Establishment of tetracycline-inducible gene expression systems in the silkworm, Bombyx mori.

Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 x tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline.

Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus.

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a "factory" for large-scale expression using the BmNPV bacmid system.

Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori.

The promoter regions of the Bombyx mori HSC70-4 and B. mori TCTP genes characterized previously were used for the construction of a series of constitutive gene expression systems active in cultured cells. The relative abilities of these promoters were evaluated by comparing those of a silkworm actin A3 (BmActin3) promoter, which is used widely as the first choice. A series of constitutive expression systems constructed were assayed for the transcription efficiency by connecting four reporter cDNAs, firefly luciferase, 3GFP, Ds-Red, and beta-galactosidase gene using the Gateway LR reaction. The insertion of an intron enhancer into the site between the TCTP promoter and gene increased the transcription of the BmTCTP promoter by 10-fold. The insertion of the IE-1 gene and HR3 enhancer to the all three promoters were found to increase the transcription up to 560 times.

Expression and characterization of a sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea.

A cDNA encoding glutathione S-transferase (GST) of the fall webworm, Hyphantria cunea, was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone (hcGST) was sequenced and deduced for amino acid sequence, which revealed 87, 59, and 42% identities to Sigma-class GSTs from Bombyx mori, Manduca sexta, and Blattella germanica respectively. A recombinant hcGST protein (rhcGST) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rhcGST retained more than 75% of its original GST activity after incubation at pHs 6 to 11. Incubation for 30 min at temperatures below 50 degrees C scarcely affected the activity. rhcGST was able to catalyze the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. We also found that as compared to B. mori Sigma-class GST, rhcGST had a higher affinity for fenitrothion, an organophosphorus insecticide.

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop.

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

Site-specific gene integration in cultured silkworm cells mediated by phiC31 integrase.

The integrase from the Streptomyces bacteriophage phiC31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the phiC31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two phiC31 attP sites was co-transfected together with a helper plasmid encoding the phiC31 integrase into a cell line in which phiC31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the phiC31 integrase. These results indicate that the phiC31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes.

Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection.

Sonoporation (ultrasound treatment) provides a new and attractive nonviral way of in vivo gene transfer. To access the applicability of this method to the silkworm, Bombyx mori, we have compared the efficiencies of gene transfer by means of lipofection (using an appropriate agent, PDD111), sonoporation (ditto, FluoroGene), and lipofection followed by sonoporation. By these methods, a luciferase expression plasmid was found to be markedly transferred into the haemocoel of newly ecdysed fifth instar silkworm larvae, and also into other tissues although with lower rates compared with the haemocoel. In terms of luciferase activity, the efficiencies of transgene by lipofection plus sonoporation were approximately 6 (hemocytes), 20 (silk glands), 8 (mid-gut), 38 (fat body), 10 (Malpighian tubules), 33 (ovaries), and 16 (testes) times as high as those by lipofection or sonoporation alone. These results demonstrated that the present method is useful to introduce the exogenous DNA into insect organs in vivo.

Cloning and characterization of a ribonuclease L inhibitor from the silkworm, Bombyx mori.

The ribonuclease L (RNase L) pathway plays an important role in the response of cells to double-stranded RNA (dsRNA) during the events such as virus infection. Ribonuclease L inhibitor (RLI) belonging to the ABC transporter family is known as a regulator of the RNase L pathway. The homologs of RLI were reported in many organisms including the fruit fly and mosquito, but their functions in insects and arthropods have not been elucidated to date. In the present study, we cloned a cDNA of a silkworm RLI homolog, termed BmRLI, and its nucleotide sequence was determined. RT-PCR analysis revealed that the expression of BmRLI mRNA was marked in the testis, ovary and fat body. From the cDNA, recombinant protein with an apparent molecular mass of 69 kDa was expressed in Escherichia coli and cultured insect cells. Although no obvious effect of up-regulation of the BmRLI expression on RNAi was observed, its down-regulation slightly reduced RNAi efficiency.

Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4.

Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues.

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.

Molecular cloning and characterization of the translationally controlled tumor protein gene in Bombyx mori.

Translationally controlled tumor protein (Tctp/p23) is known to be synthesized preferentially in cells during the early growth phase of tumors, but is also expressed in normal cells. To elucidate its molecular basis of the expression and physiological significance, a cDNA encoding for the Bombyx mori Tctp (BmTctp) was deduced by editing the partial cDNA sequences registered in a Bombyx EST database. RT-PCR analyses indicated that the BmTCTP mRNA was transcribed in all larval organs examined and was present constantly during the cell cycle of BmN4 cells. A genomic clone of 4255 nucloetide residues produced by inverse PCR contained the 5'-flanking region, two introns and three exons of the BmTCTP gene. Sequence analysis of the 5'-flanking region indicated that a putative promoter region contains several canonical transcription elements such as GATA box, CCAAT motif, MEF2, E4BP4.01 and AP-1, but lacks a TATA box element. Luciferase reporter assay of the deletion constructs of the 5'-flanking region revealed that the -676 to +66 region enhanced the promoter activity the most markedly. In addition to this, there were at least two enhancer-like elements and several repressor elements.

In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells.

Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice. The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting. In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter. The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting. Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect. The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori).

Analysis of extrachromosomal homologous recombination in cultured silkworm cells.

Double-strand breaks (DSBs) are potentially lethal lesions causing the loss of chromosomal information. Eukaryotic cells have evolved the error-free repair systems of DSBs by homologous recombination (HR) through gene conversion with or without crossing over. In this study, we have developed a rapid assay system for extrachromosomal HR events in the cultured silkworm BmN4 cells. When HR occurs within the disrupted luciferase gene, an enzymatically active luciferase is restored and expressed. Our results strongly suggest that error-prone single strand annealing (SSA) accounts for the majority of extrachromosomal recombination processes in the cells. However, upon the substrates which cannot be repaired through SSA, DSBs were efficiently repaired though gene conversion. The rapid and sensitive HR assay system developed in the present study is expected to be a powerful tool for the identification and analysis of HR-related genes in the silkworm.

Cloning and characterization of testis-specific tektin in Bombyx mori.

A testis-specific cDNA library of Bombyx mori was constructed by an mRNA subtraction technique. Several clones were randomly selected and determined for their nucleotide sequences. One of them, designated as BmTST, contained a 3'-part of an open reading frame homologous to tektin, the protein known to form filamentous polymers in the walls of ciliary and flagellar microtubules. Also isolated was a genomic fragment, which contains the 5'-part of the coding sequence of BmTST and its promoter region. As a whole, the complete open reading frame was found to encode 508 amino acid residues, whose sequence had 28, 28 and 30% identities with the Strongylocentrotus purpuratus tektins A1, B1 and C1, respectively. Expression analysis by reverse transcription polymerase chain reaction with the cDNA and Western blotting with a polyclonal antibody indicated that the BmTST gene was expressed specifically in the testis during sperm maturation. The protein was immunologically detected exclusively in the fraction expected to contain the 9 + 2 flagellar axonemes of sperms. We infer that the BmTst protein is possibly involved in the spermatogenesis of B. mori.

Translation of RNA from Eggs During Post-Diapause Development of Bombyx mori: (translation/RNA/diapause (egg)/Bombyx mori).

Eggs of Bombyx mori are aroused from diapause by long-term chilling and develop when transferred to 25°C. During the first 20 hr of post-diapause development, the polysome content and the presumed rate of protein synthesis increase about 3-fold, while the ribosome content and the total RNA content increase only 1.1-fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post-diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA-like oligonucleotides. On translation in a protein-synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post-diapause development is controlled at a translational level.

Homoeotic Gene ETc Controls the Cell Number of Embryonic Ectodermal Layers of Bombyx mori: (E-pseudoallele/homoeotic mutant/ectodermal cell number/segmental differentiation/Bombyx embryos).

The formation of segments of Bombyx larvae involves differentiation of legged and legless segments during embryogenesis. Observations by light microscopy of serial sections of developing embryos of Bombyx showed that the cell number of the ectodermal layers increased more rapidly in segments where legs were being extruded than in those where no appendages were formed. In the embryos of a homoeotic mutant for the E-pseudoallelic locus (about 0.0-VI), ETc /ETc , in which all the abdominal segments were legless, the cell number of the ectodermal layers did not increase as in normal embryos. These findings suggest that the ETc gene controls the cell number of the ectodermal layers in relation to the differentiation of abdominal segments.

INSTABILITY OF MESSENGER AND RIBOSOMAL RNA IN A GLUE-PROTEIN MUTANT OF Bombyx mori.

The mucous glands of Bombyx pupae secrete glue proteins which attach deposited eggs to the mounting sheet. A mutant of a dominant gene, named no glue (Ng), produces nonadhesive eggs which have a low capacity for glue-protein synthesis. In the present study it was shown that the mucous glands of Ng silkworms showed rapid degradation of mRNA as well as rRNA during development; this may cause the low capacity for glue-protein synthesis in the mutant organ. In contrast, the mucous glands of normal silkworms showed a significant increase in content of RNA's until the maximum rate of glue-protein synthesis was achieved. The degradation of RNA in the Ng mucous gland was inhibited by actinomycin D injected into the body fluid. Thus it is supposed that the Ng gene codes for a presumptive controller RNA, which would be the mediator of RNA instability in the mucous glands of Ng pupae.

TRANSCRIPTIONAL CONTROL OF PROTEIN SYNTHISIS IN THE DEVELOPING OVARIES OF BOMBYX MORI.

Amino acid incorporation was studied with cell-free extracts and ribosomes prepared from pupal ovaries at different ages of Bombyx mori. Poly(U)-directed 3 H-phenylalanine incorporation attained a maximum rate at a certain stage of development, but soon dropped to a low level and was replaced by 3 H-leucine incorporation, which was due to endogenous mRNA. The latter incorporation occurred at the stage when actual protein synthesis takes place in the ovaries. "Run-off" of the ribosomes which had a high endogenous activity resulted in an enhancement of the poly(U)-dependent activity. The results indicate that the protein synthesis in the ovary is mainly controlled at the level of mRNA. This was further supported by the fact that the relative amount of an ovarian poly(A)-containing "mRNA" fraction increased in parallel with the endogenous activity.