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Author(s): Kazumichi M. Nishida [1]; Kazuhiro Sakakibara [1]; Yuka W. Iwasaki [2]; Hiromi Yamada [1]; Ryo Murakami [1]; Yukiko Murota [1]; Takeshi Kawamura [3, 4]; Tatsuhiko Kodama [4]; Haruhiko Siomi [...]

The mutant forms of octopamine receptors from Bombyx mori (BmOAR1) are constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The binding and activation of BmOAR1 with octopamine (OA) is through electrostatic and hydrogen bond interactions and can be used to design new pest control chemicals.

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering. carotenoid transport | cocoon color | transgenic silkworm

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an [F.sub.1] (p50T X C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an [F.sub.1] male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an [F.sub.1] female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of [approximately equal to]3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated. silkworm | microsatellite

The concentrations of six metal elements (Na, K, Mg, Ca, Cu and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm is investigated using proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorpton spectroscopy (AAS). It is seen that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca, which decreased significantly in the anterior part.

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an 'on' rate of 0.068 [+ or -] 0.01 [micro][M.sup.-1] x [s.sup.-1]. With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is [approximately equal to] 1 ms. A pH-dependent conformational change (BmorPB[P.sup.B] [right arrow] BmorPB[P.sup.A]), associated with the release of pheromone, is a first-order reaction (k = 74.1 [+ or -] 0.32 [s.sup.-1]; [t.sub.1/2], 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity ([K.sub.D] = 105 nM) at pH 7 but low affinity ([K.sub.D] = 1,600 nM) at pH 5, when tested at both low and high KCI concentrations. A protein with a C-terminal segment deleted (BmorPBP[DELTA]P129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence. Bombyx mori pheromone-binding protein | bombykol | effect of C terminus on pheromone release | fast uptake of pheromone and delivery | mutated pheromone-binding proteins

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of [greater than or equal to] 15 bases of mononucleotide repeats and [greater than or equal to] 5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.

Byline: O. A. Lihoradova (1), E. Yu. Bachurina (1), Sh. S. Azimova (1) Keywords: recombinant DNA; M-HBsAg; baculovirus; gene engineering; baculovirus expression system; silkworm larvae Abstract: Human hepatitis B virus middle surface antigen (M-HBsAg) is an envelope protein used as a basis for constructing vaccines and test systems detecting the virus. The M-HBsAg cDNA was inserted in transfer vector pBK273 under the transcriptional control of the polyhedrin gene promoter to obtain recombinant pBHep-2. Cotransfection with pBHep-2 and the wild-type Bombix mori NPV DNA yielded a recombinant baculovirus, rBmNPVHep, with the M-HBsAg cDNA controlled by the polyhedrin gene promoter. Infection of B. mori larvae with rBmNPVHep led to expression of the foreign gene recombinant M-HBsAg accumulated predominantly (to more than 90% its total content) in the fat body of larvae. Author Affiliation: (1) Institute of Genetics and Plant Experimental Biology, Academy of Sciences of Uzbekistan, Yuqori-Yuz, Tashkent Region, 702151, Uzbekistan Article History: Registration Date: 26/09/2004

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin lightchain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 X UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

Research was conducted to determine the phi, psi torsion angles of Ala and Gly residues of Bombyx mori silk fibroin using bond orientations derived from 15N and 13C solid-state NMR spectroscopy and constraints from fiber diffraction unit cell paramaters. The best fit torsion angles (phi, psi) within the reduced conformational space were defined as (-140 degrees, 142 degrees) and (-139 degrees, 135 degrees), respectively. Results were in good agreement with data obtained from fiber diffraction.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial [Beta]-1,3 glucanases and [Beta]-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polyclonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

The structure and expression of the cDNA encoding a precursor peptide for diapause hormone (DH) from the suboesophageal ganglion (SG) of the silkworm Bombyx mori were described. It was shown that the cDNA encodes pheromone biosynthesis-activating neuropeptide and three other neuropeptides in addition to DH. The results indicate that DH, along with four other neuropeptides, is generated from a common precursor polyprotein that is encoded by a single mRNA transcribed in neurosecretory cells of SG.

The structure of actin-containing cortical cytoskeleton of developing oocytes of the silkmoths Hyalophora cecropia and Antheraea polyphemus was investigated. In addition, the process of mRNA localization in the oocytes and the role of RNA polymerase II (pol II) in determining actin filament distribution pattern were sttudied. Immunohistochemical analysis coupled with confocal laser scanning microscopy were used in the study. The results indicate that the distribution of both mRNA and pol II in the cortex depends on the integrity of the actin scaffolding in the cortex.

The conformational transition from the predominantly random-coil starting structure to the final beta-sheet-rich state happens at remarkably specific pH and ionic conditions. The results imply the important roles that pH and ion affinity play for understanding the way in which spiders and silkworms store and spin tough threads under ambient conditions.

Increasing the rate of fluid flow across a chemosensory structure, for instance, by sniffing, would be expected to counteract the tendency of sensory receptors to habituate to odorant molecules presented [...]

Coactivators MBF1 and MBF2 mediate BmFTZ-F1-dependent transcriptional activation in vitro by interconnecting BmFTZ-F1, TATA binding protein TBP, and TFIIA. Here, we analyzed temporal and spatial expression patterns of MBF2 during embryonic and larval development of the silkworm Bombyx mori. MBF2 was detected in unfertilized eggs and embryos until stage 26. In stage 22 embryos, MBF1, MBF2, and BmFTZ-F1 colocalize in neural cells. During the larval stage, MBF2 was not expressed in the fat body and trachea. In the silk gland, MBF2 mRNA was constitutively expressed, but MBF2 protein appeared in the period between the second day and the molting D3 stage in both the third and the fourth instars and then disappeared. MBF2 was also detected on the second and third days of the fifth instar. Immunostaining during the fourth molt showed that MBF1, MBF2, and BmFTZ-F1 localize in the nucleus only at the D3 stage, while the two cofactors are present in the cytoplasm at other stages. Immunoprecipitation experiments suggested that MBF1, MBF2, and BmFTZ-F1 form a complex at the D3 stage. Transient expression of these factors in Schneider cell line 2 revealed that MBF1 and MBF2 localize to the nucleus and enhance BmFTZ-F1-dependent transcription only when all three factors are present. These data illustrate the functional regulation of MBF1 and MBF2 at the step of nuclear transport and implicate MBF2 in tissue- and stage-specific transcription. [C] 2000 Academic Press Key Words: coactivator; nuclear receptor; nuclear transport.

The studies revealed that silk fibroin from the silkworm Bombyx mori is an Ala/Gly-rich protein, which is spun from aqueous solution at room temperature to produce strong and elastic fibers. The report states that the constituting 55% of the total silk fiber thus, consists of 18% distorted beta-turn, 13% beta-sheet with parallel Ala, and 25% beta-sheet with alternating Ala. Half of the local conformations in the remaining amorphous Tyr-rich region (45% of the total silk fiber) are distorted beta-turns, the other half, distorted beta-sheets.

Allometry purportedly determines physiological rates in developing as well as adult animals. Yet, the tissues and organs of developing animals must switch from hyperplastic differentiation to hypertrophic growth, and allometric principles may not apply during this transition (Burggren, 2005). We tested the allometry of VO2 and heart rate in developing silkworm larvae from 1st-5th instar and in pupae to determine if conventional interspecific allometric principles predict intraspecific VO2. VO2 at 25[degrees]C was 1.2+0.04 mlO2/mg/h in 1st instar larvae (mass 0.3mg-O.8mg, n=25), failing to 0.98+0.17 mlO2/mg/h in the 2nd instar (mass 0.3mg-1.6mg, n=8) and 0.44+0.02 mlO2/mg/h in the 5th instar (mass 780mg2200mg, n=8). VO2 in pupa was considerably lower at 0.18+0.02 mlO2/mg/h (mass 760 mg 1800 mg, n=25). Importantly, the relationship between mass-specific VO2 and body mass differed between instars: sharply negative slopes relating VO2 to body mass in instars 1-3; slightly positive slopes for instars 4-5; and no significant relationship in pupae. These data suggest that intraspecific allometric predictions for developing animals, especially in insects showing marked internal reorganization associated with metamorphosis, may depart from those derived from interspecific allometric data based on adults.