A transgenic silkworm expressing the immune-inducible cecropin B-GFP reporter gene.

To analyze cecropin B promoter (P-CecB) activity in vivo, we constructed transgenic silkworms that expressed EGFP under the control of P-CecB using the piggyBac transposable element. Genomic Southern blot analysis of the G1 and G2 generations indicated the stable insertion of EGFP in the genome. Injection of Escherichia coli cells into the larvae strongly induced EGFP expression in the fat bodies and all five hemocyte cell types. Northern blot analysis indicated that the expression kinetics of EGFP in the fat bodies following bacterial injection were correlated with that of endogenous CecB. Flow cytometric analysis of the hemocytes revealed that EGFP expression was increased by bacteria, but not by yeast. Our results indicate that the features of EGFP expression in the transgenic silkworm are equivalent to those of endogenous CecB and that P-CecB activation can be monitored by EGFP expression using transgenic silkworms.

Expression pattern of Bombyx vasa-like (BmVLG) protein and its implications in germ cell development.

Germ cell development in the silkworm Bombyx mori is interesting in that the species has no recognizable germ plasm, and its germ cells appear first on the ventral side of the embryo, not on the posterior pole as in Drosophila melanogaster. We previously reported the isolation of a vasa homologue (BmVLG) from B. mori and revealed the specific expression of transcript in the germ cells. In this paper, we describe the embryonic expression pattern of BmVLG protein. Consistent with the lack of recognizable germ plasm, the protein is not localized in freshly laid eggs, and its specific expression is first detectable several hours after energids penetrate the periplasm. This is in contrast to D. melanogaster, where germ cell lineage can be traced with anti-vasa antibody just after the formation of pole cells as they sequester vasa-positive germ (pole) plasm during cellularization. It is also revealed that, within the first few hours of their appearance when extensive cell movement does not seem to occur, stained cells are sometimes widely dispersed along the midline, which eventually may lead to the formation of ectopic germ cells. The implications of these results for germ cell development are discussed.

Role of the male BmDSX protein in the sexual differentiation of Bombyx mori.

The sex determination pathway is different between Drosophila melanogaster and Bombyx mori in the initial signal. Here we show evidence that the sex determination pathway in B. mori is similar to that of D. melanogaster at the level of the terminal regulator, doublesex (dsx), which is essential for the proper differentiation of the sexually dimorphic somatic features of D. melanogaster. In B. mori, a homolog of dsx (Bmdsx) is expressed in various tissues, and its primary transcript is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, transgenic silkworms carrying a construct with a Bmdsx male cDNA placed under the control of either an hsp70 promoter or a Bombyx actin3 promoter were generated by piggyBac-mediated germline transformation. Ectopic expression of the male cDNA in females resulted in abnormal differentiation of certain female-specific genital organs and caused partial male differentiation in female genitalia. Transgenic analysis also revealed that the expression of BmDSXM in females caused repression of the female-specifically expressed gene, the vitellogenin gene, and also resulted in activation of the pheromone-binding protein gene that is dominantly expressed in males. These results provide evidence that the role of BmDSXM includes the activation of some aspects of male differentiation as well as the repression of female differentiation. Taken together with our previous data on the function of BmDSXF, we can conclude that Bmdsx is a double-switch gene at the final step in the sex-determination cascade of B. mori.

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.

Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori.

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

Analysis of the biological functions of a doublesex homologue in Bombyx mori.

We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex ( dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of Bmdsx(F) in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between -95 and -89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm, Bombyx mori.

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.

Use of an N-terminal half truncated IE1 as an antagonist of IE1, an essential regulatory protein in baculovirus.

An immediate-early gene product of baculovirus, IE1, is essential for viral gene expression and for viral DNA replication. It has been demonstrated for Autographa californica nuclear polyhedrosis virus (AcNPV) that the C-terminal region of IE1 is required for dimerization. And the acidic N-terminal region of IE1 has been identified as the activation domain. We constructed an N-terminal 267 amino acid (a.a.) truncated mutant of Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1, which was defective as a transactivator of a viral early gene (p35) promoter. We then examined possible IE1 antagonistic functions of this defective IE1, IE1TN, in BmNPV-infected cells. A transient expression experiment demonstrated that IE1TN strongly repressed the activation of the hr5-dependent p35 promoter derived from BmNPV infection. In addition, DpnI assay elucidated an inhibitory effect of IE1TN on the hr5-dependent replication of plasmid in BmN cells induced by NPV infection. A marked reduction in the production of virus was observed when the BmN cells were infected with BmNPV after transfection with IE1TN-expression plasmids. These results suggested that IE1TN could act as an IE1 antagonist in silkworm cells infected with BmNPV. We then analyzed the ability of IE1TN to inhibit the multiplication of BmNPV using transgenic silkworms. The BmNPV-resistance of the transgenic silkworms was very weak, suggesting insufficient expression of the transgene product, IE1TN.