Molecular cloning of the mouse oua (DNA transfection/ouabain resistance/phage cloning/Na+,K+-ATPas

DNA prepared from ouabain-resistant mouse cells was able to transform ouabain-sensitive CV-1 cells to ouabain resistance after DNA-mediated gene transfer. The murine DNA fragment responsible for ouabain resistance was detected on the background of CV-1 DNA by virtue of a repeti- tive DNA sequence element that reacts positively with a mouse repeat DNA clone. CV-1 DNA is nonreactive with this probe. Southern analysis of several independently derived ouabain- resistant transformants indicates that the mouse ouaR gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5- kilobase DNA fragment was initially isolated from a X phage library made from a ouabain-resistant secondary transfor- mant and subsequently was subcloned in the plasmid vector pAT153. This plasmid was able to transform wild-type CV-1 cells to ouabain resistance at a frequency of about 10 cells per ng of DNA. secondary transformants by using a cloned mouse repetitive sequence element, suggesting that the homologous repeat se- quence can be used as a biological marker for cloning the ouaR gene. The ouaR gene has been isolated from a phage library prepared from the DNA of a secondary transformant and subcloned in the plasmid vector pAT153. When applied to CV-1 cells, this plasmid is highly active in transforming CV-1 cells to ouabain resistance. The isolation of the ouaR gene may prove extremely useful for understanding the biochemical basis of ouabain resis- tance and for elucidating the structure and function of the Na +,K+-ATPase.