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A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila 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. 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 BlO, 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.For prokaryotes, gene activity can be regulated by proteins binding to specific regulatory DNA sequences (1-6). In eukaryotes, the mechanisms of gene regulation are unknown, but we can assume that a sequence-specific interaction between regulatory molecules and the DNA is a prerequisite for regulation at the transcriptional level. Based upon this assumption, several studies have concentrated on the isolation of proteins associated with specific fractions of DNA or chromatin. Proteins associated with inactive chromatin (7) and a protein binding preferentially to a DNA satellite (8) have been purified. By use of fluorescent antibody techniques, the binding of specific proteins to a limited number of chromosomal loci or puffs in polytene chromosomes has been demonstrated (9, 10). In our laboratory, a DNA-binding protein, DB-1, has been purified which binds specifically to a cloned segment of nucleolar DNA in Drosophila (11). In this study, we describe the isolation of another DNA-binding protein, DB-2, the cloning of the DNA sequences to which it binds, and the sequence specificity of the DNA-protein interaction. METHODSPurification of DB-2. The isolation of total DNA-binding proteins from unfertilized eggs and from pupal nuclei of Drosophila melanogaster by DNA-cellulose chromatography was described earlier (11). Protein DB-2 was purified from total DNA-binding proteins by Sephadex-gel filtration and preparative NaDodSO4/polyacrylamide gel electrophoresis and separated from DB-1 by isoelectric focusing in the presence of 6 M urea essentially as described for protein DB-1 (...

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