Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

Analysis of a new type of major ampullate spider silk gene, MaSp1s.

Spider dragline silk, which is secreted from the major ampullate silk glands, is a unique fibrous protein with a combination of tensile strength and elasticity. Here, we describe a new short type of dragline silk gene, Cyrtophora moluccensis MaSp1s. The full-length gene is only 1320 base pairs (bp), which encodes 439 amino acids that includes the intact non-repetitive N-terminal (149 residues), C-terminal (98 residues) and so-called repetitive regions (192 residues); the deduced molecular weight is approximately 40 kDa. The sequence analysis demonstrated that the two termini are highly homologous to the other characterized dragline silk genes but that the so-called repetitive region is different. Our results suggest that MaSp1s is a possible new characteristic dragline gene; the discovery of this gene should enhance our understanding of the major ampullate spider silk genes.

The molecular structures of major ampullate silk proteins of the wasp spider, Argiope bruennichi: a second blueprint for synthesizing de novo silk.

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk.

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.

Characterization of a cysteine-rich protein specifically expressed in the silk gland of caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

A novel protein, Smsp-72k, was found to be selectively expressed in the silk gland of aquatic larvae of the Stenopsychid caddisfly (Stenopsyche marmorata). The protein was characterized by an abundance of cysteine (13.97%) and charged residues (47.21%). Amino acids with hydroxyl side-chains accounted for an additional 10% of the Smsp-72k protein, with serine at 4.4% and threonine at 5.6%. A cysteine-rich repetitive sequence is common to many potential and known underwater adhesive/cement proteins and cell-cell adhesion molecules. We hypothesized that Smsp-72k is an adhesive/cement protein that increases the adhesiveness of the silk fiber of S. marmorata. The hydroxyl groups of Smsp-72k might form a link with the heavy chain fibroin of S. marmorata, removing the weak boundary-water layer and allowing the spreading of the silk protein onto the surface of the substratum during the process of adhesion.

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

The silks of both Lepidoptera and its sister order Trichoptera contain a homologue of heavy chain (H-fibroin), which is assumed to determine the physical properties of the fiber, such as elasticity and toughness. The long repetitive region of the H-fibroin caddisfly Stenopsyche marmorata shows a conspicuous hierarchical structure that is composed of huge units, which are mainly constructed from four large blocks (SA, SB, SC and SD) arranged in an orderly fashion. Each block contains short, distinct motifs such as SXSXSX(SX), GPXG(X)(1-3) or triplet GGX, which also occur in lepidopteran and spider filaments. The SA, SB and SC blocks have nearly fixed amino acid numbers, while the length of the SD block varies, usually due to a variable number of GPXGXXX repeats. The multiple sandwich structure that occurs in the SB block is assumed to be unique to the caddisfly and may be related to the use of silk in an aqueous environment. The overall average of hydrophilicity in the repetitive H-fibroin region of S. marmorata is -0.609, whereas hydrophobicity prevails in most lepidopteran H-fibroins. Gly (29.51%), Pro (11.28%) and Ser (10.90%) are the three predominant amino acids of H-fibroin, and the high content of essential amino acids reflects the energy-rich food resources of the caddisfly. The H-fibroin of S. marmorata is about 400-500 kDa and expressed in both the middle and posterior silk glands, which is different from the expression pattern in Lepidoptera species.

Sequence structure and expression pattern of a novel anionic defensin-like gene from silkworm (Bombyx mori).

A defensin-like gene, BmdefA, was rediscovered in the silkworm genome and expressed sequence tags databases. The open reading frame of BmdefA encodes a prepropeptide consisting of a 22-residue signal peptide, a 34-residue propeptide, and a 36-residue mature peptide with a molecular mass of 4.0 kDa. The mature peptide possesses the characteristic six-cysteine motif of insect defensins, and its predicted isoelectric point is 4.12, indicating it is a novel anionic defensin. An intron is present in BmdefA and several cis-regulatory elements are in the regulating region. It is transcribed constitutively at a high level in the hemocyte, silk gland, head, and ovary of the silkworm larvae, and in the fat body of early-stage pupae and moth. BmdefA is also strongly induced by immune challenge. These results suggest that BmdefA plays an important role in both immunity and metamorphosis.

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.

The Pitx homeobox gene in Bombyx mori: regulation of DH-PBAN neuropeptide hormone gene expression.

The diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is expressed exclusively in seven pairs of DH-PBAN-producing neurosecretory cells (DHPCs) on the terminally differentiated processes of the subesophageal ganglion (SG). To help reveal the regulatory mechanisms of cell-specific DH-PBAN expression, we identified a cis-regulatory element that regulates expression in DHPCs using the recombinant AcNPV-mediated gene transfer system and a gel-mobility shift assay. Bombyx mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds this element and activates DH-PBAN expression. The BmPitx was expressed in various tissues, including DHPCs in the SG. Suppression of DH-PBAN expression by silencing of the BmPitx successfully induced non-diapaused eggs from a diapause egg producer. To the best of our knowledge, this report is the first to identify a neuropeptide-encoding gene as a target of the Pitx transcriptional regulator in invertebrates. Thus, it is tempting to speculate that functional conservation of Pitx family members on neuropeptide gene expression occurs through a "combinational code mechanism" in both vertebrate and invertebrate in neuroendocrine systems.

Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi.

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Unique molecular architecture of egg case silk protein in a spider, Nephila clavata.

We describe a unique silk protein secreted from the cylindrical silk glands of the spider Nephila clavata. This silk is primarily composed of three proteins, whose transcripts of approximately 16.0, 14.5 and 13.0 kb are homologous to one another in two termini and repetitive units, as determined on Northern blotting. Its overall organization shows that it is similar to other characterized silk proteins, including in the mainly central repetitive region as well as the non-repetitive N-terminal (166 residues) and C-terminal (176 residues) parts. However, up to 90% of the protein consists of highly ordered repetitive structures that are not found in other silks. The repetitive region mainly consists of several types of complexes and remarkably conserved polypeptide repeats. The assembled repeat units (A1B1) contain a high proportion of Ala (30.41%), Ser (25.15%), and residues with hydrophobic side chains (22.22% for Gly, Leu, Ile, Val and Phe combined). The presence of Ser-rich and GVGAGASA motifs suggests the formation of a beta-sheet. The repetitive region is characterized by alternating arrays of hydrophobic and hydrophilic blocks. The results suggested that this egg case silk is an exceptional protein when compared with previously investigated spider silks.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis-regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH-producing neurosecretory cells (PTPCs). The cis-element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.

We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA.

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

Cell cycles in embryos of the silkworm, Bombyx mori: G2-arrest at diapause stage.

In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G1, S and G2 phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G1, S and G2 were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G2 at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G1 from G2, suggesting that their G1 phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G1 fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G2 at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G1. Finally, most of the cells may be arrested in G1, while there is only a small fraction of cells continuously cycling.