Isolation and expression of an ecdysteroid-inducible neutral endopeptidase 24.11-like gene in wing discs of Bombyx mori.

In the process of comparison of two cDNA libraries (W0, W2), we isolated a clone from the wing discs of Bombyx mori encoding a putative neutral endopeptidase 24.11-like gene. The predicted open reading frame encoded 772 amino acid residues, having about 53% identity with Drosophila GH07643, 36% with rat NEP, and 34% with rat ECE. This is the first NEP gene isolated in invertebrate. A 3.6-kb transcript was found to accumulate in the wing disc according to the increase of ecdysteroid titer during metamorphosis. Accumulation of the transcript was induced in wing discs with 20-hydroxyecdysone about 20h after incubation, which was inhibited by cycloheximide. This gene is ecdysone-inducible, appears to encode a functional protein, and may function during wing metamorphosis.

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations.

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.

Isolation and expression of the ecdysteroid-inducible angiotensin-converting enzyme-related gene in wing discs of Bombyx mori.

We isolated a clone encoding a putative angiotensin-converting enzyme-related gene from the wing disc cDNA library of the silkworm, Bombyx mori (refer to as BmAcer). The predicted open reading frame encoded 648 amino acids with about 50% identities with the Drosophila melanogaster angiotensin-converting enzyme Ance and Acer. Northern analysis identified a 2.2-kilobase mRNA which was abundant in wing discs two days after the beginning of wandering. An accumulation of the transcript was observed approximately 2 h after 20-hydroxyecdysone (20E) exposure in vitro and was blocked slightly by a protein synthetic inhibitor. These data suggest that the transcription of the BmAcer gene is directly 20E-inducible.