High-resolution mapping of DNase I hypersensitive site in the chicken beta-tubulin gene transfected into monkey cells

In this report, we describe a method for high-resolution mapping of a nuclease hypersensitive site (HS) at the promoter region of the chicken beta 2-tubulin gene transfected into monkey Cos-7 cells. Using the indirect end-labeling method of Wu, a tissue-specific nuclease HS was located initially approximately 100 base pairs upstream of the transcription start site. For high-resolution mapping of the HS in the transfected DNA, a DNA fragment of about 400 base pairs containing the promoter region of the beta 2 gene was subcloned into M13 phage. A single-stranded DNA probe with a defined restriction enzyme cleavage site at one end was prepared from the M13 phage DNA template. The single-stranded probe and the restriction enzyme-digested DNA, prepared from nuclease-digested nuclei from transfected cells, were sequentially annealed back to the template. After ligation between the probe and the transfected DNA, the HS was determined by DNA sequencing gel electrophoresis. This method permits determination of the 3' boundary of the HS with precision at the single-nucleotide level. We mapped a major HS at -107 nucleotides of the beta 2-tubulin gene, where a CACCC-like sequence is present. The application of this method in determining HS is discussed.