Identification of multiple transcription initiation sites for the PECAM-1 gene. (A) 5′-RACE analysis. Total RNA was isolated from HUVEC, HEL, and Dami cells, all which express PECAM-1, as well as from the PECAM-1-negative Raji cell line. 5′-RACE analysis was conducted using a forward primer complementary to the anchor together with one of two different antisense primers, beginning either 63 bp upstream from the major TIS (lanes A) or 101 bp upstream from the TIS (lanes B), as described in Materials and Methods. The resulting PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (A) and (B) represent the two separate primer sets used (see Materials and Methods). As shown, HUVECs and HEL and Dami cells yielded comparably sized products, indicating that all three cell lines use the same region for transcription initiation. Note that the bands on the gel are somewhat broad, suggesting that a number of closely spaced PCR products might exist within each band. Raji cells showed no specific amplification products. (B) Sequence analysis of the 5′-RACE products. The 5′-RACE products shown in (A) were excised, subcloned, and sequenced. A summary of the 5′-termini derived from sequencing 15 individual clones is shown. The number under a specific nucleotide indicates the number of clones whose 5′-end corresponded to that particular base. The TIS, as previously determined by primer extension analysis,28 is shown above the sequence. Based on the 5′-RACE data, it appears that transcription of the PECAM-1 gene initiates at several closely spaced sites within this region.