Specific protein synthesis in cellular differentiation: IV. The chorion proteins of Bombyx mori and their program of synthesis

Detailed analysis of the chorion proteins of Bombyx mori reveals more than 70 components, each distinguished by electrophoretic mobility, relative abundance, and kinetics of synthesis. Many proteins are strain specific. A protein numbering system is established, based on isoelectric point and molecular weight. As in Antheraea polyphemus, chorion proteins are produced asynchronously, individual proteins showing characteristic developmental kinetics. The synthetic program is analyzed in detail. Stages of choriogenesis are defined according to the pattern of protein synthesis, and their relative and absolute durations are determined. In a few stages, synthesis of numerous protein cohorts is coordinately initiated.     

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.     

Effect of the pleiotropic GrB mutation of Bombyx mori on chorion protein synthesis

The Gr^B mutation has a profound pleiotropic effect, leading in the homozygous state to the absence or extreme reduction of a substantial number of chorion proteins. The effect shows developmental specificity: most of the proteins normally synthesized beginning with stage III of choriogenesis or later, but possibly none of these normally beginning with stage II, are eliminated in the mutant. More subtle quantitative effects on certain other proteins are also observed, including prolongation of synthesis of some proteins which normally terminate at stage VIII. The proteins eliminated in the mutant are present in the heterozygote at intermediate levels, quantitatively close to those in the wild-type. The differences in chorion protein composition result from correspondingly altered protein synthesis rather than from post-translational degradation or modification. The missing proteins also fall to be synthesized in vitro when total RNA from mutant follicles is translated in the wheat germ system. It appears that as a consequence of the mutation, a set of mRNAs fails to be synthesized or accumulated. These results are consistent with the possibility that Gr^B is a regulatory mutation, or a deletion eliminating multiple chorion genes, clustered predominantly according to the developmental period of their expression.     

Specific protein synthesis in cellular differentiation: II. The program of protein synthetic changes during chorion formation by silkmoth follicles,and its implementation in organ culture

During their differentiation, the follicular epithelial cells of the silkmoth, Antheraea polyphemus, produce the extracellular proteinaceous eggshell or chorion. Choriogenesis entails continuous changes in cell-specific protein synthesis; the various chorion proteins are synthesized with distinct kinetics. On the basis of protein synthetic profiles, 17 stages of choriogenesis are defined. The average duration of the stages is 3.0 hr, and thus choriogenesis lasts a total of approximately 51 hr. This program of protein synthetic changes is autonomous; i.e., it is implemented with normal kinetics by follicles cultured in isolation in a defined tissue culture medium.     

Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori

The isovaleric acid-emanating silkworm mutant skunk (sku) was first studied over 30?years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsible for the sku mutant. Because of its specific features and symptoms similar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer-monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

Post-translational processing in the synthesis of egg-specific protein in the silkworm,Bombyx mori

The post-translational processing of egg-specific protein (ESP) in developing ovarian follicles of the silkworm, Bombyx mori was analyzed using in vivo and in vitro labeling systems with some radioactive precursors. The labeling with[³⁵S]methionine revealed that ESP is first synthesized as 69 kDa peptide (69K-ESP) which is then converted to 72 kDa peptide (72K-ESP) until 2 h. Some of 72K-ESP molecules were converted to 64 kDa peptide (64K-ESP) after 10h-labeling. [¹⁴C]Mannose was incorporated into 69K-ESP and 72K-ESP. In the presence of tunicamycin, labeling with [³⁵S]methionine brought about a new 67 kDa peptide (67K-ESP) by reducing the incorporation into 69K- and 72K-ESP. [³²P]Ortho-phosphate was incorporated into only 72K-ESP by a 2 h-pulse labeling. Treatment of 72K-ESP with alkaline phosphatase converted it to 69K-ESP. These results along with the available information led to the conclusion that the primary translation product is sequentially processed to 67K-ESP by signal peptide cleavage, to 69K-ESP by glycosylation, and finally to 72K-ESP by phosphorylation as the actual product in the peptide synthesis. The limited conversion of 72K-ESP to 64K-ESP is proposed to be a post-endocytotic event.     

Gene evolution and regulation in the chorion complex of Bombyx mori. Hybridization and sequence analysis of multiple developmentally middle A/B chorion gene pairs

Twenty-two pairs of chorion genes belonging to the A and B multigene families have been characterized and mapped within two segments of a 320 kb (1 kb = 10(3) bases or base-pairs) chromosomal walk in the domesticated silkmoth Bombyx mori. Eighteen of the gene pairs belong to two groups that are typified by the previously characterized A/B.L12 and A/B.L11 chorion gene pairs, and are defined by two respective types of short (approx. 280 base-pairs) bidirectional promoter sequences. In the chromosome, the L12-like and L11-like pairs are interspersed with each other and with the remaining four gene pairs, which have unrelated promoter sequences. We have sequenced the promoter regions and adjacent small exons of all L12-like and L11-like A and B genes in the walk. The L12-like promoters are highly conserved, whereas L11-like promoters are somewhat more variable. Reconsideration of previous data on RNA accumulation and disappearance during choriogenesis, in the light of the sequences, indicates that L12-like genes are developmentally early-middle, while L11-like genes correspond to two developmental subgroups, middle I and middle II. Sequence comparisons of all these promoters, as well as the previously characterized promoters of the developmentally late HcA and HcB genes, identify short elements of possible regulatory significance. The sequences, as well as extensive cross-hybridization analysis with short probes derived from the reference A/B.L12 gene pair, under carefully controlled conditions of stringency, indicate the occurrence of sequence transfers among A or B genes. These sequence transfers, which could result from gene conversions or unequal crossovers, are less abundant than in the HcA and HcB families, but do result in a patchwork of similarities and differences in the A and B genes. The transfers appear to be least frequent between the moderately divergent A genes that belong to different temporal classes, while the L12-like and L11-like B genes appear to be extensively homogenized in sequence.     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

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.     

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.     

Structural features of B family chorion sequences in the silkmoth Bombyx mori, and their evolutionary implications.

Partial protein sequences, and DNA sequences of corresponding cDNA and genomic clones were obtained and analyzed to reveal the primary structural features of major, developmentally middle or late components of the B chorion multigene family in Bombyx mori. Comparisons with other types of sequences confirm and clarify the tripartite domain structure of chorion proteins. Glycine-, leucine- and tyrosine-containing, tandemly repetitive peptides form the bulk of the amino-terminal and carboxy-terminal domains ('arms'). Extensive sequence homologies suggest a common evolutionary origin for the amino-terminal arms of some B. mori B sequences and the corresponding portions of members of a different (A) chorion multigene family in Antheraea polyphemus, a distantly related silkmoth.     

Ligand-release pathways in the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.     

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.     

A walk in the chorion locus of bombyx mori

We have recovered overlapping clones that represent in the aggregate a contiguous segment of chromosomal DNA 270 kb in length, or probably one third of the chorion locus of Bombyx mori. Approximately 70 genes have been identified, the majority of which are arranged in coordinately expressed pairs. The nonidentical genes expressed in the late period of choriogenesis are clustered within a single, 130 kb region, which is flanked by regions containing genes that are active during the middle developmental period. The late genes encode two families of high-cysteine proteins; the evolutionarily persistent clustering of these families contrasts sharply with the extensive sequence diversification of the structural genes and their flanking DNA elements. We discuss the possible regulatory significance of the clustered arrangement, as well as certain features of multigene family evolution.     

Nutritional and non nutritional stimulations of protein synthesis in the fat body of Bombyx mori

The relations between the nutritional state and the polysome level are determined in the fat body and silk glands of the Bombyx mori larva. In the fat body, the polysome level is drastically reduced during starvation and increases rapidly 20 min after intake of food. This delay does not correspond to a digestion time or a delay of transit in the intestine. The increase occurs only if free amino acids are present in the food. The ingestion of tryptophan alone by previously starved larvae promotes the formation of heavy polysomes and an increase in the incorporation of radioactive precursors into the proteins of the fat body. Enhancement of the proteosynthetic activity of this organ can also be obtained independently of any food intake by the injection of various and unrelated substances.Tryptophan ingestion and insulin injection have no effect on the polysome level of the silk glands.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.