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 reversed 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 acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models 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 Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance 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 biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.
Borovsky
D
D
University of Florida-IFAS, Vero Beach 32962, USA.
Janssen
I
I
Vanden Broeck
J
J
Huybrechts
R
R
Verhaert
P
P
De Bondt
H L
HL
Bylemans
D
D
De Loof
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004175:Diptera / Q000737:chemistry*; D005260:Female; D006801:Humans; D007301:Insect Hormones / Q000502:physiology*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D007301:Insect Hormones; D009842:Oligopeptides; D012333:RNA, Messenger; C065612:Peptide oostatic hormone; D014357:Trypsin