In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.
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.
Vinson
C R
CR
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210.
LaMarco
K L
KL
Johnson
P F
PF
Landschulz
W H
WH
McKnight
S L
SL
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
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA