Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects.

The cytoplasmic actin genes BmA3 and BmA4 of Bombyx mori were found clustered in a single genomic clone in the same orientation. As a similar clustering of the two cytoplasmic actin genes Ha3a and Ha3b also occurs in another lepidopteran, Helicoverpa armigera, we analyzed the sequence of the pair of genes from each species. Due to the high conservation of cytoplasmic actins, the coding sequence of the four genes was easily aligned, allowing the detection of similarities in noncoding exon and intron sequences as well as in flanking sequences. All four genes exhibited a conserved intron inserted in codon 117, an original position not encountered in other species. It can thus be postulated that all of these genes derived from a common ancestral gene carrying this intron after a single event of insertion. The comparison of the four genes revealed that the genes of B. mori and H. armigera are related in two different ways: the coding sequence and the intron that interrupts it are more similar between paralogous genes within each species than between orthologous genes of the two species. In contrast, the other (noncoding) regions exhibited the greatest similarity between a gene of one species and a gene of the other species, defining two pairs of orthologous genes, BmA3 and HaA3a on one hand and BmA4 and HaA3b on the other. However, in each species, the very high similarities of the coding sequence and of the single intron that interrupts it strongly suggest that gene conversion events have homogenized this part of the sequence. As the divergence of the B. mori genes was higher than that of the H. armigera genes, we postulated that the gene conversion occurred earlier in the B. mori lineage. This leads us to hypothesize that gene conversion could also be responsible for the original transfer of the common intron to the second gene copy before the divergence of the B. mori and H. armigera lineages.

Chest wall pain and possible rhabdomyolysis after chloropicrin exposure. A case series.

Three cases are summarized in which persistent chest wall pain, as well as elevations of creatine phosphokinase (CK) levels, occurred after exposure to chloropicrin vapor in an agricultural chemicals facility. Both the severity of the symptoms and the degree of biochemical abnormality appeared to follow a dose-response relationship. Myoglobinuria, on the other hand, was not found. Elevation of CK after chloropicrin exposure has not previously been reported and may represent low-grade rhabdomyolysis. Workups performed after symptomatic chloropicrine exposure should include CK-level determination.

Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA.

The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure of the Ser1 gene. From these and previous data, it was possible to deduce the sequence of the sericins 1 and to predict the secondary structure and physiochemical properties of the different regions of the proteins.

A strong inhibitory element down-regulates SRE-stimulated transcription of the A3 cytoplasmic actin gene of Bombyx mori.

To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.

Two alternative promoters drive the expression of the cytoplasmic actin A4 gene of Bombyx mori.

By screening cDNA and genomic libraries, we have cloned A4, the fourth and last actin gene of Bombyx mori, which encodes a typical cytoskeleton actin and is expressed in all larval tissues. A4 is closely related to A3, another cytoplasmic actin gene of the silkworm, in its encoded amino-acid sequence, and the location as well as the sequence of a single intron. Both A3 and A4 have possibly arisen from the recent duplication of an intron-containing ancestral gene. The two genes display different organization of their 5' untranslated and flanking sequences. In contrast to A3, which harbours a single promoter, A4 exhibits two leader exons transcribed by the use of alternative promoters. A3 and A4 actins differ only by two amino acids at positions known to vary among cytoplasmic actins of other species, and are likely to be functionally equivalent. We speculate that transcriptional constraints are actually the target of a selective pressure that maintains two distinct cytoplasmic actin genes in insects, as well as in other animals.

Regulation of the P25 gene transcription in the silk gland of Bombyx.

The gene encoding the silk protein P25 is specifically transcribed in the posterior silkgland of Bombyx during larval intermoults and is repressed during moults. By performing in vitro DNA-protein interactions, at least five putative regulatory elements were localized in the 5' flanking region of the gene. The most proximal element, close to the TATA box, interacts with SGFB, a silkgland-specific factor which could be involved in the tissue-specific expression of the gene. A more upstream sequence is recognized by an ubiquitous factor, BMFA, which exhibits cyclical modifications in relation to the moulting cycle and could thus be involved in the temporal control of the gene during the development. A construct containing a reporter gene fused to 1450 bp of P25 5' flanking sequences was integrated into the Drosophila genome and shown to be specifically expressed in the larval salivary gland, the organ homologous to the silkgland. Recurrent deletions of this construct showed that the proximal 254 bp contain all the sequences required for this specific expression. Similar foreign constructs introduced in the silkgland in vivo by a particle delivery system were specifically transcribed in the posterior silkgland but remained silent in the middle silkgland as the endogenous genes. This methodology will be used to assay the function of the defined cis-acting elements in the spatial regulation of expression of P25.

Structural features of mag, a gypsy-like retrotransposon of Bombyx mori, with unusual short terminal repeats.

Mag is a retrotransposon found as an insert in the Sericin 2 gene. It is present in a few copies--4 to 15--dispersed in the genome of different strains of Bombyx mori as well as in Bombyx mandarina. Flanked by a 5 bp target sequence with no sequence specificity, it is bordered by direct repeats of 77 nucleotides. Despite their unusual short size, these terminal repeats and their immediately adjacent sequences present all the signals necessary for transcription into genomic RNA and for reverse transcription. Mag contains two overlapping open reading frames which are organized as the gag and pol genes of retroviruses and encode putative nucleic acid binding peptide, protease, reverse transcriptase, RNase H and endonuclease in this order. Sequence comparison of these proteins places mag within the gypsy group of LTR retrotransposons next to the echinoderm element SURL.

Differential transcription of multiple copies of a silk worm gene encoding tRNA(Gly1).

Ten different tRNA(Gly1) genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNA(Gly1) genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmi1, pBmp1, pBmh1, pBmt1) and low to barely detectable levels (e.g., pBms1, pBmj1 and pBmk1). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A+T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNA(Gly1) genes in vivo.

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins.

Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.

Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori.

Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore, the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.

Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland.

Four mRNA of 10.5, 9.0, 4.0, and 2.8 kb are made from the sericin Ser1 gene by alternative maturation of a unique mRNA precursor. By means of RNA blots and in situ hybridization, we investigated variations in the distribution of these mRNA during the last larval instar in different territories of the middle silkgland. Taken together, the results from these two techniques show that 150 out of the 266 cells of this region of the organ express the Ser1 gene, but accumulate distinct mature mRNA species. Of these 150 cells 42 are specialized in a processing pathway resulting in the production of the 2.8-kb Ser1 mRNA throughout the larval instar. The 108 others perform successively three distinct splicing pathways leading to a development-dependent accumulation of, respectively, the 4.0-, the 10.5-, and the 9.0-kb mRNA. This suggests the occurrence of two switches in the splicing capacities of these cells during the fifth instar. The middle silkgland cells also express another sericin gene (Ser2) which encodes two mRNA of 5.4 and 3.1 kb, also arising by differential splicing. At the beginning of development, all the middle silkgland cells express this gene but, as development proceeds, expression becomes restricted to only the anterior cells. The biological consequence of this topological and temporal regulation of the mode of expression of these two genes is the sequential secretion and layering of the different sericins around the silk thread.

A single gene produces multiple sericin messenger RNAs in the silk gland of Bombyx mori.

The sericins are a family of major cocoon proteins specifically synthesized in the middle silk gland of the silkworm Bombyx mori. The 5' part of one sericin gene had been cloned and described by Okamoto et al. (1982, J. Biol. Chem. 257, 15192-15199). Using a differential screening procedure of Bombyx genomic libraries, we obtained the 3' part of this gene. We demonstrate that it consists of a single gene extending over 24 kb, present in two allelic forms in hybrid strains. This gene encodes for four mRNAs which result from a unique transcript by an alternative splicing mechanism. This explains, at least partially, the diversity of the sericins found in the cocoon.

Isolation of actin genes in Bombyx mori: the coding sequence of a cytoplasmic actin gene expressed in the silk gland is interrupted by a single intron in an unusual position.

To study the regulation of the gene(s) coding for the actin present in the microfilaments involved in the secretion of silk, we have probed a Bombyx mori genomic library with a Drosophila actin cDNA clone and selected 16 recombinant phages. They correspond to 3 different genomic fragments each containing a distinct actin coding sequence. Southern blots of genomic DNA probed with the cloned genes show that in Bombyx mori, there are at least 5 different actin genomic sequences. Two cloned genes A1 and A2 hybridize to a 1.7 kb long mRNA abundant in the carcass of the larva and thus probably code for muscle type actin. The third cloned gene, A3, hybridizes to two mRNAs of about 1.8 kb present in the silk gland and thus probably encodes a cytoplasmic actin. The coding sequence of this gene has been sequenced: it is almost identical to the Drosophila cytoplasmic actin genes but it has a single intron of 92 nucleotides within the codon 116, a position not observed in any other organism.

Structural organization of the P25 gene of Bombyx mori and comparative analysis of its 5' flanking DNA with that of the fibroin gene.

We have cloned a large portion of the P25 gene of Bombyx mori encoding the 25,000 dalton polypeptide which associates with fibroin to constitute the major silk protein. Its structure has been investigated by restriction mapping R-loop analysis, S1 nuclease protection experiments and nucleotide sequencing of the region spanning the 5' end of the gene and its flanking DNA. This has permitted a comparative sequence analysis of the DNA from the P25 and fibroin genes. The genes demonstrate no relatedness in their coding regions but they exhibit large blocks of sequence homology in their 5' flanking regions. In particular, the DNA upstream of the P25 gene possesses a sequence very similar to a region of fibroin 5' flanking DNA that is known to possess transcription modulation signals. The functional significance of these homologous regions is discussed with regard to the highly coordinated expression of these two genes.

Actin microfilaments and fibroin secretion in the silkgland cells of Bombyx mori. Effects of cytochalasin B.

Bombyx mori posterior silkgland cells exhibit an impressive microfilament apparatus located at the cellular apex. It consists of bundles of packed, long microfilaments of 50-70 A diameter running along circumferences delimiting the lumen of the gland, perpendicularly to the flow of luminal silk. Microfilaments are closely associated with microtubules of the cytoplasmic 'radial microtubule system'. Immunolabelling with purified antihuman actin antibodies was used to demonstrate their actin-like nature. Apical microfilaments are sensitive to cytochalasin B (CB) which selectively inhibits the secretion of fibroin. Following the removal of the drug, microfilaments recover their normal morphology and secretion resumes. The possible implication of contraction of microfilaments in the process of secretion is discussed.

Developmental variations of a nonfibroin mRNA of Bombyx mori silkgland, encoding for a low-molecular-weight silk protein.

The characterization of a new silk protein mRNA (P25 mRNA) in posterior silkgland cells (PSG) and the developmental variations of its cell molecular concentration versus that of fibroin mRNA are described. A 80% pure P25 cDNA was obtained by class separation of total nonfibroin cDNA from PSG and used to identify the mRNA in blotted PSG mRNA as a single 1100 nucleotide long species. When purified from agarose gel and translated in a reticulocyte cell-free system, P25 mRNA yielded a 25-kD polypeptide (P25), identical to a 25-kD protein of the cocoon in terms of pI value and partial peptide mapping pattern. Moreover, this protein comigrated with an abundant polypeptide of the posterior silkgland (PSG) and of the middle silkgland (MSG). When tritiated leucine was injected in vivo, labeled P25 showed up in the PSG after a 2-hr pulse but appeared in the MSG only after 24 hr of labeling. Since MSG cells were found to be devoid of P25 mRNA, we concluded that P25 is exclusively synthesized in the PSG, that it accumulates in the MSG lumen and that it is spun out in the same way as fibroin. Specific probes were used to measure the concentrations of P25 mRNA and also fibroin mRNA in PSG total RNA by hybridization with an excess of cDNA. Both species are highly degraded in the few hours following the physiological arrest of feeding which precedes the fourth molting period. Their subsequent accumulation during the fifth intermolt is triggered by food uptake and proceeds in such a way that a constant 1:1 molar ratio is maintained during the period of silk secretion.