Mapping of human transcription initiation sites by RLM-RACE. (A) Agarose gel electrophoresis of the initial PCR reaction products obtained from the RLM-RACE procedure. Molecular size markers are in the middle well and sizes (base pairs) are indicated on the right. Arrows on the left mark the major PCR products obtained using RNA isolated from MCF7 cells and the arrow on the right marks the major product obtained using RNA from MRC5 cells. (B) Agarose gel electrophoresis of the nested PCR reaction products obtained from the RLM-RACE procedure. (C) Sequence of the human p27Kip1 gene upstream of the translation initiation codon. The large vertical arrow above the sequence indicates the major transcription start site identified by cloning and sequencing the RLM-RACE products shown in B. The small arrows indicate minor transcription start sites identified by RLM-RACE. Horizontal arrows below the sequence indicate the gene specific primers used for the RLM-RACE procedure. The start codon is boxed. The sequence shown is derived from Genbank accession number AB003688.