Molecular cloning of genes coding for three inducible antibacterial proteins of Hyalophora cecropia

Abstract: The humoral immune system in the pupa of the giant silk moth Hyalophora cecropia can be induced by an injection of non-pathogenic bacteria. Three families of antibacterial proteins are synthesized, namely lysozyme, the attacins and the cecropins. This thesis deals with the isolation and characterization of cDNA and genomic clones coding for these proteins.Lysozyme shows a high degree of homology with chicken type lysozymes. In the Cecropia population there are probably three allelic forms which can be explained by point mutations. Attacins comprise six forms of molecules which are the products of two related genes. Two cDNA clones have been isolated which correspond to the two main forms, basic and acidic attacins. Comparison of their primary structure revealed up to 76% homology in coding regions suggesting that they must have originated from a common ancestral gene. At least basic attacin is synthesized in a pre-pro form. The heterogeneity found in attacins isolated from hemolymph is probably due to secondary modifications of the two precursor molecules. Cecropin B cDNA clones have been isolated and sequenced. The deduced amino acid sequence corresponds to a precursor molecule which is 62 amino acids long. A glycine terminates the coding region. It was proposed that this residue is the donor of the amide group for the amidation of the C-terminal of the protein. The precursor of cecropin B is therefore processed at both ends. The deduced structure for cecropin B was confirmed by solid phase synthesis, because the natural and the synthetic compounds were indistinguishable.The chromosomal organization of these genes has been investigated by construction of a genomic library and the characterization of genomic clones which include genes coding for the three classes of antibacterial proteins. The transcription unit of a cecropin B gene is analyzed in detail. The gene is approximately 0.9 kb long and is interrupted by a single intron. Indications were found for the presence of multigene families and multiple copy genes.