Male germ cells of Bombyx mori (Lepidoptera) have three cell kinds such as spermatogonia, eupyrene spermatocytes and apyrene spermatocytes. We observed spindle structure and the distribution of γ-tubulin in spermatogenesis. The microtubules (MTs) of mitotic apparatus in Bombyx spermatogonia were similar to that of general mammalian cells. In eupyrene spermatocytes, the asters were separated from the spindle pole, and then the spindle was transformed into a barrel shape at metaphase. In apyrene spermatocytes, the feeble-looking spindle body was formed, and asters were not separated at metaphase. The barrel-shaped spindle was not formed in apyrene metaphase cells. The chromosome arrangements on the equatorial plate were quite rough at metaphase and the movements toward the poles during anaphase were not synchronous. In all the three kinds of cells, spots of γ-tubulin were observed at centrosomes during cell division. Only in eupyrene spermatocytes, however, γ-tubulin was also disclosed in the spindle region from metaphase to anaphase. Many newly formed non-kinetochore-MTs appeared in the spindle interzonal region when the kinetochore-MTs disappeared and chromosomes reached the flat edge of spindle pole. It is indicated that non-kinetochore-MTs are built up by the action of spindle γ-tubulin. On the other hand, the distribution of γ-tubulin in apyrene spermatocytes appeared similar to the spermatogonia that performed regular chromosome separation. Therefore, we consider that the feeble spindle and the missegregation of chromosomes in apyrene meiosis are not caused by the γ-tubulin in the centrosomes.

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A histone variant H2A.Z cDNA has been cloned and sequenced from the silkworm, Bombyx mori. The predicted amino acid sequence of this cDNA showed a high homology with H2A.Zs from Anopheles gambiae, Drosophila melanogaster and Homo sapiens. The positions of introns in the H2A.Z genes were conserved among these species. Analysis of B. mori (Bm) H2A.Z gene expression indicated that this gene is expressed tissue-specifically in the fat body and hemocytes and life stage-specifically during 5th instar and wandering stages. Injection of 20-hydroxyecdysone into 5th instar larvae decreased expression of this gene, suggesting that this hormone plays a role to some extent in the novel expression pattern of the BmH2A.Z gene.

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Replication protein A (Rpa) is a heterotrimeric protein complex, composed of three tightly associated subunits of RPA70 (Rpa1), RPA32 (Rpa2) and RPA14 (Rpa3) in the case of human cells. The Rpa is known to be required for almost all aspects of cellular DNA metabolism. In the present study, we isolated and characterized the cDNAs orthologous to the Homo sapience RPA2 and RPA3, BmRPA2 and BmRPA3, respectively, from the silkworm, Bombyx mori. Each gene has a single conserved OB-fold domain. Although there was a higher level of homology among Rpa2s from different species, those of Rpa3s were very low. Furthermore, the analysis using yeast two-hybrid system revealed that the BmRpa2 and BmRpa3 form a tight heterotrimeric complex with BmRpa1. In addition, we confirmed the silkworm Rpa complex binds single-stranded DNA, but not double-stranded DNA.

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Hsp90-Cdc37 chaperone complex facilitates the folding and activation of numerous protein kinases. In this report, we have isolated a cDNA clone coding for the Bombyx mori Cdc37 homologue, BmCdc37, and determined its nucleotide sequence. Its mRNA encodes a polypeptide of 373 amino acid residues, which shares 52% amino acid identity with Drosophila melanogaster Cdc37. RT-PCR analysis revealed that the expression of BmCDC37 mRNA occurred mainly in the testis. Direct interaction of the HA-tagged BmCdc37 with endogenous BmHsp90 was demonstrated by co-immunoprecipitation assay from the cell lysates. Subcellular localization site of HA-BmHsp90 and HA-BmCdc37 was exclusively cytoplasmic. However, anomalous nuclear localization of DsRed-BmHsp90, probably due to interaction with EGFP-fused BmCdc37, was re-adjusted to cytoplasmic localization by heat stress. These results suggested that BmCdc37 interacts with BmHsp90 in vivo and tends to be transported to cytoplasm by stress-induced cellular mechanisms.

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We constructed a new piggyBac vector using the Bombyx kynurenine 3-mono oxygenase gene under the control of the cytoplasmic actin gene promoter (A3KMO). The vector contains an Upstream Activation Sequence (UAS) from yeast GAL4 with the terminator of the SV40 early gene and A3KMO. We tested whether the new vector could be applied to construct transgenic silkworms by inserting a green fluorescent protein (GFP) gene downstream of UAS. The result showed that the vector could be used for making transgenic silkworms. The transgenic first instar larvae were easily visible to the naked eye due to their brown integuments. Strong GFP expression was observed in the eggs and larvae when a G1 silkworm was crossed with a strain showing strong GAL4 expression in all stages and tissues.

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The gene for carotenoid binding protein (CBP) in the silkworm, Bombyx mori, was mapped. The linkage group for the CBP gene was identified by scanning linkage analysis of the restriction fragment length polymorphism (RFLP) pattern, and its genetic mapping with respect to known molecular markers within the group was established. The CBP gene was found to be linked to RFLP linkage group 19 which is equivalent to the phenotypic linkage group 2. The linkage map at 55cM in length was constructed for this linkage group, with CBP mapped at 21cM in length. When compared to a phenotypic map of chromosome 2, this result suggests that the gene that encodes CBP is the Y- gene.

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To increase the efficiency of producing transgenic silkworms (Bombyx mori), we developed a new DNA injection system for injecting silkworm eggs at the pre-blastodermal stage. In the new system, the tip of a glass capillary can be precisely guided into a hole made with a tungsten needle and inserted into the egg. This way, DNA can be injected into eggs more quickly and easily compared to previously used injection methods. In addition, it allows for a more precise injection into the primordial germ cell. Therefore, our system greatly increases the efficiency of transgenic silkworm production.

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The bacmid system first established for Autographa californica nucleopolyhedrovirus (AcNPV) is a powerful tool for generating recombinant viral DNA in E. coli, which allows one to produce knockout viruses (even viruses missing essential genes) independent of viral replication in host cells. We constructed a bacmid system for the type strain (T3) of Bombyx mori NPV (BT3Bac) and analyzed the functional necessity of a delayed-early gene he65 using the polyhedrin-gene-rescued BmNPV/T3 bacmid (BT3Bac^+polh). An apparent difference in the viral replication profile judging from numbers of polyhedra-forming cells and amounts of viral DNA in the culture medium was not observed between BmNPV/T3 and BT3Bac^+polh. On the other hand, the accumulation of viral DNA was delayed by about 12 hrs in he65-knockout BT3Bac (BT3Bac^+polh-he65KO) compared to those in BT3Bac^+polh or BmNPV/T3, indicating that he65 is not essential for BmNPV replication but critical for an optimal replication in BmN cells. The BmNPV/T3 bacmid system constructed here is useful for the functional analysis of BmNPV genes.

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