We describe a procedure for detecting highaffinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.DNA sequences required for the regulation of eukaryotic gene expression have now been identified, largely through in vitro mutagenesis and subsequent analysis of the transcriptional activity of the mutagenized genes. What remains undetermined in most of these cases is the identity of specific transcription factors that interact with these DNA sequences to direct gene expression. The interaction of specific proteins with DNA regulatory sequences probably represents a fundamental process in controlling gene activity, and thus, the overall complement of sequence-specific proteins in the cell nucleus may largely determine the growth or differentiation state of the cell.The existence of site-specific DNA-protein interactions in eukaryotes has been demonstrated by several approaches. These include nitrocellulose filter-binding assays (1-4), detection of discrete DNase-hypersensitive sites in chromatin (5-7), exonuclease III digestion of chromatin (8), affinity chromatography (9, 10), and in vitro transcription (11)(12)(13)(14)(15)(16)(17)(18)(19). A limitation of these techniques is that they identify the site of protein binding within the DNA but do not identify the specific proteins involved. Only after extensive purification procedures can information be obtained about the nature of the proteins involved. Bowen et al. (20) have introduced a procedure that allows detection of DNA-binding proteins by blotting electrophoretically separated proteins on nitrocellulose. This procedure, however, has not proven to be effective for directly identifying site-specific DNA-binding proteins without partial purification (21-23) or heat-inactivation (3) of crude nuclear extracts. We describe here a procedure that uses protein blotting and that requires no prior purification steps, which has the potential for directly identifying and studying the regulation of proteins that interact with a target DNA sequence. We have used this procedure to identify a group of proteins that appear to bind with specificity and high affinity to the promoter region of the gene encoding the transferrin receptor. MATERIALS AND METHODSPreparation of Nuclear Extract. Cells were grown, at 370C in a 10% CO2 atmosphere in roller bottles, in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ,ug/ml), Confluent cultur...

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Abstract. A 365-bp fragment from the 5' region of the human transferrin receptor gene has been subcloned and sequenced. This fragment contains 115 bp of flanking sequence, the first exon, and a portion of the first intron. It contains a TATA box, several GC-rich regions, and is able to efficiently promote expression of the bacterial CAT gene in mouse 3T3 cells. Sequence comparisons demonstrate that this DNA segment has homology to the promoter regions of the human dihydrofolate reductase gene and the mouse interleukin 3 gene, as well as to a monkey DNA sequence that has homology to the SV40 origin and promotes expression of an unidentified gene product. Several high molecular mass proteins that interact with the transferrin receptor gene promoter have been identified. The activity of these proteins is transiently increased in 3T3 cells that have been stimulated by serum addition. This increase precedes a rise in transfen-in receptor mRNA levels in the cytoplasm, which in turn precedes entry of the cells into S phase. DNase I footprinting of the transferrin receptor promoter reveals several protein binding sites. Two of the sites are within the conserved GC-rich region of the promoter. One of these binding sites probably interacts with Spl, while the second interacts with an uncharacterized protein.

Following the earlier demonstration that iodo-Hoechst 33258 sensitizes DNA and cells to UVA, presumably mediated by formation of a carbon-centred radical on the ligand upon dehalogenation, three isomeric analogues of iodo-Hoechst 33258 have now been studied. The isomers differ in the location of the iodine atom in the phenyl ring of the ligand, relative to the site of attachment of the bibenzimidazole moiety, and are accordingly denoted ortho-, meta- and para-iodoHoechst. Comparison of the ligands with respect to induction of DNA ssb in pBR322 DNA revealed a wide range of activity; (D37's vary by a factor of 37), decreasing in the order: ortho- > meta- and para- > iodoHoechst 33258. Preliminary dehalogenation studies suggest that the higher activity of the ortho isomer results more from increased cross-section for dehalogenation than from increased efficiency of strand breakage per dehalogenation event. However, the chemistry of strand breakage by the ortho-isomer is distinctive, and tentatively assigned to initial attack at the 1'-deoxyribosyl carbon; the other two isomers, like iodo-Hoechst 33258, attack the 5'-carbon. The results are discussed in terms of the spectrum of DNA strand breakage chemistry associated with ionizing radiation, and the potential of DNA strand breaking agents such as the iodoHoechst compounds to study the chemical and biological consequences of the different subclasses of initial DNA damage.

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