MOLECULAR SEQUENCING AND MODELING OF NEOBELLIERIA-BULLATA TRYPSIN EVIDENCE FOR TRANSLATIONAL CONTROL BY NEOBELLIERIA TRYPSIN-MODULATING OOSTATIC FACTOR

Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reverse transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid amplification-of-cDNA-ends method , cloned and sequenced. The deduced protein sequence of 254 amino acid s exhibited 46% identity to Drosophila trypsin and 32% identity to Ano philine trypsin and Aedes trypsin. Three-dimensional models of Neobell ieria trypsin and Drosophila trypsin were built and compared. Both mod els contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp276 sits at the bottom of the specificity pocket. Southern blot an alysis suggested that Neobellieria trypsin is encoded by one gene. Nor thern blot analysis showed that an early trypsin transcript is found i n the midgut of sugar-fed females. This message disappeared after a li ver meal, and was replaced by a late transcript. Injection of trypsin- modulating oostatic factor (TMOF) at 10(-9) M prevented the disappeara nce and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the bio logical signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.