Organization and developmental timing of the Bombyx mori chorion gene clusters in strain C108

We have standardized the map of chorion structural gene clusters in Bombyx mori strain C108 by analyzing quantitative and qualitative chorion electrophoretic markers in recombinant progeny from four independent crosses. In all we assigned 22 markers to three gene clusters, representing about one-third of the total number of chorion genes: 2 to Ch 1, 9 to Ch 2, and 8 to Ch 3. Three additional markers belong either to Ch 7 or Ch 2. By referring to published chorion protein synthesis patterns, we show that the clusters are restricted in their developmental specificities: Ch 3 appears to be an early locus, carrying all of the mapped early markers (4) and half of the early middles (3/6), while Ch 1 and Ch 2 carry predominantly middle (4/5) and all late, Hc (6) markers, along with some early middle markers (3). We cite evidence to show that Ch 1 and Ch 2 compose the left and right halves of a single gene cluster, which we formally designate as Ch 1–2.     

Mining single nucleotide polymorphisms from EST data of silkworm, Bombyx mori, inbred strain Dazao

We made use of 81,635 expressed sequence tags (ESTs) derived from 12 different cDNA libraries of the silkworm, Bombyx mori, inbred strain Dazao (P50), to identify high-quality candidate single nucleotide polymorphisms (SNPs). By PHRAP assembling, 12,980 contigs containing 11,537 contigs assembled by more than one read were obtained, and 101 candidate SNPs and 27 single base insertions/deletions were identified from 117 contigs assembled from 1576 high-quality reads base-called with PHRED and screened on the basis of the neighborhood quality standard (NQS). Simultaneously, we also predicted 40 SNPs in coding regions (cSNPs), of which 26 were predicted to lead to amino acid non-synonymous variations and 14 synonymous substitutions. Also, the 1.66:1 ratio of transition/transversion is different from that of other insects. As the first SNP analysis of a Lepidoptera, B. mori, the single nucleotide polymorphic density is estimated to be 1.3 x 10(-3) by sequence diversity. This analysis shows that expressed sequences from multiple libraries may provide an abundant source of comparative reads to mine for cSNPs from the silkworm genome.     

家蚕核糖体蛋白L7基因cDNA序列的克隆和分析

为了研究家蚕孤雌生殖的调节机制,应用二维凝胶电泳(2DE)技术分离正常生殖的家蚕卵与孤雌生殖家蚕卵的差异蛋白质,在蛋白质水平上筛选与家蚕孤雌生殖过程相关的重要蛋白质.利用MALDI-TOF-TOF MS分析这些差异蛋白,获得了大量小肽的序列特征.BLAST搜索本实验室构建的cDNA文库,获得了1个与家蚕孤雌生殖相关的核糖体蛋白L7基因.根据已有的cDNA文库,采用RACE方法克隆得到该核糖体蛋白基因的全长cDNA.利用生物信息学的方法和工具,对这个基因在核酸水平和蛋白质水平分别作了详细的分析和讨论并进行蛋白结构预测.结果表明,核糖体L7基因的cDNA全长为858 bp,编码区包含6个外显子,共编码268个氨基酸残基,蛋白的疏水性平均值为-0.586,分子量大小为30 kD,极性的最大值为 39.616,最小值为0.451,等电点为10.52.分子系统分析显示,该蛋白与Apis, Lysiphlebus 和Meladema中的核糖体蛋白L7具有较高的同源性.     

Molecular analysis of the GrB mutation in Bombyx mori through the use of a chorion cDNA library

To study the molecular basis of the Gr^B mutation, which prevents the synthesis of many stage-specific chorion proteins, a cDNA library has been constructed from wild-type chorion mRNA of Bombyx mori strain 703. By differential screening of the library with +/+ and B/B mRNAs, under appropriately stringent conditions to minimize cross hybridizations of related chorion sequences, we have selected several distinct clones corresponding to RNA sequences which are affected by the mutation (that is, are represented only in +/+ mRNA) or are unaffected (that is, are represented in both +/+ and B/B mRNAs). We show by Southern analysis that, whereas unaffected gene sequences are represented in both +/+ and B/B chromosomal DNA, affected sequences have been deleted from B/B DNA. The organization and regulation of developmental stage-specific chorion genes are discussed in light of these findings and the known effects of Gr^B on stage-specific protein synthesis.     

Specific protein synthesis in cellular differentiation: VI. Temporal expression of chorion gene families in Bombyx mori strain C108

Detailed patterns of expression for putative members of 5 major chorion gene families have been obtained by separating labeled proteins using two-dimensional polyacrylamide gel electrophoresis. Proteins fall into 4 temporal cohorts called early, early middle, middle, and late on the basis of when they initiate and terminate synthesis. Proteins synthesized during the early, early middle, and late periods are highly synchronous, exhibiting abrupt onset times and relatively uniform termination times. Middle proteins begin synthesis in small groups at staggered times over a relatively long period, but most cease translation as the late proteins turn on. This data is correlated with a previous follicle staging system based on separation of newly synthesized chorion proteins by isoelectric focusing alone. The absolute timing of choriogenesis was determined in vivo, using trypan blue dye to mark vitellogenic follicles. The relative age difference between chorionating follicles was 2.2–2.6 hr; chorion biosynthesis took 4 days in all. These data are discussed in terms of patterns of activity of chorion gene families, the functions of the temporal cohorts, and regulation of the chorion multigene system.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

Characterization of strain specific typing antisera for genetic monitoring of inbred strains of rats

Strain-specific typing antisera (SSTA) were prepared for six inbred strains of rats by using a pooled immunization protocol. The SSTA were used in both a haemagglutination assay and a complement dependent microcytotoxicity assay to compare the usefulness of the two test systems. Both assays were simple, reliable and repeatable, and each system had distinct advantages and disadvantages. The haemagglutination assay was faster and required less specialized equipment than the microcytotoxicity assay. On the other hand, interpretation of results in the microcytotoxicity assay was easier and more objective. It was concluded that SSTA could be used with the microcytotoxicity assay and/or the haemagglutination assay to provide a simple and effective genetic monitoring method for inbred strain of rats.     

Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis.     

Cloning and Sequence Analysis of cDNA for Diuretic Hormone Receptor from the Bombyx mori

The insect diuretic hormone (DH) binds to their receptor in malpighian tubules, and stimulates water secretion and cAMP synthesis. Complementary DNA encoding a diuretic hormone receptor was cloned from the malpighian tubules of Bombyx mori. The cloned cDNA encodes a protein consisting of 391 amino acid residues with the seven transmembrane domains. The receptor protein is homologous with that of other insects, and is structurally related to G-protein coupled receptors such as corticotropin relating factor (CRF), secretin, and vasoactive intestinal peptide.     

Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae

To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD) was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development.     

Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.     

Characterization of Argonaute family members in the silkworm,Bombyx mori

Abstract The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5′ end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.     

Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori

The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL (Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.     

Functional Characterization of a Cactus Homolog from the Silkworm Bombyx mori

A cDNA encoding an IκB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IκB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A gultathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.     

Characterization of a gene encoding prohibitin in silkworm, Bombyx mori

Background

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.

Methods

To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry.

Results

BmPHB gene has an ORF of 825 bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth.

Conclusions

The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm.

General significance

Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori.     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。     

RFLP analysis of chromosomal fragments in genetic mosaic strains of Bombyx mori

Mottled striped (p^Sm), a genetic mosaic strain for larval body marking of the silkworm, Bombyx mori, contains a small chromosomal fragment generated by breakage near the end of the 2nd chromosome. This fragment carries the striped marking (p ^S ) gene and part of the chorion gene clusters. To determine the structural features of this fragmented chromosome, we studied the organization of the chorion genes on the fragment using restriction fragment length polymorphism (RFLP) analysis. Tow of three classes of chorion gene family probes detected RFLPs in the two phenotypes, p^Sm (p/p/p ^S ), and p (p/p), which had lost the fragment, segregated among the siblings of a mottled striped strain. Although hybridization patterns were basically identical between them, one or two additional bands were always observed associating with the mosaic phenotype (p^Sm). This suggests that the additional bands correspond to extra copies of chorion genes on the chromosomal fragment, which have a different structure from ones on the intrinsic 2nd chromosomes. Such heterogeneity of chorion genes may have been maintained since the beginning of mosaic induction, due to the absence of recombinational events between the two chromosomes. We are unable to detect any RFLPs by hybridization with the early class of chorion genes, implying that chromosomal breakage might have occurred between the two chorion clusters, Chl-2 and Ch3, which are located approximately 4 cM apart from each other. Based on RFLP analyses for two independent mosaic strains (788 and 872), we postulate a common chromosomal origin with independent breakpoints and construct structural models for the two kinds of chromosomal fragments.