Mapping and recombination analysis of two moth colour mutations, Black moth and Wild wing spot, in the silkworm Bombyx mori.

Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing- and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to ~2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing- and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera.

Linkage and mapping analysis of a non-susceptibility gene to densovirus (nsd-2) in the silkworm, Bombyx mori.

Nonsusceptibility to Bombyx mori densovirus type 2 (BmDNV-2) is controlled by a recessive non-susceptibility gene, nsd-2 (non-susceptibility to DNV-2) in B. mori. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 (BF1) progeny were used for linkage analysis and mapping of nsd-2 using silkworm strains C124 and 902, which are classified as being highly susceptible and non-susceptible to DNV-2, respectively. BF1 larvae were inoculated twice with DNV-2 virus at the first and second instar stages. DNA was extracted from each of the surviving fifth instar larvae and analysed by RFLP inheritance patterns using probes specific to each of the 28 linkage groups of B. mori. Our results indicated that the non-susceptibility gene was linked to linkage group 17, since all surviving larvae showed the homozygous profile of strain 902 in their genotype. The other linkage groups showed mixtures of heterozygous and homozygous genotypes, indicating an independent assortment. A linkage map of 30.6 cM was constructed for linkage group 17 with nsd-2 mapped at 24.5 cM and three closely linked cDNA markers were identified.

Linkage analysis of maternal EST cDNA clones covering all twenty-eight chromosomes in the silkworm, Bombyx mori.

A cDNA library of maternal messages was constructed from non-fertilized Bombyx mori eggs collected just after oviposition without mating. cDNA clones were identified by their nucleotide sequences and evaluated as probes for RFLP linkage analysis. Back crossed F1 segregants - by crossing an F1 female (RF02 x RF50) and a male (RF02) - were used, which takes advantage of the phenomenon that no crossing over occurs in Bombyx females. Fifteen ordered BF1 segregants gave either homozygous (homo) or heterozygous (hetero) RFLP patterns with each cDNA probe. cDNA probes on the same linkage group gave the same homo/hetero order. One hundred and fifty one out of 248 cDNA clones showed polymorphisms between RF02 and RF50, and were therefore suitable as probes for RFLP linkage analysis in the present BF1 cross. They were sorted into twenty-seven linkage groups and one independent group, by the homo/hetero pattern matrix, covering all twenty-eight chromosomes in B. mori.

Distinctive structural and kinetic properties of an unusual juvenile hormone-hydrolyzing esterase.

The insect juvenile hormone specific esterases (JHEs), related to acetylcholinesterases but exhibiting substrate specificity for juvenile hormone (JH), are essential enzymes for normal insect development, making them attractive targets for biorationally designed, environmentally safe pesticides. We examine here a new enzyme, JHER, related to, but yet structurally, biochemically, and kinetically distinct from, the classical JHE. Both classical JHE and baculovirus-expressed JHER hydrolyze JH show disproportionately higher catalytic rates at higher substrate concentrations (in contrast to substrate inhibition reported for acetylcholinesterase) and are similarly inhibited by an organophosphate. However, JHER, which possesses an unusual cysteine residue at +1 to the catalytic serine, is less sensitive to trifluoromethyl ketone transition state analogs designed around the structure of JH. We propose a model in which JHER is expressed just prior to metamorphosis for hydrolysis of a JH-like substrate with hydrophobic backbone, a proximal ester, and a terminal expoxide or related substitution.

Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases.

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, Rad and Gem-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Biochemical characterization of the Ras-related GTPases Rit and Rin.

We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.

Dinocampus (=Perilitus) coccinellae teratocyte-specific polypeptide: its accumulative property, localization and characterization.

Dinocampus (=Perilitus) coccinellae (Braconidae: Hymenoptera) teratocytes synthesize a teratocyte-specific polypeptide (TSP) with a high molecular weight of 540kDa. The TSP has a tendency to accumulate in the teratocyte cells without release after synthesis ([Okuda and Kadono-Okuda, 1995]), which was confirmed in this study. Pulse-chase fluorography indicated that teratocytes at a younger stage (6 days after parasitization)secreted negligible TSP into the medium after synthesis, while teratocytes at an older stage (11 days after parasitization)secreted the synthesized products into the medium, although the amount released was still low. Western blot with anti-TSP serum showed that only a small amount of TSP appeared in the parasitized host hemolymph, even when TSP synthesis by teratocytes was actively taking place, which also supported the accumulative nature of TSP. The immunoelectronmicroscopic studies revealed that the TSP was localized specifically in high electron-dense vacuoles. Lectin blot analysis identified TSP as a high mannose glycoprotein. The amino acid composition of the major subunit of the TSP was quite similar to that of nutritive proteins such as vitellogenin and storage proteins of some insects. These characterization data, together with the accumulation property of the TSP indicates that Dinocampus teratocyte primarily plays a nutritive role for the developing parasitoid larvae. TSP exhibited esterase activity, which indicates that TSP may have an additional function in the host-parasitoid reaction.

Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.

An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay.

We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [alpha-32P]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

Structure of two cecropin B-encoding genes and bacteria-inducible DNA-binding proteins which bind to the 5'-upstream regulatory region in the silkworm, Bombyx mori.

Two genomic DNAs encoding cecropin B (CecB), an antibacterial protein from Bombyx mori, were cloned and sequenced. The number of CecB genes was estimated to be more than four copies per haploid by genomic Southern blotting. Two genes, CecB1 and CecB2, were located tandemly within 12 kb in the same orientation. These two genes encoded identical amino acids, though 15 nucleotides (nt) were different in the coding region and the intron size varied. About 90% of the nt spanning 800 bp in the 5'-untranslated region (UTR) were identical between the two genes. This 5'-flanking region contained characteristic sequences such as a repetitive element of B. mori (Bm1), an interleukin-6 response element (IL-6 RE), and two putative lipopolysaccharide (LPS) response elements (LPS RE). An electrophoretic mobility shift assay (EMSA) showed that the fat body contains at least three different nuclear proteins inducible by bacteria which bind to the 5'-UTR, suggesting that these proteins may be involved in CecB expression triggered by bacteria.

cDNA cloning and gene expression of lebocin, a novel member of antibacterial peptides from the silkworm, Bombyx mori.

A cDNA encoding lebocin, a novel member of insect antibacterial peptides, was isolated from the fat body cDNA library of Bombyx mori larvae immunized with Escherichia coli. The cDNA was 844 bp long and had an open reading frame (ORF) containing a probable signal peptide (16 amino acids), a putative prosegment (104 amino acids) and a mature peptide (32 amino acids) followed by 27 additional amino acids at its carboxyl-terminus. Northern blot analysis showed that lebocin gene expression was inducible by bacterial injection, occurred tissue-specifically in the fat bodies and continued at least for 48 h post-infection. These results suggest that lebocin has a unique precursor structure and shows typical gene expression pattern as insect antibacterial peptide.

Baculovirus-mediated production of the human growth hormone in larvae of the silkworm, Bombyx mori.

To express the cDNA encoding human growth hormone (hGH) in larvae of Bombyx mori, B. mori nuclear polyhedrosis virus (BmNPV) was employed as an expression vector. For the construction of the recombinant virus, the hGH cDNA was inserted into the downstream of the strong polyhedrin promoter to achieve a high level expression. Immunoblot analysis revealed that the virus-mediated hGH was synthesized in the larvae and secreted into the hemolymph. The yield of the recombinant hGH synthesized in the larvae reached to a level of 160 micrograms/ml of hemolymph after purification. The purified recombinant hGH was confirmed to have both the same molecular weight and amino acid sequence at its N-terminal region as those of the natural counterpart. In addition, the biological activity of the recombinant hGH was comparable to that of the natural hGH in the growth-stimulating effect on rat Nb 2 Node lymphoma cells.

Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins.

A Bombyx mori cDNA was cloned that hybridized with Hyalophora cecropia attacin probe and its nucleotide sequence was determined. This cDNA consisted of 846 nucleotides and the deduced amino acid sequence showed that the cDNA encodes an attacin precursor protein. The putative mature protein of B. mori attacin had 70.4, 68.3 and 18.8% identity in amino acid sequences with that of H. cecropia acidic and basic attacins and Sarcophaga peregrina sarcotoxin IIA, respectively. B. mori and H. cecropia attacins and S. peregrina sarcotoxin IIA had two subdomains in each G domain, suggesting that common amino acid residues in the subdomains are conserved during evolution and plays an important role in the activity of the antibacterial proteins. Expression of B. mori attacin gene was rapidly induced by the injection of Escherichia coli cells into B. mori larvae and continued at least for 48 h mainly in fat bodies and hemocytes.

Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor.

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristics features of the carbohydrate-recognition domain of C-type animal lectin were revealed at C-terminal sequence of hemocytin. When cDNA encoding this region was introduced into baculovirus vector, hemagglutinating activities were detected in the culture fluid of a recombinant virus-infected cells. These activities were inhibited by D-mannose, N-acetyl-D-galactosamine, and D-maltose which are haptenic saccharides of authentic hemocytin. Analysis of dot and Northern blot hybridization revealed that hemocytin gene was transcribed in hemocytes of the silkworm at larval-pupal metamorphosis and/or after the injection of Escherichia coli and lipopolysaccharide. After silkworm larvae were injected with C-terminal portion of hemocytin, aggregation of hemocytes was observed in the hemolymph. Hemocytin has significant homology with mammalian von Willebrand factor which involves in platelet adhesion to subendothelium. Also, hemocytin has a homologous region with coagulation factor V and VIII. These results suggest that hemocytin molecule is an adhesive protein and relates to hemostasis or encapsulation of foreign substances for self-defense.

Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori.

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals.

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.