Daisuke Funabara

Molecular Cloning and Characterization of Two Isoforms of Trypsinogen from Anchovy Pyloric Ceca

Abstract: Complementary DNA clones encoding two isoforms of trypsinogen were isolated from the pyloric ceca of anchovy by rapid amplification of cDNA ends (RACE). Nucleotide sequences of isolated clones encoded, in addition to characteristic signal and activation peptides, two isoforms of trypsin containing 220 and 221 amino acid residues. Both enzymes contained the catalytic triad of a serine protease, together with the residues determining substrate specificity. The anchovy trypsins showed a high amino acid identity of about 80% to those of other fish species. Southern blot analysis with a probe cross-reactive to both isoforms showed a complex genomic pattern. Northern blot analysis with the same probe revealed the highest expression of meassenger RNA in the pyloric ceca. Structural parameters possibly involved in higher catalytic properties of fish trypsin were examined by three-dimensional modeling, which included deletion in the autolysis loop, lack of Tyr-151 at the entrance of the S1 pocket, and distribution of charged residues.

Screening for Genes Participating in the Formation of Prismatic and Nacreous Layers of the Japanese Pearl Oyster Pinctada fucata by RNA Interference Knockdown

Many genes have been identified to participate in the shell formation so far. Nevertheless, the whole picture of the molecular mechanisms underlying the shell formation has remained unknown. In our previous study, we analyzed comprehensively genes expressed in the shell-producing tissues and identified 14 genes to be involved in the shell formation by the RNA interference (RNAi) method. In the present study, we performed further screening to find additional novel genes involved in the formation of the nacreous and prismatic layers. We here selected 80 genes from the EST data as candidates to function in the shell formation, conducted knockdown experiments by the RNAi method, and observed surface appearances on the nacreous and prismatic layers. We newly identified 64 genes that could participate in the shell formation. Taken together with our previous study, 78 genes were supposed to function in the shell formation. These findings indicate that the combination of transcriptome and knockdown analyses is a powerful tool to screen novel genes involved in the shell formation.