Figure 3.

R2C nuclear proteins occupy cis-elements in the MIS type II receptor promoter. DNase I footprinting was done with ³²P-labeled fragments PCR of the rat MISRII promoter incubated with 25, 50, and 75 μg of protein and digested with DNase I. The digested products were electrophoresed on an acrylamide gel alongside a sequencing reaction (not shown) performed with the same oligonucleotide primer to precisely map the footprinted nucleotides in the fragment. The lines indicate the DNA fragment, and the open boxes indicate the footprinted region. The wedges above the digests indicate increasing amounts of nuclear protein. The nucleotides protected from DNase I digestion are indicated in Fig. 1. (A) Fragment made from the proximal 144 bases of the MISRII promoter shows footprints in the GC box or SP1 site and to previously unidentified sites (nucleotides are shown in Fig. 1) which are labeled with a question mark. The GATA site is indicated where there appears to be increased digestion. (B) Fragments made with the distal SF-1 site (SF-1 250) and with the proximal SF-1 site (SF-1 200) are shown with their corresponding footprints. (C) The proximal SF-1 probe did not contain the distal SF-1 site.