Analysis of transcriptional activity of the 5′-flanking region of the PECAM-1 gene. HEL, Dami, and Raji cells were transfected with PECAM-1 promoter/luciferase reporter constructs (shown schematically on the left) containing selected segments of the 5′-flanking region of the PECAM-1 gene. Luciferase assays were conducted as described in Materials and Methods to assess transcriptional activity, which was normalized to β-galactosidase activity derived from equal transfection of a separate reporter plasmid encoding lacZ. The boundaries of the constructs are numbered relative to the transcription initiation site, and the relative 5′ → 3′ orientation of each construct is indicated by the direction of the arrow. Normalized luciferase activity present in each transfected cell line is indicated on the right. The data shown represent the mean ± SEM of three independent experiments, each performed in duplicate. (A) Orientation-specific transcriptional activity of the 5′-flanking region of the PECAM-1 gene. Note that both the longer 4,500-bp construct, as well as the shorter 1,100-bp construct are capable of driving orientation- and lineage-specific transcription of luciferase activity. (B) Localization of transcriptional activity to a core region of the PECAM-1 promoter. 5′-serially-truncated PECAM-1 promoter/luciferase constructs were assayed for luciferase activity to assess PECAM-1 promoter activity in HEL, Dami, and Raji cells as described in the text. Note that the region encompassing nucleotides −163 to +204 retains transcriptional activity in the PECAM-1–positive HEL and Dami cells and that all constructs failed to drive transcription in Raji cells.