Shiro Tomino

Molecular properties and biosynthesis of major plasma proteins in Bombyx mori

Abstract In the silkworm, Bombyx mori, a group of structurally related proteins referred to as ‘30K proteins’ comprises the major plasma proteins of the last instar larvae. Four protein components consisting of 30K proteins were purified to homogeneity from the larval hemolymph and designated Component 1, 2, 3 and 4, respectively. Close similarity in amino acid composition was noticed between Components 1 and 3, and between Components 2 and 4. Rabbit antibody prepared against Component 4 crossreacted with Component 2 as well as Component 4 but not with Components 1 or 3. In a cell-free translation system, RNA isolated from the fat body of the last instar larvae directed the synthesis of proteins reactive with anti-Component 4 antibody. Developmental change in the hemolymph concentration of 30K proteins well reflected the level of functional mRNA for these proteins in the fat body, indicating that the biosynthesis of 30K proteins is regulated during development at pre-translational level.

Purification and molecular properties of vitellin from the silkworm, Bombyx mori

Abstract Vitellin was purified from the eggs of the silkworm, Bombyx mori by a simple method which included a specific precipitation at pH 6 under low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy and ultracentrifugation. Vitellin was defined as glycolipoprotein with a sedimentation coefficient (S 20, W ) of 13.5S and a molecular weight of 440,000. The molecule was almost spherical in shape with a diameter of 13 nm. The molecule contained 3% mannose and 7.5% total lipids which comprised triacylglycerol, diacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The amino acid composition displayed a high content of glutamic and aspartic acids and a low content of methionine. The molecule was composed of two non-identical subunits with molecular weights of 180,000 and 42,000, and the native molecule was assumed to be a tetramer composed of two molecules of each of these subunits. Separation of the two subunits was achieved, and mannose was covalently associated only with the heavier subunit. The rabbit anti-egg vitellin antibody cross-reacted with the haemolymph vitellogenin but not with other haemolymph proteins, nor with the vitellogenin from Locusta migratoria . The antibody also reacted with the haemolymph vitellogenin of the silkworm, Philosamia cynthia .

Structure and expression of mRNA for vitellogenin in Bombyx mori

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

Vitellogenin gene of the silkworm, Bombyx mori: Structure and sex-dependent expression

Vitellogenin of Bombyx mori is a precursor of major yolk protein synthesized in the female fat body at larval—pupal ecdysis. The gene for B. mori vitellogenin is composed of seven exons interspersed by six introns. Developmental profile of the primary transcript of the gene indicated that the biosynthesis of B. mori vitellogenin is regulated transcriptionally in a sex‐ and stage‐dependent manner in the fat body. The Arg‐X‐Arg‐Arg sequence, which conforms to the recognition site of mammalian furin, occurs in a region just upstream of the putative proteolytic cleavage site of B. mori previtellogenin.

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm, Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.

Structure and expression of mRNA for a pupal cuticle protein of the silkworm, Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.

Molecular cloning and characterization of cDNA for insect biogenic peptide, growth-blocking peptide

Growth‐blocking peptide (GBP) is an insect biogenic peptide that prevents the onset of metamorphosis from larva to pupa. A cDNA coding for GBP is described. Mixed oligonucleotides derived from a GBP peptide sequence were used to generate amplified DNA by the polymerase chain reaction (PCR). Based on the sequence of the amplified DNA, a 41 bases oligonucleotide was designed for screening a cDNA library which was constructed from the armyworm Pseudaletia separata larvae parasitized with the parasitic wasp Cotesia kariyai. The cloned cDNA for GBP was 809 base pairs in length. An open reading frame of 429 base pairs encodes a pre‐pro‐peptide of 143 amino acid residues in which GBP is localized at the C‐terminal region, and other three peptides including a putative signal peptide and appropriate processing sites for endoproteolytic cleavage precede the GBP sequence. Northern blot analyses demonstrate the presence of a 800‐base mRNA transcript in fat body and 2.5‐kilobase transcript in brain and nerve cord, suggesting the possibility that the transcription of GBP gene is regulated in a tissue‐dependent manner. This interpretation was supported by isolating a GBP cDNA fragment from cDNA pool of brain‐nerve cords. GBP mRNA is constantly expressed in both parasitized and non‐parasitized last instar larvae and there is no difference in the levels of the mRNA between both larvae, thus indicating that parasitism may effect on translational or posttranslational level to elevate plasma GBP concentration.

Translation of fat body mRNA from the silkworm, Bombyx mori

Abstract The amount of m RNA in the fat body of the silkworm, Bombyx mori was studied by use of heterologous translation systems. The amount of functional m RNA relative to total RNA in the fat body declines during the larval-pupal transformation. The fat body m RNA from the fifth instar larvae directed the synthesis of storage proteins in a cell-free system derived from wheat germ, whereas that from female pupae, immediately after larval-pupal ecdysis, encoded a subunit of vitellogenin in a wheat germ system and in Xenopus oocytes.

A defective non-LTR retrotransposon is dispersed throughout the genome of the silkworm, Bombyx mori

The presence of long repetitive sequences is demonstrated in the genome of the silkworm, Bombyx mori. Members of this BMC1 family reveal several features typical of the L1 (long interspersed sequence one) family of mammals, except for species specific elements. The number of BMC1 elements is estimated to be approximately 3500 per haploid genome. Elements containing the full length unit of 5.1 kb are dispersed throughout the genome and their restriction sites are conserved, although most members are preferentially truncated to varying extents at their 5′ ends. DNA sequencing indicates that this element contains six tandem repeats of 15 bp CpG-rich sequence in the 5′ proximal region. It terminates with a 3′ oligo(A) stretch, and is flanked at both ends by a 7–10 bp target sequence duplication. In addition, there is significant evidence for amino acid sequence homology with reverse transcriptase domains of other L1 families, especially F, Doc and lockey of Drosophila melanogaster. No large open reading frame is present. The BMC1 element is suggested to be dispersed in the genome by a transposition mechanism involving RNA intermediates.

Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx mori

The nucleotide sequence (1974 bp) of cDNA coding for membrane-bound alkaline phosphatases (m-ALP) of Bombyx mori was isolated. The cDNA clone contained an open reading frame encoding a polypeptide (547 amino acids), which contains a hydrophobic signal peptide of 36 amino acids and the mature protein of 511 amino acids (Mr = 56,163). We found a highly hydrophobic domain presumed to be a membrane anchoring region at the C-terminus. Comparing analysis between Bombyx m-ALP and mammalian and Escherichia coli ALPs suggested an evolutionary relationship of sharing a common ancestral gene.