Isolation of cDNAs for trypsinogen from the winter flounder, Pleuronectes americanus

Knowledge of the timing of digestive enzyme expression in developing larvae is essential for evaluating the appropriateness of formulated larval diets . Since little is known at the molecular biological level about the ontogen y of digestive enzyme function in flatfish, we have isolated cDNA clones fo r key digestive enzymes such as trypsinogen. Portions of trypsinogen genes have been amplified from winter flounder cDNA libraries by PCR using primer s based on sequence motifs conserved among trypsinogen genes from other org anisms. The PCR products were sequenced, cloned, and used as radioactively labeled probes to screen the libraries. Three distinct trypsinogen cDNAs ha ve been isolated, representing the first cDNAs for winter flounder digestiv e enzymes. The first type (Flounder 1) is very similar to a trypsinogen seq uence reported from a related flounder, Pleuronectes platessa, The second t ype (Flounder 2) is related to sequences reported from Atlantic salmon, cod , and the Antarctic fish, Paranotothenia magellanica. The third type (Floun der 3) shows Limited similarity to the Antarctic fish trypsinogen sequence. All three cDNAs encode a cleavable signal sequence at the amino-terminal e nd, and Flounder 1 and 2 contain the characteristic acidic sequence for act ivation of the trypsinogen proenzyme to trypsin. Southern hybridization exp eriments indicate that Flounder I and 3 are single-copy genes, whereas Flou nder 2 may be present in more than one copy. In addition, Flounder 2 detect s homologs in a wide variety of other fish species. The sequence informatio n will be used to establish RT-PCR assays for larval winter flounder (Pleur onectes americanus) digestive enzyme expression.