This chapter focuses on the experimental approach that uses DNA-mediated gene transfer for isolating the DNA sequence coding for the Na+, K+-ATPase subunit responsible for ouabain resistance. The plasma membrane Na+, K+-ATPase is the enzymatic activity responsible for the maintenance of the high internal K+ and low internal Na+ concentrations characteristic of most living cells. The ability to isolate the gene coding for the ATPase represents an important technical step toward elucidating the primary structure of the Na+,K+-ATPase. The approach considered for isolating the Na+,K+-ATPase gene is essentially based on three main considerations: (1) the cardiac glycoside ouabain is a specific inhibitor of the Na+,K+-ATPase and cell lines of different species vary considerably with respect to ouabain sensitivity, (2) ouabain-resistant mutants exhibit decreased affinity for ouabain, and (3) ouabain resistance can be transferred from ouabain-resistant to ouabain-sensitive cells by direct DNA transfer, metaphase chromosome transfer, or by microcell fusion. This approach takes the advantage of the species-specific differences in ouabain sensitivity in order to develop a gene transfer and selection system for isolating the mouse gene responsible for ouabain resistance. The chapter outlines the methodologies used to isolate the ouabain resistance gene and explores how this DNA sequence can be used to facilitate genetic studies of the Na+,K+-ATPase and the ouabain resistance locus.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology