CLONING AND SEQUENCING OF A GENE ENCODING A 21 KDA TRYPSIN-INHIBITOR FROM THEOBROMA-CACAO L

A cocoa library was constructed in a bacteriophage LambdaGem-11 vector and screened with the protein-coding region of a cocoa trypsin inhibi tor cDNA. Fourteen of 20,000 clones screened were positive. One positi ve clone was purified, subcloned into a pBluescript phagemid vector, a nd sequenced. Sequence analysis revealed a single open reading frame s tarting with an AUG initiation codon and ending with a TAA termination codon. The predicted encoded protein was 221 amino acids long and inc luded a 26 amino acid signal peptide. The 5' noncoding region had a pu tative TATA box, TATAAAT, at position -65, and an AGGA box, AAAAGAA, a t position -117 with respect to the initiation codon. The 3' noncoding region revealed two putative polyadenylation signals, AATAAA, 66 and 194 base pairs down-stream from the first termination codon, TAA. The genomic clone contained no introns, and Southern blot analysis showed it to be a member of a multigene family.