Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.

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Abstract
A DNA-binding protein (DB-2) was isolated from unfertilized D. melanogaster eggs by DNA-cellulose chromatography. 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. To investigate the specificity of the interaction between DB-2 and the DNA, isolation of the DNA sequences to which DB-2 binds was attempted. DB-2 was used as a probe to screen a gene bank established by inserting randomly sheared fragments of D. melanogastor 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, 2 plasmids, A17 and B10, were isolated which bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects 2 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 by EM. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.