Analysis of the nucleotide sequence of the p35 promoter. (A) Nucleotide sequence of the human IL-12(p35) proximal promoter region (accession no. AF050083). Nucleotides corresponding to the nuclease-hypersensitive site in untreated cells are underlined. Nucleotides encompassed by nuc-2 appear in bold. The putative Sp1-binding sites (Sp1#1 and Sp1#2, indicated by boxes) were identified by computer analysis (MatInspector V2.2). Nucleotide +1 denotes the S1 initiation site in monocytes as determined by Hayes et al.10 A putative TATA box (–32) is underlined. (B) Mutagenesis of the p35 Sp1-binding sites. The 2 oligonucleotides used as probes in EMSA (named Sp1#1wt and Sp1#2wt) encompass several putative binding sites for Sp1 (these sites are indicated by underlined nucleotides). The recognition core sequence 5′-GG-3′ of each potential Sp1 site is indicated by a thicker bar. Nucleotides that differ from the Sp1 consensus sequence are marked by an asterisk. For Sp1#1mut and Sp1#2mut oligonucleotides, only the bases that are changed compared with the wt sequence are indicated (substitution of the GC cores with the dinucleotide TT). The Sp1 consensus DNA-binding site is shown.
