5′ RACE analysis of the transcriptional start site of the endoglin gene. (A) Electrophoresis analysis of nested PCR amplification reactions. cDNA was synthesized from total RNA of phorbol ester-stimulated U-937 cells or poly (A)⁺ RNA from human placenta using reverse transcriptase. An adaptor was then ligated to both ends of cDNA and two sets of succesive PCR amplifications were performed: first with the anchor oligonucleotide AP1 in the presence of the oligonucleotide PE#4, and second with AP1 and PE#2 oligonucleotides. PCR products were analyzed by electrophoresis in a 3% agarose gel. Bands corresponding to U-937 (a and b) or placenta (c and d) were purified for cloning purposes and analyzed in a different gel. Lane M is a size ladder marker. Bands were visualized by ethidium bromide staining. (B) Nucleotide sequence of the cloned 5′-RACE products. PCR products (bands a through d from A) were isolated from the gel, cloned into plasmids, and sequenced. The complementary sequences for the anchor and nested primers used for the PCR reaction are underlined. The sequence of the commercial adaptor linked to the endoglin cDNA is overlined.