| Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. I have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a conceptual protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in chitinases from plants, yeast, bacteria, fungi and nematodes, and also in a mouse secretory protein. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis and partial genomic DNA sequencing indicated that at least two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest during about days 5-7 of the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA. |
