The chromatin structure of the promoter region of the human DHFR gene was studied using DNaseI, micrococcal nuclease, exonuclease III, Bal 31 and various restriction enzymes. Mapping of endonuclease sensitive sites and an exonuclease protection assay showed that the DNA fragment between -670 and +150 base pairs (bp) from the initiation site of the DHFR gene is nucleosome free. Five discrete DNaseI hypersensitive sites were mapped in this region. A 170 bp fragment that extends from -340 to -170 was shown to be resistant to both endo- and exonucleases, suggesting that multiple proteins are bound to this region. Another protein binding site was mapped at the initiation site of the gene. DNA mediated gene transfer using a series of deletion mutants of a DHFR minigene showed that a minimal promoter segment containing a TATA box is sufficient for gene expression. Methylation of the DHFR minigene in vitro reduced transforming frequency to 10-20% of control. In cells transformed with the methylated gene only a region corresponding to the initiation site was specifically demethylated. The DHFR minigene was found to be amplified up to 500 fold in CHO cells. Amplification occurred within a few weeks after transfection in the absence of MTX, and the amplified genes were maintained stably without selection.
