The nucleotide and deduced amino-acid sequences of minigene-1 encoding the CTL-2A12 mHA epitope. (A) Exon 2 encoding the original start codon in HMSD was deleted. After searching all frames, 2 reading frames in the HMSD-v transcript shown here were found to be able to encode polypeptides longer than 9 aa's starting with an ATG codon. Polypeptides longer than 9 aa's are all indicated. Asterisks indicate a stop codon. The start of exon 3 and exon 4B are indicated with horizontal arrows. The epitope recognized by CTL-2A12 is underlined (see Figure 4). (B) Six small minigenes with 9 aa's overlapping derived from the longest 53-mer polypeptide and downstream 3 minigenes (shown in panel A) were expressed in 293T/B*4403 cells and cocultured with CTL-2A12. Production of IFN-γ was similarly measured by ELISA. Release of IFN-γ is expressed in arbitrary units (AUs) corresponding to optical density at 630 nm. (M) indicates an artificially added methionine as a start codon.