MOLECULAR ANALYSIS OF NA+/K+-ATPASE

Project: Research project

Description

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.
StatusFinished
Effective start/end date12/1/846/30/93

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $180,509.00
  • National Institutes of Health

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Adenosine Triphosphatases
Ouabain
Messenger RNA
Protein Isoforms
Cardiac Glycosides
sodium-translocating ATPase
Complementary DNA
Genes
Genetic Techniques
Enzymes
Site-Directed Mutagenesis
Rodentia
Cell Survival
Bacteriophage lambda
Physiological Stress
Peptides