Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.
A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila eggs by DNA-cellulose chromatogrphy. In competition assays with DNA from other species, DB-2 preferentially binds to Drosophila DNA. This binding protein can also be isolated from pupal nuclei and comprises only a small fraction ( < 0.01%) of the total nonhistone chromosomal proteins. In order to investigate the specificity of the interaction between DB-2 and the DNA, we attempted to isolate the DNA sequences to which DB-2 binds. DB-2 was used as a probe to screen our gene bank established by inserting randomly sheared fragments of Drosophila DNA into bacterial plasmids. Groups of plasmids were tested for binding to DB-2 by a filter binding assay. The plasmids bound to the nitrocellulose filter were eluted and used for bacterial transformation. After several cycles of transformation and cloning, two plasmids, A17 and B10, were isolated that bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects two short DNA segments of approximately 13 and 30 base pairs from digestion by DNase I. By filter hybridization according to Southern, these sequences were mapped to a defined restriction fragment. Further evidence for the binding specificity was obtained by visualizing the protein-DNA complex in the electron microscope. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D004247:DNA