We analyzed expression patterns in nine transcription factor genes and their isoforms in the posterior silk gland (PSG) of the silkworm Bombyx mori during the fourth and fifth instars. Two subtypes (-A and -B1) of EcR mRNA were expressed as almost corresponding to the change in ecdysone levels, but two subtypes (-1 and -2) of usp mRNA were controlled differently. The expression of usp-1 mRNA was suppressed at the peak of the ecdysone level and also at the expression peaks of EcR-A and usp-2 RNAs during the fourth instar. The β-isoform-specific region of the FTZ-F1 gene was identified, and distinct temporal expression patterns of the α-and β-isoforms of FTZ-F1 mRNA were detected using isoform-specific probes. The β-isoform peaked in the fourth molting stage, while the α-isoform was increased after the fourth ecdysis. The expression patterns of the βFTZ-F1 and BR-C genes showed the best reversed images relative to those of the silk protein genes. The genes for transcription factors (Fkh/SGF-1, FMBP-1, Awh and Ubx) predicted to be involved in the activation of the fibroin gene were expressed not only in the feeding stages but also in the fourth molting stage when the fibroin gene is suppressed. In addition, we found that the Bombyx Arrowhead (Awh) gene generated a male-specific splice variant in the PSG and gonad. These findings provide new insights into regulatory mechanisms for the silk protein genes.

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An entomoparasitic fungus, Paecilomyces tenuipes, was harvested from dried and dead pupae of the silkworm, Bombyx mori. Hot-water extracts of P. tenuipes (PTE) were administered orally to mice for 5 weeks after 8 weeks of subcutaneous injection of d-galactose. d-galactose-treated mice were used to examine the effect of supplementation with PTE on brain aging and age-related disease. A step-through passive avoidance test and Morris water maze test were also performed to examine the learning and memory capacities of these mice. The PTE group increased step-through latency and improved spatial learning and memory ability than the aging controls. Furthermore, in the mice aged with d-galactose, Holzer staining revealed histochemical evidence of gliosis in the hippocampal CA3 area. The PTE group of d-galactose-treated mice showed a decrease in gliosis. These results indicated that PTE may cause neural improvement in the aged brain.

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The genes encoding two subunits (α and β) of phenylalanyl-tRNA synthetase (PheRS), which catalyzes aminoacylation of tRNAPhe with L-phenylalanine (Phe), were cloned from the silk glands of the domesticated silkworm, Bombyx mori, and their full-length cDNA sequences were identified. The deduced amino acid sequences of the B. mori PheRS (BmPheRS) showed a high similarity with its counterparts from other organisms. A comparison with the previous mutation studies suggested that the Ala residue at the amino acid position 450 in the α-subunit of BmPheRS would be one of the key determinants for discriminating Phe from its amino acid competitors. To relax the amino acid specificity of BmPheRS, Ala to Gly mutation was introduced at the residue 450 of its α-subunit to generate the αA450G BmPheRS mutant. In vitro activity assay demonstrated that the recombinant αA450G BmPheRS mutant catalyzed the aminoacylation of the synthesized B. mori tRNAPhe with p-chloro- and p-bromo-substituted Phe analogs which were not recognized by the wild-type BmPheRS. This finding opens up the possibility of developing a novel type of genetically-modified silkworm which can utilize unnatural amino acids as protein building blocks.

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The relationship between two linkage groups, 24th and 27th, of the standard linkage maps in the silkworm, Bombyx mori, was revaluated using four visible mutants, Sel, tyw (from the 24th linkage group) Xan, and l-li (from the 27th linkage group), as markers. All cross experiments indicated that the 4 marker genes were located on the same linkage group. These data indicate that the two original linkage groups should be integrated as a revised 24th linkage group.

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Two types of calcium oxalate (CaOx) crystal, spherical druse and prismatic forms, were found in mulberry mesophyll cells near veins, which were identified by X-ray microanalysis and crystal morphology. The content of CaOx per leaf increased according to the leaf position from top, while CaOx content on dry weight basis was fairy constant regardless of the leaf position. To survey effects of CaOx on feeding the silkworm, Bombyx mori, the 5th instar larvae were reared on an artificial diet enriched with CaOx crystals. No change in the larval growth pattern occurred even when higher amounts of CaOx were incorporated into the diet.

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