The Cloning of Homologous Na+/K+-ATPase Genes From New Species by a PCR Based Approach

The investigation of new models for the study of the mechanisms of regulation of ion transport in new species such the European eel (Anguilla anguilla), requires the use of gene probes to enable the measurement of mRNA levels using nucleic hybridisation techniques. One possibility, for proteins such as Na+/K+-ATPase, is to use genes already available from other species for this purpose. However, the cross reactivity of these genes, with diverse species such as the eel is likely to be low, especially in the case of the Na+/K+-ATPase β (β1) subunit gene. Consequently, the only alternative is to clone the genes concerned from the model organism. Normal cloning procedures requiring the screening of up to 107 colonies are both expensive and time consuming. However, using the recently developed PCR (polymerase chain reaction) technology (Perkin Elmer/Cetus), fragments of genes such as Na+/K+-ATPase, (where regions of amino acid homology are known to exist between species) can be quickly and easily cloned, and used as gene probes. This poster illustrates the methods used for the cloning of the Na+/K+-ATPase a and β genes from the European eel. These methods are however, easily adapted to any gene where known regions of amino acid or nucleotide homology exist.