1 1985

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Molecular Cloning
Complementary DNA
Brain
Ouabain
Clone Cells
Kidney
HeLa Cells
Messenger RNA
Galactosidases
Cell Line
Sodium-Potassium-Exchanging ATPase
Gene Dosage
sodium-translocating ATPase
Antibodies
Liver
Human Genome
Fluorescein
Gene Library
Immunoblotting
Base Pairing

All Science Journal Classification (ASJC) codes

  • General

Cite this

Schneider, J. W. ; Mercer, R. W. ; Caplan, M. ; Emanuel, J. R. ; Sweadner, K. J. ; Benz, E. J. ; Levenson, R. / Molecular cloning of rat brain Na,K-ATPase α-subunit cDNA. In: Proceedings of the National Academy of Sciences of the United States of America. 1985 ; Vol. 82, No. 18. pp. 6357-6361.
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Molecular cloning of rat brain Na,K-ATPase α-subunit cDNA. / Schneider, J. W.; Mercer, R. W.; Caplan, M.; Emanuel, J. R.; Sweadner, K. J.; Benz, E. J.; Levenson, R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 82, No. 18, 01.12.1985, p. 6357-6361.

Research output: Contribution to journalArticle

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AU - Schneider, J. W.

AU - Mercer, R. W.

AU - Caplan, M.

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N2 - We have isolated a cDNA clone for the rat brain Na,K-ATPase α subunit. A λgt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, λrb5, containing a 1200-base-pair cDNA insert expressed a β-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the α subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of λrb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney > brain > liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to λrb5 cDNA were amplified ≃40-fold. Analysis of the λrb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase α-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the λrb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase α subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.

AB - We have isolated a cDNA clone for the rat brain Na,K-ATPase α subunit. A λgt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, λrb5, containing a 1200-base-pair cDNA insert expressed a β-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the α subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of λrb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney > brain > liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to λrb5 cDNA were amplified ≃40-fold. Analysis of the λrb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase α-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the λrb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase α subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.

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