Yukihiro Sato

Molecular characterization of ovary trehalase of the silkworm, Bombyx mori and its transcriptional activation by diapause hormone.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

PHE-X-PRO-ARG-LEU-NH2 PEPTIDE PRODUCING CELLS IN THE CENTRAL NERVOUS SYSTEM OF THE SILKWORM, BOMBYX MORI

Members of the neuropeptide family having Phe-X-Pro-Arg-Leu-NH(2) (FXPRLamide; X=Ser, Thr, Val, or Gly) at the C-terminus serve as regulators of oviduct and visceral muscle contraction, sex pheromone production, and diapause induction. Antibody raised against Bombyx mori diapause hormone recognized a variety of FXPRLamide peptides. Using this antibody, the antigen was immunocytochemically localized in the central nervous system (CNS) of the silkworm, Bombyx mori. Immunoreactive somata were observed in all ganglia of the CNS including the brain. Twelve somata localized at the midline of the suboesophageal ganglion (SG) were most intensely stained, and their neurite projections reached the retrocerebral complex. Thus, these cells in the SG exhibited typical features of neuroendocrine neurons. Marked reduction in immunoreactivity was observed in a pair of neurosecretory cells in the labial neuromere in SG of diapause type pupae, which indicates an active release of FXPRLamide peptides from these cells. No clear connection to neurohemal sites were observed in immunoreactive cells in the brain, thoracic or abdominal ganglia, suggesting that the immunoreactive peptides in these organs are likely to serve as neurotransmitters or neuromodulators.

Purification, cDNA cloning and Northern blot analysis of trehalase of pupal midgut of the silkworm, Bombyx mori☆

Trehalase (alpha-glucoside-1-glucohydrolase, EC 3.2.1.28) was purified from silkworm pupal midgut to homogeneity by DEAE-Sepharose CL-6B and hydroxyapatite chromatography, and native gel electrophoresis. The enzyme had a molecular mass of 70 kDa. The N-terminal amino-acid sequence of the intact trehalase and its three fragments by V8 proteinase digestion was determined. Based on the amino-acid sequence, degenerate oligonucleotides were synthesized and used as primers in a polymerase chain reaction (PCR). Using a 0.8 kb PCR product as a hybridization probe, trehalase clones were isolated from the pupal midgut cDNA library. Sequence analysis revealed that the isolated trehalase cDNA contains 3103 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis clearly showed a 3.0 kb transcript in midgut, and Malpighian tubule, but not in fat body, silk gland, ovary, trachea, brain and suboesophageal ganglion.

Induction of embryonic diapause and stimulation of ovary trehalase activity in the silkworm, Bombyx mori, by synthetic diapause hormone

Abstract Diapause hormones, BomDH-I[19-Cys] and BomDH-I[19-Trp], of the silkworm, Bombyx mori were chemically synthesized and used to confirm the physiological function proposed in our previous experiments performed on the partially purified hormone preparation. Injection of the synthetic hormone into day-4 pharate adults caused the moths to lay diapause eggs and the dose for a half maximal activity was estimated to be 10 pmol/pupa. The induced diapause eggs ceased embryogenesis at stage 8 as observed for natural diapause embryos. Embryogenesis was resumed by chilling at 5°C for 60 days or by HCl treatment, which was effective to break down natural diapause induced by the implantation of an active subesophageal ganglion. Synthetic diapause hormone stimulated trehalase activity in developing ovaries in both in vivo and in vitro conditions according to the same time course. The stimulated activity was comparable to that found in ovaries which developed in situ with the endogenous hormone. This diapause hormone action was suppressed by the addition of cycloheximide and actinomycin D to the incubation medium. Addition of EGTA to the medium reduced the hormonal stimulation of trehalase activity, whereas Ca 2+ addition rescued the hormone action on trehalase activity in ovaries. These results confirmed that diapause hormone is responsible for both the induction of embryonic diapause in laid eggs as well as the stimulation of trehalase activity in developing ovaries of silkworms.

Structure and expression of a gene coding for egg-specific protein in the silkworm, Bombyx mori

Egg-specific protein (ESP) is a yolk protein which is synthesized in developing ovarian follicles of the silkworm, Bombyx mori. We have isolated a gene coding for ESP from a genomic DNA library using cDNA as a probe and sequenced about 2.9 kb including 5′- and 3′-flanking regions. Three closely located sites were identified as transcription initiation sites by primer extension analysis. In the 5′-upstream region, there are several short sequences which are homologous to the regulatory elements of other genes: short sequences homologous to the ecdysteroid response elements of the heat shock protein genes and the glue protein genes, cis-acting elements of chorion genes, and a protein factor binding site of the fibroin gene. Southern hybridization analysis revealed the presence of a single copy of the ESP gene per a haploid genome. The amino acid sequence was highly homologous to the human gastric and rat lingual lipases. Northern hybridization analysis of RNA prepared from various tissues at different developmental stages showed that the ESP gene is expressed only in follicles undergoing vitellogenesis. Injection of 20-hydroxyecdysone into the isolated pupal abdomen stimulated the expression of the ESP gene with a lag period of 2 days.

Induction of non-diapause eggs by injection of anti-diapause hormone rabbit serum into the diapause type of the silkworm, Bombyx mori

Abstract Polyclonal antiserum was raised by immunizing rabbits with the synthetic diapause hormone (BomDH-I[19-Cys]) of the silkworm, Bombyx mori . By immunological analyses, the antiserum was demonstrated to specifically recognize the diapause hormone. The antiserum was injected into larvae, pupae and pharate adults of the Daizo strain that were destined to lay diapause eggs, to see whether the serum is able to act in vivo as an anti-hormone agent. Injection of the antiserum at various stages from the fourth larval instar to the early pharate adult stage induced moths to lay non-diapause eggs. The effect of the antiserum injection declined suddenly from the middle of the pharate adult stage, when diapause hormone is secreted actively. The dose-response curve demonstrated the maximal dose to be a 1.0 μl injection of the antiserum and a half-maximal dose of 0.1 μl. When neutralized in vitro with the synthetic diapause hormone, the antiserum lost its ability to induce non-diapause eggs, which indicates that the antiserum inactivates diapause hormone through immunoneutralization. A transplantation experiment using suboesophageal ganglia preexposed to the antiserum indicated that the antiserum had no cytotoxic effects. Following injection of the antiserum trehalase activity and glycogen content in developing ovaries were reduced to the levels found after removal of the suboesophageal ganglion. The results indicate the potential for using rabbit IgG as a simple tool for the control of neuropeptide hormone titers in insect hemolymph.

Synthesis and secretion of egg-specific protein from follicle cells of the silkworm, Bombyx mori

The synthesis and secretion of egg-specific protein (ESP) were investigated using the follicle cells isolated from the developing ovary of the silkworm, Bombyx mori. The follicle cells were isolated manually from a follicle into a cell layer by thoroughly extruding the oocyte contents through a small hole. Whole follicles and isolated follicle cells were incubated in vitro with [35S]methionine, and ESP and its precursors were immunochemically isolated using antiserum raised to ESP. The isolated follicle cells incorporated label into ESP but the incorporation rate was about one-fifth of that found in whole follicles. About 20% of the total radioactivity of ESPs were recovered from the incubation medium of the isolated follicle cells while only trace activity (<2%) was found in the incubation medium of whole follicles. These results clearly showed that follicle cells synthesize and release ESP to be taken up by the developing oocyte.

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: a unique role for adult activity proposed from gene expression and production at the terminal phase of metamorphosis

A unique hydrophobic peptide (VAP-peptide) isolated from male adult heads of the silkworm, Bombyx mori, has been shown to act as a synergist to the diapause hormone when administered exogenously. Here, we investigated the true role of the endogenous VAP-peptide on differentiation and development of adult organs in the silkworm. By northern blot analyses, the VAP-peptide gene was shown to be exclusively expressed at the terminal phase of adult development in epithelial tissues, especially in the wing and the thoracic integument. In situ hybridization analysis revealed that the gene was highly expressed in the epidermal cells of the wing vein and the thoracic integument. The stage- and tissue-dependent gene expression were clearly correlated to the accumulation profile of VAP-peptide. In the adult thoracic integument, VAP-peptide was predominantly deposited in the cuticle layer. Affinity chromatography indicated the ability of VAP-peptide to bind to chitin. Based on its expression patterns, localization, and chemical properties, VAP-peptide is conceived to be a structural protein that participates in mechanical strengthening of specific cuticle structures, supporting their physical requirements in the adult life of the silkworm.

Linkage analysis of the gene encoding precursor protein of diapause hormone and pheromone biosynthesis-activating neuropeptide in the silkmoth, Bombyx mori.

We have determined the map position of the gene encoding a common precursor protein for diapause hormone and pheromone biosynthesis-activating neuropeptide (the DH-PBAN gene, Dh) in the silkmoth, Bombyx mori. First we compared the structure of introns in the DH-PBAN gene by the polymerase chain reaction, and found that the Dh locus carried three alleles, DhA1, DhA2 and DhB. The DhA1 and DhA2 alleles contained a fourth intron consisting of 740 bp, whereas DhB had a longer fourth intron of 770 bp. DhA1 and DhA2 contained a fifth intron consisting of 940 bp, whereas the fifth intron in DhB was much longer and consisted of 1700 bp. DhA1 was distinguished from DhA2 by an RFLP in the fifth intron after digestion with Rsa I. Linkage analyses using these polymorphisms showed that Dh was linked to the bp gene on chromosome 11, and independent of markers on chromosomes 1, 2, 3, 4, 5, 6, 7 and 13. To determine the map position, we obtained F1 hybrids between the n501 strain (K DhA1) and the w30 strain (+K DhB), and backcrossed the F1 hybrid to females of the w30 strain. From the segregation of K and Dh in 864 individuals in the next generation, the recombination value was calculated as 25.5% between K and Dh. Similarly we obtained backcross progeny between the No. 744 strain (Bu DhA1) and the w30 strain (+Bu DhB), and calculated the recombination value between Bu and Dh as 30.4% from 487 progeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-translational processing in the synthesis of egg-specific protein in the silkworm, Bombyx mori

Abstract The post-translational processing of egg-specific protein (ESP) in developing ovarian follicles of the silkworm, Bombyx mori was analyzed using in vivo and in vitro labeling systems with some radioactive precursors. The labeling with[ 35 S]methionine revealed that ESP is first synthesized as 69 kDa peptide (69K-ESP) which is then converted to 72 kDa peptide (72K-ESP) until 2 h. Some of 72K-ESP molecules were converted to 64 kDa peptide (64K-ESP) after 10h-labeling. [ 14 C]Mannose was incorporated into 69K-ESP and 72K-ESP. In the presence of tunicamycin, labeling with [ 35 S]methionine brought about a new 67 kDa peptide (67K-ESP) by reducing the incorporation into 69K- and 72K-ESP. [ 32 P]Ortho-phosphate was incorporated into only 72K-ESP by a 2 h-pulse labeling. Treatment of 72K-ESP with alkaline phosphatase converted it to 69K-ESP. These results along with the available information led to the conclusion that the primary translation product is sequentially processed to 67K-ESP by signal peptide cleavage, to 69K-ESP by glycosylation, and finally to 72K-ESP by phosphorylation as the actual product in the peptide synthesis. The limited conversion of 72K-ESP to 64K-ESP is proposed to be a post-endocytotic event.