FIG. 4.

The mouse p18 gene contains two promoters. DNA fragments containing mouse p18 genomic sequences were subcloned into the pGL2-Basic luciferase reporter plasmid, which lacks eukaryotic promoter and enhancer sequences. The restriction sites used in the construction of these plasmids are indicated. The A nucleotide in the ATG codon has been arbitrarily set to 1. The promoter activity of each construct was determined by measuring the luciferase activity of each construct 24 h after transient transfection into proliferating NIH 3T3 cells. Transfection efficiency was normalized by including a CMV-LacZ expression plasmid. Relative luciferase activity was normalized to β-galactosidase activity, and the normalized luciferase activity of the promoterless pGL2-Basic plasmid was set to 1. The data are averages of three independent experiments. The transcription initiation site of the p18(S) transcript, localized by promoter activity assays between the SmaI (nt 854) and HindIII (nt 355) restriction sites (highlighted with a dashed line), has not been precisely determined. (A) Localization of a second mouse p18 promoter; (B) comparison of two promoter strengths.