Cyclic nucleotide-dependent screening of a lamda expression library for nucleic acid binding activities.
Here we describe a modified ligand screening strategy for the expression cloning of mammalian proteins that require the activation of protein kinase cascades to activate ligand binding activity. The manipulation of prokaryotic signaling pathways by the application of appropriate inhibitors or agonists to the nitrocellulose filter during functional screening of standard bacteriophage cDNA expression libraries can permit detection of activities that would not otherwise be found in their active state. We have applied this strategy to a A expression library to clone a novel renal cDNA that exhibits cAMP-dependent RNA binding activity when subsequently tested by ectopic expression in mammalian cells.
Loflin
Paul
P
University of Texas-Houston Medical School, 77225, USA.
Lever
Julia E
JE
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D000242:Cyclic AMP / Q000235:genetics*; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods*; D007763:Lac Operon; D008322:Mammals; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012313:RNA; D000242:Cyclic AMP