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.

Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L.

Changes in the translational machinery components of the Bombyx mori posterior silk gland were analysed during starvation and refeeding and compared to the regularly fed larvae. During starvation, tRNA and ribosomal RNA synthesis are stopped. The amounts of different RNA classes and of the different tRNA species slow down at the same rate. Thus various tRNA show similar half-lifes and the preexisting tRNA adaptation to fibroin mRNA translation persists during starvation. Similarly, the tRNA/rRNA ratio is constant during starvation and refeeding (12 tRNA molecules for one ribosome) as in silk glands of control animals. Aminoacyl-tRNA synthetases and tRNA charging levels are decreased during starvation. The maximal tRNA charging level obtained during maximal protein synthesis in control animals is regained after 24 h refeeding of starved larvae. Changes observed in the free amino acid pool are not similar from one amino acid to another and levels reached after starvation do not differ strongly from the controls. Our results suggest that the production of translation apparatus components is coordinated and adjusted to the protein synthesis activity. Whether this coordination occurs in the silk gland is discussed on the basis of the "metabolic regulation", primarily described in prokaryotes and Yeast. Transfer RNA charging levels seem to play a key role in the process of regulation and could be implicated in the mechanism of tRNA adaptation if this phenomenon results as expected from a transcriptional control.

Does quantitative tRNA adaptation to codon content in mRNA optimize the ribosomal translation efficiency? Proposal for a translation system model.

Neither a dynamic nor an energetic approach of the translation process has taken into account that intracellular levels of iso-tRNA species are adapted or adjusted to the codon frequency of mRNA being decoded (Bombyx mori silk gland, rabbit reticulocyte). A critical study of available experimental data suggests that the average elongation rate of a protein is maximized in the presence of an adapted tRNA population, usually an homologous tRNA. In addition, the amount of synthesized protein parallels that of corresponding mRNA. Other evidences--including in vitro and in vivo elongation assays with fibroin mRNA--show that individual elongation rates are not uniform. Pauses occur at certain sites of the mRNA chain. The relative lifetime of these pauses depends on the tRNA pool used. Finally, it appears that translation accuracy also depends on the balanced tRNA population. We propose to explain these different effects by using a codon-anticodon recognition model, called "trial and error system" based on a stochastic processing of the ribosome. Accordingly, various acylated tRNA species which surround a ribosome randomly encounter the receptor A site. Every trapped tRNA species is tested for a proper pairing with the codon to be recognized at the level of a comparator or discriminator function. If the pairing is correct, transpeptidation becomes irreversible. If not, the aminoacyl-tRNA is rejected and another randomly trapped tRNA is processed in turn. Mathematical analysis of this model shows that the mean number of trials used for translating the whole sequence of a mRNA is minimized when the proportion of different iso-tRNA species is correlated with the square root of codon frequency. Quantitations of reticulocyte tRNA support such a parabolic relation. Our translation system model brings some light into the role of tRNA adaptation for optimizing translation efficiency, i.e. maximizing both speed and accuracy. Some consequences of the model are discussed.

Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system.

Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.

The biolistic method as a tool for testing the differential activity of putative silkmoth chorion gene promoters.

Bombyx mori unpaired early chorion gene copies 6F6.1,.2 and.3 are exceptions to the typical organization and distribution pattern of known early ErA/ErB, middle A/B and late HcA/HcB divergently transcribed gene pairs. Contrary to such pairs, the boundaries of the 6F6 regulatory sequences are not easily defined; moreover, they share common sequence elements with the regulatory sequences of middle and late genes. In order to perform a functional study of the tissue and temporal specificity of the 6F6 putative promoter region, we decided to apply biolistics. In the present work, use of a region from the 6F6.2 5' untranslated sequence, spanning nucleotides -138 to the cap site, gave an expected expression pattern of a lacZ reporter gene. Temporal specificity was further verified by control experiments using the cloned intergenic sequence of the late gene pair HcA/B.12, which resulted in lacZ expression in late choriogenic follicles. At present, despite the recent successful germinal transgenesis of Bombyx mori, the biolistic transient expression system seems to be the most rapid technique to pursue the functional study of the promoter region of early chorion genes, including the three unconventional early 6F6 genes.

3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards.

Transgenesis was recently achieved in Bombyx mori L., but it has proved difficult and time-consuming to screen the numerous progeny to identify the transgenic individuals. As the 3xP3-EGFP marker has been shown to be a suitable universal marker for transgenic insects (Nature 402 (1999) 370), we evaluated its use for embryonic-stage screening for B. mori L. germline transformation. Using the piggyBac-derived vector pBac[3xP3-EGFPaf], we were able to isolate four transgenic individuals from about 120,000 embryos (560 broods). The screening was straightforward due to EGFP production in the G1 embryonic stemmata, which was visible through the translucent egg chorion. EGFP was produced in the stemmata and central and peripheral nervous systems from the fifth day of embryonic development. It persisted at high levels in the stemmata throughout the larval stage, and was also present in the compound eyes and nervous tissues of the pupae and the compound eyes of the moths.

PREPARATION AND PROPERTIES OF NUCLEI FROM THE POSTERIOR SILK GLANDS OF THE SILKWORM BOMBYX MORI.

An original method to isolate nuclei from the posterior part of the silk glands has been developed. After a collagenase and Triton X-100 treatment, silk glands were filtered through a steel sieve. This step, which is the most efficient one for the purification, is followed by several washings. The preparation of nuclei is fairly pure, RNA: DNA ratio being 0.3 at the end of the whole procedure, and the final DNA recovery quite satisfactory (40-60%). Although chromatin of the purified nuclei is unusually condensed, and in spite of RNase activity, RNA transcription measured in vitro is quantitatively significant (0.01 % of the total DNA). This transcription, resulting from the activity of endogenous RNA polymerases, reaches a maximum when nuclei were extracted from animals on the 4th day of the fifth instar. Differences with results obtained in vivo are discussed.

A helium burst biolistic device adapted to penetrate fragile insect tissues.

To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast. The efficiency of the gene gun bombardment was reinforced by the addition of a focusing nozzle. The reduced blast effect allowed us to carry out high-pressure shootings to small organs with improved penetration. This system allowed potentially all the internal embryonic tissues to be transfected with optimal survival rates. The new module was effective on tissues that are difficult to transfect, such as the epithelial wing disk that is covered by a peripodial membrane, and the ovarian follicle cells that lie under the ovariole cell membrane. The new macrocarrier allowed both an aqueous delivery of particles and an ethanolic dry delivery. No significant differences were noted between these two modes of delivery. The major improvement is the possibility of high pressure shooting correlated with appreciable penetration and a weak blast effect.

Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms.

RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.

[tRNA adaptation and the optimization of translation]. / Adaptation des tRNA et optimisation de la traduction.

The intracellular level of each tRNA species is adjusted to the codon frequency of the mRNA being decoded. This was first observed in such highly differentiated cells as the silk gland of Bombyx mori, which produces fibroin and sericin, and the rabbit reticulocyte. tRNA adaptation also occurs in other cell types from E. coli to mammalian cells. Regardless of the mechanism regulating tRNA biosynthesis, we believe that tRNA adaptation is the basic step optimizing chain elongation at the ribosomal level. We propose the system of trial and error as a working model for the ribosome. This model clarifies the correlations between iso-accepting tRNA levels and codon frequencies, as well as the effect of tRNA pool balance on mean elongation rate and non-uniform individual elongation rate (depending on whether codons are rare or abundant) for fibroin mRNA translated in a reticulocyte cell-free system.

Stimulation of adenosine 3':5'-cyclic monophosphate accumulation in anterior pituitary gland in vitro by synthetic luteinizing hormone-releasing hormone.

A near-maximal dose (20 ng/ml) of synthetic luteinizing hormone(LH)-releasing hormone/follicle-stimulating hormone(FSH)-releasing hormone added to incubated anterior pituitary tissue of male rats leads to concomitant increases of intracellular concentrations of adenosine 3':5'-monophosphate and of release of both LH and FSH. The stimulatory effect of LH-releasing hormone/FSH-releasing hormone is observed after a lag period of about 90 min and is progressive at later time intervals; a 3-fold stimulation of cAMP accumulation over control is seen after 210 min of incubation. Half-maximal stimulation of cAMP accumulation is observed between 0.1 and 1.0 ng/ml (0.1-1 nM) of LH-releasing hormone/FSH-releasing hormone. In the presence of 10 mM theophylline, the stimulatory effect of LH-releasing hormone/FSH-releasing hormone on cAMP accumulation is similar to that observed in the absence of the inhibitor of cyclic nucleotide phosphodiesterase, indicating that the releasing hormone exerts its effect by specific activation of adenylate cyclase in LH- and FSH-secreting cells rather than by inhibition of cyclic nucleotide phosphodiesterase. Since the release of growth hormone, thyrotropin, prolactin, and adrenocorticotropic hormone is not affected by LH-releasing hormone/FSH-releasing hormone, and since cAMP stimulates the release of all six adenohypophyseal hormones. the observed changes of cAMP concentrations indicate specific stimulation of adenylate cyclase activity in LH-and FSH-secreting cells of the adenohypophysis.