Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa ten and Agrotis ipsilon produce STI-i nsensitive trypsins in the midgut following ingestion of dietary plant prot einase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997 ) 855-874]. In this paper, the effects of dietary STI on a related family o f midgut serine proteinases, the chymotrypsins, were investigated. STI-inse nsitive midgut chymotrypsins were detected in larvae of H. tea and A. ipsil on feeding on diets containing 1% STI while STI-sensitive chymotrypsins wer e present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin, Four midgut cDNA libraries were c onstructed from larvae of each species fed either 0% STI or 1% STI diets. S ix full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showe d that some chymotrypsin mRNA were detected at higher levels while others w ere down-regulated when comparing insects reared on 0% STI and Ilo STI diet s. Southern hybridizations suggested that (like mammals) both species conta ined several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. tea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymo trypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus m otifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a comple x response mechanism linked to ingestion of proteinase inhibitors. (C) 2001 Elsevier Science Ltd. All rights reserved.