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Related Experiment Videos

Gene isolation by direct in situ cAMP binding.

M L Lacombe1, D Ladant, R Mutzel

  • 1Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.

Gene
|January 1, 1987
PubMed
Summary

Researchers developed a novel screening method to detect cyclic adenosine monophosphate (cAMP) binding activity directly on phage plaques. This technique successfully identified clones expressing the regulatory subunit of cAMP-dependent protein kinase.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The regulatory subunit of cAMP-dependent protein kinase in Dictyostelium discoideum shows high affinity for cAMP.
  • Screening methods for identifying specific protein-ligand interactions in expression libraries are crucial for molecular discovery.

Purpose of the Study:

  • To develop and validate a screening procedure for detecting in situ cAMP-binding activity directly on phage plaques.
  • To identify clones expressing the cAMP-binding regulatory subunit from a Dictyostelium discoideum cDNA library.

Main Methods:

  • Synthesis of highly radioactive [32P]cAMP using [alpha-32P]ATP and Bordetella pertussis adenylate cyclase.
  • Screening of a lambda gt11 cDNA library by incubating filter replicas with [32P]cAMP and washing.

Related Experiment Videos

  • Detection of positive clones via hybridization with a specific DNA probe and characterization of cAMP binding using analogue displacement assays.

    • Main Results:

    • Successfully identified three clones expressing the regulatory subunit of cAMP-dependent protein kinase from 1.2 x 10^5 plaques.
    • Demonstrated the ability to detect fmol levels of the regulatory subunit within phage plaques.
    • Confirmed cAMP binding specificity through analogue displacement, consistent with known affinities.

    Conclusions:

    • The developed screening method effectively detects in situ cAMP-binding activity in phage plaques.
    • This technique is sensitive enough to identify proteins with high affinities for cyclic nucleotides, applicable to libraries from various organisms.