Figure 1.

The mouse cad promoter displays E box-dependent growth-regulated transcriptional activity. (A) Sequence analysis of the mouse cad-promoter region from −112 to +129. Numbering of nucleotides is based on the putative start of transcription at +1 (indicated by the bent arrow). Transcription factor-binding sites, which are conserved between the murine and hamster cad promoters are indicated as follows: Sp1-binding sites are double underlined, the initiator element is underlined, and the E box is boxed. (B) Expression of cad mRNA displays late serum-response kinetics in mouse NIH 3T3 cells. Cytoplasmic RNA (10 μg), harvested at the indicated times after serum stimulation of quiescent NIH 3T3 cells or tRNA (10 μg) was hybridized to a murine cad riboprobe. Unprotected RNA was removed by digestion with RNase A. Protection of murine cad mRNA results in a 154-nt product, as indicated by the arrow. (C) Activated transcription from the mouse cad promoter in S phase requires the E box. Murine cad-promoter sequences from −105 to +84 or from −105 to +57 (indicated in bold in A) were fused upstream of the luciferase cDNA and transiently transfected into NIH 3T3 cells. After transfection, cells were incubated in starvation media (DMEM + 0.5% calf serum) for 48 hr, stimulated with the addition of 10% calf serum for 16 hr (corresponding to S phase of the growth cycle), and harvested for measurement of luciferase activity. The fold induction at 16 hr is reported as the ratio of luciferase activity from serum stimulated cells to the activity of the same promoter construct in serum-starved cells. Average fold induction and standard error was calculated from three independent experiments.