cDNA and genomic structures of arrowhead proteinase inhibitors

Arrowhead double-headed proteinase inhibitors A and B consist of 150 amino acid residues with three disulfide bonds. Based on their primary structures, three cDNA fragments of the inhibitors were amplified in vitro by the PCR method using a constructed arrowhead cDNA library as a template. With the overlapping sequences, the full-length cDNA sequences of the inhibitors were then ascertained. The open reading frame encodes a pre-inhibitor, including a 24 residue signal peptide. The deduced amino acid sequences are consistent in principle with those determined by primary structure analysis, except that there are seven extra residues in the C-terminal part of the inhibitors, which might be cleaved off by proteinase post-processing immediately after protein synthesis. It is worth pointing out that cDNAs of both inhibitors A and B contain an 87 bp intron in the AAG codon of residue Lys-97. According to the elucidated cDNA sequences, the structural genes of inhibitors A and B were amplified using the total cDNA or genomic DNA of arrowhead as a PCR template. It was indicated that both the cDNA and genomic structures of inhibitors A and B have the same sequences.