Two distinct genes encode two major isoelectric forms of insecticyanin in the tobacco hornworm, Manduca sexta

Insecticyanin is a blue pigment found in the epidermis and hemolymph of the tobacco hornworm, Manduca sexta. Two distinct full-length cDNA species were isolated and shown to encode the two major isoelectric forms of insecticyanin, the a form (INS-a) and b form (INS-b). Sequence analysis of the two cDNA clones, pE1-I1 and Pe3-I2, reveals that both contain an 618-bp open reading frame which predicts an 189-amino-acid protein and an 17-amino-acid signal peptide. Comparison of the deduced INS-a and INS-b proteins show 13 amino acid differences, of which six are conserved. Three amino acid substitutions are also found between the deduced INS-b sequence and the sequenced INS-b protein isolated from the hemolymph. Isolation and characterization of five genomic clones revealed that pE1-I1 and pE3-I2 come from two different genes. Both INS-a and INS-b genes have four exons interrupted by three introns at the same positions. The two genes share 93% nucleotide similarity in the coding region. Moreover, the 'TATA box' and 'CAAT box' are completely conserved in the putative promoter regions of the two genes. Primer extension revealed that both INS-a and INS-b genes begin their transcription at position -52 relative to their translation initiation codon, ATG. We conclude that the two genes are the result of gene duplication.