Molecular cloning of the mouse ouabain-resistance gene

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 repetitive 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 oua(R) gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5-kilobase DNA fragment was initially isolated from a λ phage library made from a ouabain-resistant secondary transformant 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.