Larval legs of mulberry silkworm Bombyx mori are prototypes for the adult legs.

Morphological diversity of leg appendages is one of the hallmarks of developmental evolution. Limbs in insects may develop either from their embryonic prototypes or from imaginal discs harbored inside the larva. Bombyx mori (B. mori), a Lepidopteran insect, develops adult wings from larval wing imaginal discs. However, it has been debated whether the adult legs of B. mori arise from imaginal discs or from the larval legs. Here we addressed how the larval legs relate to their adult counterparts. We present the morphological landmarks during early leg development. We used expression of developmental genes like Distalless and extradenticle to mark leg primordia. Finally, we employed classical excision approach to develop a fate map of the adult leg. Excision and ablation of thoracic legs along proximo-distal axis at various times during larval development resulted in the loss of corresponding adult leg segments. Our data suggest that B. mori legs develop from larval appendages rather than leg imaginal discs.

Phylogeny of the insect homeobox gene (hox) cluster.

The homeobox (Hox) genes form an evolutionarily conserved family encoding transcription factors that play major roles in segmental identity and organ specification across species. The canonical grouping of Hox genes present in the HOM-C cluster of Drosophila or related clusters in other organisms includes eight "typical" genes, which are localized in the order labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominalA (abdA), and AbdominalB (AbdB). The members of Hox cluster are expressed in a distinct anterior to posterior order in the embryo. Analysis of the relatedness of different members of the Hox gene cluster to each other in four evolutionarily diverse insect taxa revealed that the loci pb/Dfd and AbdB, which are farthest apart in linkage, had a high degree of evolutionary relatedness, indicating that pb/Dfd type anterior genes and AbdB are closest to the ancestral anterior and posterior Hox genes, respectively. The greater relatedness of other posterior genes Ubx and abdA to the more anterior genes such as Antp and Scr suggested that they arose by gene duplications in the more anterior members rather than the posterior AbdB.

Molecular cloning and expression pattern of an armadillo homologue from the mulberry silkworm Bombyx mori.

Armadillo/beta-catenin, encoded by the segment polarity gene armadillo functions as a downstream effector of canonical Wnt signals. The expression patterns of Bm wnt-1, -Ci and -engrailed, suggested the presence of Wnt-1 network in the middle silkglands of Bombyx mori. To test this, a homologue of armadillo from B. mori (Bm arm) was cloned by PCR using degenerate primers, designed based on the conserved regions of the protein, and characterized. The cloned region harboured one complete and two incomplete Armadillo (Arm) repeat motifs and was highly conserved across species. Genomic Southern analysis suggested Bm arm to be a multi-copy gene. The expression of Bm arm RNA was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The RNA was expressed uniformly in the embryos, whereas the Arm protein was localized in a segmentally reiterated striped pattern, in conformity with its predicted segment polarity nature. Bm arm was also expressed in the entire silkgland and the transition from third to fourth and fifth larval intermoults was accompanied by an increase in the transcript levels. However, the Arm protein was predominantly localized to the middle silkglands, especially the middle and posterior sub-compartments. The silkglands represented a novel expression domain for arm in Bombyx, and the results were consistent with the existence of a canonical Wnt network in the middle silkglands.

Expression profiling of homeobox genes in silk gland development in the mulberry silkworm Bombyx mori.

The homeobox (Hox) genes constitute an evolutionarily conserved family encoding transcription factors which play major roles in segmental identity and organ specification, across species. The expression patterns of three Hox genes, Antennapedia (Antp), Ultrabithorax (Ubx) and even-skipped (eve) were analyzed during silk gland development in Bombyx mori. Antp followed a middle silk gland (MSG) restricted pattern of expression, whereas Ubx was specifically expressed in the posterior silk gland (PSG) during embryonic and larval developmental stages. Eve protein, on the other hand, was expressed in both MSG and PSG. We have also identified and characterized a novel Pax-like mRNA that is expressed exclusively in the PSGs during embryonic and larval development. The expression of Antp, Ubx and Pax-like reached maximum levels in the fifth larval intermoult and no expression was detected during the intervening moults. The region-specific expression of certain Hox genes appears to be responsible for the specification of silk gland compartments, whereas other Hox genes may play a global role in controlling the expression of genes encoding silk proteins.

Spatio-temporal expression of wnt-1 during embryonic-, wing- and silkgland development in Bombyx mori.

A homologue of the segment polarity gene wnt-1 from Bombyx mori (Bmwnt-1) has been characterized. The segmentally reiterated pattern of Bmwnt-1 transcrip9t distribution in B. mori embryos suggested its segment polarity function. Maximal levels of Bmwnt-1 RNA during embryonic development were reached by stage 21A. In the larval stages, Bmwnt-1 was expressed in the fore- and hindwing discs, ovaries, testes and gut, reminiscent of the expression domains in Drosophila. Bmwnt-1 was expressed in the wing-margin area of both the fore- and hindwing discs. The pattern of wnt-1 expression in the hindwing discs was similar to that in the butterfly Precis coenia but subtle differences existed in forewing discs of the two species, which correlated well with the absence of proximal bands of pigmentation in the adult Bombyx wings. In addition, Bmwnt-1 was expressed in the silkglands and the expression was confined to the anterior sub-compartment within the middle silkglands throughout development from the embryonic to late larval stages. This domain of Bmwnt-1 expression overlapped with those of Cubitus interruptus (BmCi) and sericin-2 but excluded the Engrailed expression domain viz. the middle and posterior sub-compartments of middle silkglands. Bmwnt-1 expression was detected only during the intermoults and not in the moulting periods.

Cell cycle events during the development of the silk glands in the mulberry silkworm Bombyx mori.

Silk glands of the mulberry silkworm Bombyx mori are long and paired structures originating from the labial region and are anatomically and physiologically divided into three major compartments, the anterior, middle and posterior silk glands. The silk gland morphogenesis is complete by 8 days post egg laying. Extensive growth of silk glands during the larval stages is due to increase in tissue mass and not cell number. The cells in a completely formed silk gland pursue an endoreplicative cell cycle, and the genome undergoes multiple rounds of replication without mitosis or nuclear division. The expression patterns of cyclin B (mitotic cyclin) and cyclin E (G1 cyclin, essential for G1/S transition in both mitotic and endoreplicative cell cycles) in the course of silk gland development revealed that mitotic cell divisions take place only in the apex of the growing silk gland. However, the persistence of another mitotic focus in the middle silk gland even when the growing apex has moved well past this zone suggested the continued operation of mitosis for a while in this restricted region. The lack of cyclin B expression and abundance of cyclin E in the rest of the areas confirmed an alternation of the G1 and S phases of the cell cycle without an intervening mitotic phase. No expression of cyclin B was noticed anywhere in the silk glands after stage 25 of embryogenesis, indicating a complete switch over to the endomitotic mode of the cell cycle. The onset of expression of various genes encoding different silk proteins correlated with the onset of endomitotic events.

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages.

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus ( BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori.

A homologue of the segment polarity gene Cubitus interruptus from Bombyx mori, (BmCi) has been cloned and characterized. This region harbouring Zn(2+) finger motif is highly conserved across species. In B. mori, BmCi RNA expression was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The segmentally reiterated striped pattern of transcript distribution in stage 21C embryos was in conformity with its predicted segment polarity nature. BmCi was expressed in the fore- and hind-wing discs, ovaries, testes and gut during fifth larval intermolt, reminiscent of its expression domains in Drosophila. Besides, BmCi expression was seen in the anterior part of the middle silkglands in late embryonic stages, and this pattern was maintained during larval development. The transition from third to fourth and fifth larval intermolts was accompanied by an increase in the transcript levels in the middle silkglands. Our results demonstrate the presence of a novel expression domain for Ci in Bombyx.

The wings of Bombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report.

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm, Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development in B. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike in Drosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adult Drosophila wings. Partially excised wing discs did not show in situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early in B. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades in B. mori unlike evagination of only the pouch region as wing blades seen in Drosophila.

Differential expression of individual gene copies from within a tRNA multigene family in the mulberry silkworm Bombyx mori.

In mulberry silkworm Bombyx mori, tRNA1(Gly) constitutes a multigene family from which the individual members are transcribed at different levels in vitro in homologous nuclear extracts. We report here the quantification of functional transcripts of these gene copies in vivo in B. mori-derived BmN cells based on a suppression assay. The gene copies were converted to encode suppressor tRNAs and co-transfected into cell lines with reporter gene(s) harbouring one or more nonsense mutations and the reporter gene activity was quantified. Individual members of the gene family were transcribed to very high-, medium- and very low-levels, following the same pattern as in vitro. All these gene copies were maximally expressed in Bm cells as compared to other insect cell lines.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm.

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44kDa were detected in silk glands and BmN cells at comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells.

Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein.

The gene encoding the green fluorescent protein (gfp) under the control of the highly expressed Autographa californica nucleopolyhedrovirus (AcMNPV)-polyhedrin promoter has been introduced into the polyhedrin (polh) locus of Bombyx mori nucleopolyhedrovirus (BmNPV) by homologous recombination. The insect host larvae and the cultured cells infected with this recombinant virus (vBmGFP) showed high levels of expression of gfp. The larval tissues permissive to virus multiplication could be readily visualized using the tagged recombinant virus, thus providing a direct approach to study the progress of virus infection or its control in the animal host. The highly expressed recombinant protein, GFP, could be easily solubilized from fat bodies. Thus, the caterpillar-based expression could serve as an economic alternative method for the large-scale production of recombinant proteins, even when they are nonsecretory in nature. Further, if the recombinant vBmGFP is used as a parent in generating other recombinants, conversion of the fluorescent plaques to colorless plaques serves as an easy means for screening recombinants. Such a method is especially helpful for BmNPV-recombinant selections in the absence of the other simplified techniques as are available for the prototype baculovirus AcMNPV system.

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication.

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Expression of individual members of a tRNA(Gly)1 multigene family in vivo follows the same pattern as in vitro.

Individual members of a tRNA(Gly)1 multigene family from Bombyx mori are transcribed to different levels in vitro in homologous nuclear extracts but their transcription status in vivo is not known. Two sets of tRNA(Gly)1 belonging to the extreme groups of highly transcribed and barely transcribed copies have been examined for their expression patterns in vivo in B. mori-derived cell lines following transfection. We have developed a sensitive and reliable method for directly quantifying the transcription levels of transfecting tRNA genes without relying on the biological activity of the transcript. The strategy involved the insertion of synthetic oligodeoxyribonucleotide sequences into the coding region of the transfecting gene and monitoring the transcripts in an RNase protection assay using an antisense probe that clearly distinguished them from the endogenous tRNAs. The oligonucleotide insertion did not significantly affect the transcriptional status of the genes, even though the distance between the A and B boxes was enhanced by 10-15 nt. In vivo also the transcription of tRNA(Gly)1 reached very high levels, whereas the transcripts arising from tRNA(Gly)1-6:7 accounted for only 2-5% of the former, closely resembling the transcription patterns in vitro. These individual gene copies having identical coding sequences and consequently the same internal conserved regions, differed only in their flanking sequences which modulate their transcription levels.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae.

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at + 35 nt of the polh.

Transcriptional silencing of a tRNA1Gly copy from within a multigene family is modulated by distal cis elements.

Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.

Role of TATATAA element in the regulation of tRNA1Gly gene expression in Bombyx mori is position dependent.

Transcription of tRNA genes by RNA polymerase III is controlled by the internal conserved sequences within the coding region and the immediate upstream flanking sequences. A highly transcribed copy of glycyl tRNA gene tRNA1Gly-1 from Bombyx mori is down regulated by sequences located much farther upstream in the region -150 to -300 nucleotides (nt), with respect to the +1 nt of tRNA. The negative regulatory effect has been narrowed down to a sequence motif 'TATATAA', a perfect consensus recognised by the TATA binding protein, TBP. This sequence element, when brought closer to the transcription start point, on the other hand, exerts a positive effect by promoting transcription of the gene devoid of other cis regulatory elements. The identity of the nuclear protein interacting with this 'TATATAA' element to TBP has been established by antibody and mutagenesis studies. The 'TATATAA' element thus influences the transcription of tRNA genes positively or negatively in a position-dependent manner either by recruitment or sequestration of TBP from the transcription machinery.

Expression of human growth hormone in silkworm larvae through recombinant Bombyx mori nuclear polyhedrosis virus.

We have generated a recombinant Bombyx mori nuclear polyhedrosis virus, vBmhGH, harboring the full length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletely processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 x 10(4) U/mg. The recombinant hGH retained the immunological and biological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Cloning and characterization of mycobacteriophage I3 promoters.

Restriction fragments of mycobacteriophage I3 DNA capable of initiating transcription have been cloned into a promoter selection vector of Escherichia coli, and selected on the basis of development of resistance to chloramphenicol. The growth pattern of these 'promoter clones' on a concentration gradient of chloramphenicol and the biochemical assays of the chloramphenicol acetyl transferase have permitted the assessment of their relative promoter strengths. DNA sequence analysis revealed significant homology of these promoters to the -35 regions of the mycobacterial--and E. coli promoter consensus, but less so the -10 region. Based on the sequence of phage I3 promoters identified here and the reported sequences of mycobacterial promoters, a promoter consensus for mycobacteria has been generated.