title: In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.
abstract: We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.
authors.affiliation: Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210.
authors.forename: C R
authors.initials: CR
authors.lastname: Vinson
authors.forename: K L
authors.initials: KL
authors.lastname: LaMarco
authors.forename: P F
authors.initials: PF
authors.lastname: Johnson
authors.forename: W H
authors.initials: WH
authors.lastname: Landschulz
authors.forename: S L
authors.initials: SL
authors.lastname: McKnight
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA