Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.
We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes.
Boffini
A
A
Department of Molecular Biology, University of Geneva, Switzerland.
Prentki
P
P
D001426:Bacterial Proteins / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D010583:Bacteriophage mu / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034982:Integration Host Factors