The objective of the proposed project is to characterize and use as an analytical tool the DNA sequence coding for the mouse Na+/K+ ATPase subunit which is responsible for ouabain resistance. We have recently isolated and cloned a DNA sequence from ouabain resistant mouse cells which can transform ouabain sensitive cells to ouabain resistance. We presume that this DNA sequence represents the gene coding for the Alpha-subunit of the Na+/K+ ATPase. A major goal will be to confirm this assumption. Direct comparison between the DNA sequence of a full length cDNA clone of the ouaR gene and ATPase Alpha-subunit amino acid sequence will allow us to confirm whether the Na+/K+ ATPase Alpha subunit gene has been cloned. Alternative strategies for the isolation of the Alpha-subunit gene, including the construction and screening of a Lambda phage cDNA expression library, are considered in the event our original assumption proves incorrect. The availability of a hybridization probe for the Na+/K+ ATPase will permit us to address basic issues with respect to control mechanisms which affect Na+/K+ ATPase. These include: a) Analysis of ATPase mRNA levels in various tissues of the mouse. We will use quantitative hybridization techniqes to determine whether ATPase mRNA levels vary from tissue to tissue in the mouse. b) We will scan various tissues of the mouse to determine whether Alpha-subunit molecules of differing primary sequence are present in different tissues or within the same tissue of the mouse. Our approach to address this issue will be to employ Sl nuclease mapping techniques to scan tissues for variant forms of Alpha-subunit mRNA. C) Analysis of regulation of ATPase mRNA levels during growth of tissue culture cells. We will measure ATPase mRNA levels in tissue culture cells subjected to physiological stress to determine whether physiological stress leads to regulation of ATPase activity via alterations in ATPase mRNA levels. d) An important objective of this proposal will be to develop the capacity to introduce the ATPase gene into mammalian cells under the control of an inducible promoter. To accomplish this goal, we hope to construct a recombinant molecule between a Na+/K+ ATPase full length cDNA clone and a vector which has an inducible promoter. The purpose of these studies will be obtain basic information about the assembly of the ATPase and to develop the capability of altering the level of the Na+/K+ ATPase in a cell in response to an exogenous signal. This approach will also be used to introduce the inducible ATPase gene into transformed cells in order to study the relationship between alterations in ATPase activity and cellular transformation. A long term outgrowth of the ability to transfer an ion transport gene into and out of mammalian cells will be the feasibility of experimentally manipulating ion levels in cells and studying the effects of these alterations on cellular physiology and metabolism.
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