Mori Embryos Research Articles

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Expression of the Wolbachia male-killing factor Oscar impairs dosage compensation in lepidopteran embryos.

Wolbachia are intracellular bacteria in insects that can manipulate the sexual development and reproduction by male killing or other methods. We have recently identified a Wolbachia protein named Oscar that acts as a male-killing factor for lepidopteran insects. Oscar interacts with the Masculinizer (Masc) protein, which is required for both masculinization and dosage compensation (DC) in lepidopteran insects. Embryonic expression of Oscar inhibits masculinization and causes male killing in two lepidopteran species, Ostrinia furnacalis and Bombyx mori. However, it remains unknown whether Oscar-induced male killing is caused by a failure of DC. Here, we performed a transcriptome analysis of Oscar complementary RNA-injected O. furnacalis and B. mori embryos, and found that Oscar primarily targets the Masc protein, resulting in male killing by interfering with DC in lepidopteran insects.

Homeobox protein A1-like and DNA methylation regulate embryo-specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori.

DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

Interacting C/EBPg and YBP regulate DNA methyltransferase 1 expression in Bombyx mori embryos and ovaries

DNA methylation is an important epigenetic modification. DNA methyltransferases (Dnmts), which catalyze the formation of 5-methylcytosine, play a role in ovarian and embryonic development in some insects. However, the underlying mechanism of Dnmt in mediating ovarian and embryonic development remains unclear. In this study, the regulation and function of Bombyx mori Dnmt1 were investigated. By progressively deleting the sequence upstream of Dnmt1, a region located between −580 and −560 region from the transcription initiation site was found to have the most transcriptional activity. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that transcription factor Y box binding protein (YBP), a homolog of human Y box binding protein 1 (YBX1), bound to the −580 to −560 region. YBP knockdown and overexpression in a Bombyx cell line indicated that YBP activates Dnmt1 expression. Furthermore, GST-pulldown and co-immunoprecipitation demonstrated that YBP and ovarian CCAAT/enhancer binding protein (C/EBPg) could bind each other. Simultaneous knockdown of C/EBPg and YBP was more effective than single-gene RNAi in inhibiting Dnmt1 expression and reducing the hatching rate. These results demonstrated that the interaction of C/EBPg and YBP activated Dnmt1 expression. Correlated with the expression profiles of the studies genes, our results suggest that high-level expression and interaction of C/EBPg and YBP in ovaries and embryos enhance the expression of Dnmt1, thus ensuring high reproduction rate in B. mori.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Oral Vaccination of Grass Carp (Ctenopharyngodon idella) with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Estrogen-related receptor participates in regulating glycolysis and influences embryonic development in silkworm Bombyx mori.

Estrogen-related receptors (ERRs) play indispensable roles in development, energy metabolism, and cancers and are metabolic switches in Drosophila. However, the mechanism underlying their metabolic role is unknown in insects. This study analysed the expression profiles of Bombyx mori ERR (BmERR), hexokinase (BmHK), pyruvate kinase (BmPK) and phosphofructokinase (BmPFK) during embryonic development. The expression of BmERR tended to be similar to that of the other genes. We observed a regulatory association between BmERR and glycolytic rate-limiting enzymes by BmERR overexpression, RNA interference (RNAi), and ERR inhibitors in B. mori embryo cells. Subsequently, ERR cis-regulation elements (ERREs) were predicted and identified in the BmPFK promoter. Transfection assays, electrophoretic mobility shift assays and chromatin immunoprecipitation showed that BmERR can bind to one of these elements to regulate the expression of BmPFK. ERREs were also predicted in the BmHK and BmPK promoters. In the eggs, the expression of glycolytic rate-limiting enzyme genes was suppressed when the expression of BmERR was interference by double-stranded BmERR, the glucose levels also was increased. Meanwhile, the development of silkworm embryos was delayed by about 1 day. These results indicate that BmERR can bind to the ERREs of glycolytic gene promoters and regulate the expression of glycolytic genes, ultimately affecting embryonic development in silkworms.

An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites

Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.

Sexually biased transcripts at early embryonic stages of the silkworm depend on the sex chromosome constitution

In the silkworm, Bombyx mori, females are heterogametic (WZ) whereas males have two Z chromosomes. Femaleness of B. mori is determined by the presence of the W chromosome, suggesting that there is a dominant feminizing gene on this chromosome. Recently, by transcriptome analysis of B. mori embryos, we discovered that a single W-chromosome-derived PIWI-interacting RNA (piRNA) is the long-sought primary determinant of femaleness in B. mori. However, sexual bias in the transcriptome of B. mori early embryos has not yet been well characterized. Using deep sequencing data from molecularly sexed RNA of B. mori embryos, we identified and characterized 157 transcripts that are statistically differentially expressed between male and female early embryos. Most of the female-biased transcripts were transposons or repeat sequences that are produced presumably from the W chromosome. Bioinformatic analysis revealed that these repetitive sequences are piRNA precursors. In contrast, male-biased genes were frequently transcribed from the Z chromosome, suggesting that dosage compensation in Z-linked genes does not occur or is incomplete at early embryonic stages. Our analysis has drawn a picture of a global landscape of sexually biased transcriptome during early B. mori embyogenesis and has suggested for the first time that most sexually biased embryonic transcripts depend on sex chromosomes.

Expression of recombinant proteins by BEVS in a macula-like virus-free silkworm cell line

We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells. These findings indicate that persistent infection with BmMLV has no discernible effect on BmNPV-mediated protein production in B. mori cells.

Influence of Sudden Thermal Stresses on Growth Rate and Enzymatic Identification of Bombyx mori L. Larvae

Article History The purpose of this study is to determine the impact of different thermal stresses for different periods on univoltine Bombyx mori embryos and grown larvae in terms of larval growth rates, effective rate of rearing and the enzymatic proliferation of larval haemolymph. Egg groups was exposed to different temperatures; 0°C for one hour, 0°C for two hours, 40°C for one hour and 40°C for two hours, followed by 2 hours recovery at room temperature in addition to a control group, the same was applied to the grown larvae. Larval haemolymph of tested groups were subjected to enzyme electrophoretic analysis by native polyacrylamide gel electrophoresis (PAGE) for phosphatases and esterases identification. Exposure to sudden thermal stress of 0°C for one hour at larval stage recorded the highest growth rate. The sudden thermal stresses did not significantly affect the effective rate of rearing. However, thermal stresses increased ACPase and ALKPase activities and decreased α and β esterases activities comparing to control. These results suggest applying sudden heat thermal proteins in breeding strategy for inducing robustness in productive breeds.

Efficient TALEN Construction for Bombyx mori Gene Targeting

Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments.

Impact of thermal stress on the haemolymphal proteins, biological and economical characters of the silkworm, bombyx mori L.

The purpose of this study is to determine the impact of different thermal stresses on univoltine Bombyx mori embryos and grown larva in the terms of larval haemolymph proteins , biological and economical characters. The experiment was designed to expose egg groups to different temperatures as follows; a group exposed to 0°C for one hour, a group exposed to 0°C for two hours, a group exposed to 40°C for one hour and a group exposed to 40°C for two hours, followed by 2 hours recovery at room temperature in addition to a control group, the same was applied to the grown larval groups. The larval haemolymph of tested groups were subjected to protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Data revealed that considerable alteration in temperature hamper the protein activities which affects the embryonic development and hatching of eggs. 40oC for two hours was lethal dose for embryo. The highest measured parameters were recorded for eggs exposed to 40oC for one hour. While for grown larvae, the highest results were recorded for larvae exposed to 0oC for 1 hour. Thermal stresses induced protein changes with the appearance of new bands. The epigenetic degree of similarity recorded 50% between the control egg group and the egg groups exposed to 0oC for 2 hrs and 40oC for 1 hrs. as well as, for grown larva, between control larval group and larval group exposed to 40oC for 2 hrs.

Cloning and Characterization of the Caspase Family Member BmDronc from Bombyx mori (Lepidoptera: Bombycidae) Embryo Cells

Abstract Caspases are a family of cysteinyl aspartate proteinases known for their essential roles in the regulation of apoptosis. To date, ≈20 caspases have been identified, although few have been cloned and characterized in Bombyx mori (L.). Two caspase-9 variants from the B. mori embryo cell line (BmE), designated BmDroncL and BmDroncS, were cloned. BmDroncL has three classic domains: a caspase recruitment domain and large and small subunits, and is a homologue of Drosophila Dronc, human caspase-9, Aedes Dronc, and fish caspase-9, whereas BmDroncS lacks the large subunit. Instantaneous expression of BmDroncL caused typical apoptosis in Sf-9 cells. Moreover, BmDronc gene expression increased markedly during the silkworm metamorphosis at the larva-pupa and pupa-moth stages, which suggested that BmDronc plays a crucial role during B. mori metamorphosis.

Shotgun proteomic analysis on the embryos of silkworm Bombyx mori at the end of organogenesis

Embryonic development of silkworm, Bombyx mori is a process of systematical expression of genes and proteins which is dominated by complex regulatory networks. To gain comprehensive insight into the molecular basis of embryonic development and its regulation mechanisms, the proteome profile of the B. mori embryos at the end of organogenesis (tubercle appearance stage, TA) was characterized using LTQ-Orbitrap mass spectrometer. Totally 963 proteins were identified with a false discovery rate (FDR) of 0.12%. They were involved in embryonic development, chemoreception, and stimuli response and so forth. The proteins with the largest number of identified unique peptides, implying their possibly higher abundance, were involved in heat shock response, lipid transport and metabolism, and apoptosis. It was consistent with the physiological status of embryo at the end of organogenesis. Many functionally important proteins were identified for the first time in B. mori embryo such as the progesterone receptor membrane component 2, antennal binding protein, sericotropin, and molting fluid carboxypeptidase A (MF-CPA). 253 (26.27%) specific proteins in TA versus labrum appearance stage (LA, four days before TA) embryos were identified, which were mainly associated with musculature, nervous system, and chemoreception system. They disclosed the differential temporal and spatial expression of proteins in the process of organogenesis. The relative mRNA levels of fifteen identified proteins in the two experimented stages were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. Gene Ontology (GO) annotation of the identified proteins showed that the most proteome representations were in the categories of “binding” and “catalytic” in molecular function, and “cellular process” and “metabolic process” in biological process.

Shotgun proteomics approach to characterizing the embryonic proteome of the silkworm, Bombyx mori, at labrum appearance stage

The shotgun approach has gained considerable acknowledgement in recent years as a dominant strategy in proteomics. We observed a dramatic increase of specific protein spots in two-dimensional electrophoresis (2-DE) gels of the silkworm (Bombyx mori) embryo at labrum appearance, a characteristic stage during embryonic development of silkworm which is involved with temperature increase by silkworm raiser. We employed shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to analyse the proteome of B. mori embryos at this stage. A total of 2168 proteins were identified with an in-house database. Approximately 47% of them had isoelectric point (pI) values distributed theoretically in the range pI 5-7 and approximately 60% of them had molecular weights of 15-45 kDa. Furthermore, 111 proteins had an pI greater than 10 and were difficult to separate by 2-DE. Many important functional proteins related to embryonic development, stress response, DNA transcription/translation, cell growth, proliferation and differentiation, organogenesis and reproduction were identified. Among them proteins related to nervous system development were noticeable. All known heat shock proteins (HSPs) were detected in this developmental stage of B. mori embryo. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed energetic metabolism at this stage. These results were expected to provide more information for proteomic monitoring of the insect embryo and better understanding of the spatiotemporal expression of genes during embryonic developmental processes.

Biomolecular changes and somatic mutations induced by UV laser irradiation at embryonic stage of Bombyx mori

Purpose: To determine the impact of ultraviolet (UV) laser radiation on Bombyx mori embryos in terms of its effect on embryonic and larval haemolymph proteins and morphological traits.Materials and methods: The eggs of silkworm strain NB4D2 were exposed to third harmonic laser pulses at 355 nm from a Nd:YAG laser for different durations of 30, 60, 90, 120, 150, or 180 sec. Morphological changes induced by the UV laser were analysed at larval, pupal and adult stages. The eggs exposed to UV laser irradiation at different developmental stages were subjected to protein analysis by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph derived from irradiated and control larva was also analysed by SDS-PAGE.Results: UV laser irradiation resulted in various structural polymorphisms. Asymmetrical fusion of segments was not confined to larva but persisted throughout pupal and adult stages. Development of extra caudal horn, unequal size and lack of antenna, retarded thoracic legs and variation in larval markings were observed. Comparatively, the effect of the UV laser on 8- and 16-h old embryo was greater than on the other stages. The changes in protein pattern were not distinct until the 5th day of embryogenesis as revealed by SDS-PAGE. A 178 kiloDalton (kDa) protein resolved into 198, 184 & 169 kDa polypeptides and 154 kDa new protein band along with other proteins of 110, 45, 41 & 38 kDa were noticed in irradiated eggs at the 6th day. Further, 33, 32, and 6.2 kDa new protein bands were observed in the haemolymph of 5th instar silkworm larvae derived from UV laser irradiated embryos.Conclusions: A comparative analysis of the present study revealed that UV laser not only induced continuous structural polymorphisms (somatic mutations), but also induced protein changes with the appearance of new protein bands in embryonic and haemolymph protein. The UV laser could be a potential tool for biochemical genetics and genome analysis in B. mori.

Analysis of the Formation and Autonomous Replication of an Extrachromosomal Mouse Transgene

A stable autonomously replicating shuttle transgene, pr8a was, previously isolated from Bombix mori and characterized. Autonomous replication of pr8a and its derivatives was observed in yeast cells, B. mori embryos, and transgenic mice. To continue the previous studies, transgenic mice of several generations were examined. DNA analysis revealed a course of pr8a rearrangements resulting in extrachromosomal transgene p8-2 of F2 transgenic mice. Consecutive directional elimination of vector DNA fragments was characteristic of rearrangements affecting autonomous transgenes in mouse cells. Analysis of the data obtained with transgenic mice and other organisms made it possible to identify the pr8a fragment that acts as minimal ARS in transformed yeast cells and transgenic mice. Another pr8a region was assumed to be a component of a pr8a ARS module involved in regulating autonomous replication. Inserted in two different integrative vectors, minimal ARS ensured their autonomous state in transformed yeast cells and transgenic mice. Transgenes pr8a and p8-2, along with some other well-known constructs, were considered as prototype autonomously replicating vectors suitable for studying the mechanism of autonomous replication and solving some problems of gene therapy.

Localization, expression, and secretion pathway of diapause hormone in embryo and larva of the silkworm, Bombyx mori

In this paper, the distribution and expression of diapause hormone (DH) at mRNA and protein levels in the central nervous system of Bombyx mori embryo and larva were studied using whole-mount in situ hybridization and immunocytochemistry. Whole-mount immunocytochemistry revealed that the distribution of the DH-like immunoreactivity was throughout the central nervous system including the brain, suboesophageal ganglion (SG) and thoracic ganglia (TG); and that the corpus cardiacum and terminal abdominal ganglion may be the site for DH release due to the presence of strong immunoreactivity. In situ hybridization with the probe labeled by digoxigenin shows that the Bom-DH mRNA was also localized in the mandibular, maxillary, labial cell clusters. In addition, a pair of lateral neurons in the SG and a pair of ventral midline neurons in each TG expressing the Bom-DH transcript were also identified. These results were consistent with the localization of Bom-DH mRNA in larva by in situ hybridization and the distribution of the gene by RT-PCR, which is some different from the results reported previously.

Spatio-temporal expression of wnt-1 during embryonic-, wing- and silkgland development in Bombyx mori

A homologue of the segment polarity gene wnt-1 from Bombyx mori ( Bmwnt-1) has been characterized. The segmentally reiterated pattern of Bmwnt-1 transcrip9t distribution in B. mori embryos suggested its segment polarity function. Maximal levels of Bmwnt-1 RNA during embryonic development were reached by stage 21A. In the larval stages, Bmwnt-1 was expressed in the fore- and hindwing discs, ovaries, testes and gut, reminiscent of the expression domains in Drosophila. Bmwnt-1 was expressed in the wing-margin area of both the fore- and hindwing discs. The pattern of wnt-1 expression in the hindwing discs was similar to that in the butterfly Precis coenia but subtle differences existed in forewing discs of the two species, which correlated well with the absence of proximal bands of pigmentation in the adult Bombyx wings. In addition, Bmwnt-1 was expressed in the silkglands and the expression was confined to the anterior sub-compartment within the middle silkglands throughout development from the embryonic to late larval stages. This domain of Bmwnt-1 expression overlapped with those of Cubitus interruptus ( BmCi) and sericin-2 but excluded the Engrailed expression domain viz. the middle and posterior sub-compartments of middle silkglands. Bmwnt-1 expression was detected only during the intermoults and not in the moulting periods.