To analyze the functional conservation between the fibroin H chain gene promoters in insects of the Bombycidae and Saturniidae, we constructed transgenic silkworms expressing yeast GAL4 under the control of the Bombyx mori or Antheraea yamamai fibroin H chain gene promoter (BmFibH-GAL4 and AyFib-GAL4) and examined their EGFP expression patterns after mating with a homozygous UAS-EGFP strain. Those silkworms carrying the BmFibH-GAL4 and UAS-EGFP constructs showed strict tissue- and stage-specific EGFP expression: expression was observed only in the posterior division of the silk gland after the third day of the fifth instar and then gradually increased as the larvae developed. Similarly, those silkworms carrying AyFib-GAL4 and UAS-EGFP also showed EGFP expression in the posterior division of the silk gland after the third day of the fifth instar; however, some strains expressed EGFP in other tissues as well. Thus, the basic function of the promoter is conserved between the two families, but the regulation of the A. yamamai fibroin gene promoter is seemed to be not as strict as that of the B. mori fibroin H chain promoter when it is used in B. mori.

View full abstract

Water-soluble sericin powder was prepared from the cocoons in Antheraea yamamai after elimination of the pigments and inorganic compounds. Polypeptide with apparent molecular mass 41kDa, which was isolated from the powder by gel filtration HPLC and electrophoretic analysis accelerated significantly proliferation of the insect Schneider S2 cells and of the mouse lymphocytes. These results indicate that the peptides derived from A. yamamai sericin have the potential for biopharmaceutical uses similar to the currently used sericins of Bombyx mori and A. mylitta.

View full abstract

Nosema bombycis is a highly virulent microorganism causing pebrine disease in the silkworm, Bombyx mori, and damage due to this disease has recently been successively reported in not only Japan, but also other Asian countries. Conversely, a low-virulent strain, N. bombycis Sd-NU, isolated from lawn grass cutworm (Spodoptera depravata) has been reported to exhibit no strong virulence against silkworms, unlike a highly virulent strain, N. bombycis NIS-001, derived from silkworms. There is no morphological difference between these 2 strains, and only their virulence against silkworms is different. In this study, we investigated differences in the genomic constitution between these 2 strains showing different virulence against silkworms by genome subtraction, and obtained several DNA fragments specific to the highly virulent strain. These N. bombycis NIS-001-derived DNA sequences were subjected to primer design, followed by PCR, and sequences with different sizes between N. bombycis NIS-001 and Sd-NU were amplified. The amplified sequences were not consistent between the strains on sequence analysis, clarifying that they were distinctly different. Based on these findings, Southern hybridization was performed using the DNA sequences derived from N. bombycis NIS-001 as probes, and no signals were detected in N. bombycis Sd-NU. These findings revealed that the sequences obtained by genome subtraction are present only in N. bombycis NIS-001.

View full abstract

Characterization of mulberry bending mutant was performed. Mulberry bending mutant ‘Garyu’ has a relatively short internode compared with the wild genotype ‘Ichibei’. The stem bends at the nodal position, and the bending is more apparent in the apical part of the stem. Histological observation showed that the bending occurs from the early developmental stage. Gibberellin (GA₃), natural-type abscisic acid ((S)-(+)-ABA) and their mixture were applied to the winter-dormant stem cutting of ‘Garyu’ and three normal straight-stem types. Dormancy break and growth of stem cutting was not affected by these hormones in ‘Garyu’ and ‘Shin-ichinose’, the hybrid between ‘Ichinose’ and ‘Kokuso 21’, but were affected in straight-stem types ‘Ichibei’ and ‘Ryoumenguwa’. Molecular analysis was done by using PCR with GAI degenerate and specific primers producing a fragment which turned to be 765 bp in length after sequencing. The fragment gene has homology to GRAS (GAI, RGA and SCR) transcription factor gene family, which relates to gibberellin response modulation function. We found two substitutive point mutations in GAI-like gene of mulberry ‘Garyu’ in comparison to mulberry ‘Ichibei’, ‘Shin-ichinose’, ‘Kokuso 16’ and other GRAS family gene on other plants. These mutations have the consequence to the replacement in the amino acid sequence and might have changed in protein function.

View full abstract

Double-stranded RNA (dsRNA) induces various biological responses in eukaryotic cells. Sequence-specific RNA interference (RNAi) is highly conserved throughout eukaryotic organisms. In mammalian cells, long dsRNA is known to have sequence-nonspecific gene silencing effect by activating several cellular pathways. As for insect cells, only the smallest amount of information has been reported so far. Here, we investigate the sequence-nonspecific effect of dsRNA on gene expression in a lepidopteran cell line, silkworm Bombyx mori BmN4. Cells were cotransfected with long dsRNA and a reporter plasmid expressing luciferase or β-galactosidase, and measured for the activities of each enzyme. dsRNAs which are not homologous to the target gene reduced the reporter activity in a concentration-dependent manner, albeit its effective concentration was considerably higher than that caused by dsRNA homologous to the target gene. The sequence-nonspecific repression by non-homologous dsRNAs was further confirmed using dsRNAs complementary to several genes from some organisms such as silkworm, fruit fly and human, as well as a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)). Since the sequence-independent effect by long dsRNA was also observed in another lepidopteran cell line, fall armyworm Spodoptera frugiperda Sf21, it may be one of the conserved immune responses among lepidopteran insects.

View full abstract

BmTRN-1, an RNA-binding protein homologous to mammalian TIA-1/TIAR, which possesses three RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain was shown to be involved in several mechanisms of RNA metabolism including the regulation of transcripts in the cells of the silkworm, Bombyx mori. The present gene analysis revealed that, in addition to previously reported mRNA, another isoform of mRNA with an alternatively spliced exon coding for an additional 14 amino acids in the auxiliary domain, was transcribed. Reporter protein production from the introduced plasmid was shown to be inhibited in the cultured cells by overexpressing both GFP-fused isoforms of BmTRN-1 with significant poly(U)-binding activity, but not by overexpressing the truncated mutant, such as RRM1-3 and the auxiliary domain. This indicates that the entire sequence of BmTRN-1 including both RRM1-3 and the auxiliary domain is necessary for the regulation of transcripts. Furthermore, analyses of the subcellular distribution of BmTRN-1 indicated that full-length BmTRN-1 is a shuttling protein present in both the nucleus and cytoplasm. However, BmTRN-1 was strongly distributed in the nucleus, especially when the cells were infected by the baculovirus, Autographa californica nucleopolyhedrovirus (AcNPV). Production of the glycoprotein of AcNPV, in the cytoplasm of infected cells, was clearly inhibited by the strong presence of both the full-length BmTRN-1 isoforms. In addition, the domains of BmTRN-1 related to its nuclear export and nuclear accumulation during viral infection were found to be RRM2 and RRM3, respectively. Thus, these findings indicate that BmTRN-1 with the domain responsible for its nuclear accumulation caused by the viral infection, was possibly related to the cellular function of eliminating baculovirus transcripts in the nucleus.

View full abstract

The gross and cell morphology and ultrastructure of larval labial glands of the sweet potato hornworm, Agrius convolvuli, were investigated by light, fluorescent and transmission electron microscopy. The labial gland is a narrow tubular epithelium that can be divided into three regions composed of hexagonal cells, each with a large branched nucleus. The ultrastructure of these cells was similar to that of silk gland cells in silkworms, characterized by well-developed rough endoplasmic reticulum and Golgi bodies. Agrius larvae spin small amounts of fibrous material immediately after hatching and at the wandering stage. Several bands (approx. 30, 67, 70, 116, 200kDa) were detected by SDS-PAGE from the labial glands throughout the fifth instar stage. Interestingly, most peptides disappeared at around the onset of the wandering stage, replaced by two proteins of approximately 180 and 220kDa. Two major polypeptides with similar molecular mass (180, 220kDa) were also detected in fibrous materials spun during the wandering stage. These results suggest that a switchover of protein synthesis occurs in the glands before metamorphosis.

View full abstract