Gene isolation by direct in situ cAMP binding.
The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.
Lacombe
M L
ML
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Ladant
D
D
Mutzel
R
R
Véron
M
M
D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics; D005800:Genes, Fungal; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism
D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases