We have been applying a technique developed by us for the rapid identification of sequence specific DNA binding proteins to examine mammalian transcriptional regulatory factors. By combining this technique with a sensitive exonuclease footprinting assay, DNA-protein complexes can be formed, rapidly separated from the vast majority of protein and competitor nucleic acids, centrifugally concentrated and """"""""footprinted"""""""". This approach has led to the identification of the existence of a cellular factor which binds to the enhancer of the gibbon ape leukemia virus (GALV). The binding of this factor is eliminated in competitive binding assays only by nucleic acids which contain sequences highly homologous to the 48 bp repeated element present in the LTR of the Seato strain and which display enhancer activity in vivo. Another application of this method has been in the study of the regulation of the human c-myc oncogene, in collaboration with the laboratory of J.M. Bishop at the University of California, San Francisco. Using a similar approach, we have demonstrated that there is a region upstream of the c-myc oncogene which negatively regulates transcription from promoter P1 and P2. The same region which negatively regulates, as measured in transfection assays, binds a factor which can be measured and enriched by the procedure outlined above.
