Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae.
cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter. The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation. Total RNA from the transformed strains was isolated and analyzed by Northern hybridization. The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA. 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit. A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-). Experiments to detect an alpha specific translation product are in progress.
Horowitz
B
B
University of Southern California School of Medicine, Los Angeles 90033.
Farley
R A
RA
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005796:Genes; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase