Figure 1. Determination of the transcription starting site (TSS) of βIGH3 mRNA

A: A major product corresponding to 91 bp and two minor bands with sizes of 88 and 87 bp were observed from primer extension reactions of mRNA from A549 (20 μg, lane 2). Lane 1, IR-700 dye labeled size markers (Li-Cor). Schematic description (B) and results (C) of nested PCR from the human heart CapSite cDNA library. The PCR products were analyzed by 1.2% agarose gel electrophoresis. Lane 1 shows the molecular size marker. Lane 2 shows the first round of PCR with 1RC and HβIGH3-B.3 primers. Lane 3 shows the second round of PCR with 2RC and HβIGH3-A.3. The DNA fragment having a size of about 100 bp was gel-purified and sequenced. D: The transcription of βIGH3 mRNA starts from "ACT TCC CT..." following the 2RC primer sequence ("...CAC AGC GTA TGA TGC") as confirmed by the sequencing results. It corresponds to the -65 bp position in the 5'-flanking region of the βIGH3 gene indicated by an asterisk in Figure 2A.