The biosynthesis of transfer RNA in insects. II. Isolation of transfer RNA precursors from the posterior silk gland of Bombyx mori.
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The occurrence of precursors to tRNA in the post-polysomal fraction of the posterior silk gland of Bombyx mori was demonstrated by pulse-chase labeling and DNA-RNA hybridization competition experiments. These precursors had molecular sizes ranging from 4S to 5S on polyacrylamide gel electrophoresis. Analysis of the incorporation of the methyl group from [methyl-14C]methionine revealed that a radioactive peak on polyacrylamide gel appeared in the 4.5S region during brief labeling. This suggested that some methylation occurred at the 4.5S precursor step. (+info)
Comparison of Bombyx mori and Helicoverpa armigera cytoplasmic actin genes provides clues to the evolution of actin genes in insects.
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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. (+info)
Active transport of calcium across the isolated midgut of Hyalophora cecropia.
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1. The net flux of 45Ca from lumen to blood side across the isolated and short-circuited Cecropia midgut was 1-9 +/- 0-2 muequiv. cm-2h-1 in 8 mM Ca and the flux ratio was as high as 56 to 1. 2. The calcium influx was depressed by anoxia; 73% after 30 min. 3. The kinetics of Ca transport were anomalous; the apparent Km varied with Ca concentration from less than 0-2 to greater than 5-6 mM Ca and the apparent Vmax varied from less than 1-3 to greater than 3-3 muequiv. cm-2h-1. 4. The calcium influx showed a delay before the tracer steady state was attained, indicating the existence in the transport route of a calcium pool equivalent to 5-7 muequiv/g. wet weight of midgut tissue. 5 High calcium (16 mM) depressed the short-circuit current and potassium transport from blood to lumen side across the midgut. 6. Calcium depressed magnesium transport, from lumen to blood side across the midgut, and magnesium depressed the calcium transport. 7. Ca transport by the midgut does not regulate the Ca level in the haemolymph in vivo; it merely aids the diffusion of calcium down its electrochemical gradient. However, Ca transport may assist the uptake of the nutrients from the midgut contents. (+info)
Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.
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Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid. (+info)
Gene targeting in the silkworm by use of a baculovirus.
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The Bombyx mori fibroin light (L)-chain gene was cloned and the green fluorescent protein (GFP) gene inserted into exon 7. The chimeric L-chain-GFP gene was used to replace the polyhedrin gene of Autographa californica nucleopolyhedrovirus (AcNPV). This recombinant virus was used to target the L-chain-GFP gene to the L-chain region of the silkworm genome. Female moths were infected with the recombinant virus and then mated with normal male moths. Genomic DNA from their progenies was screened for the desired targeting event. This analysis showed that the chimeric gene had integrated into the L-chain gene on the genome by homologous recombination and was stably transmitted through generations. The chimeric gene was expressed in the posterior silk gland, and the gene product was spun into the cocoon layer. (+info)
Characterization of the 25K FP gene of the baculovirus Bombyx mori nucleopolyhedrovirus: implications for post-mortem host degradation.
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Mutagenesis experiments on the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2'-deoxyuridine generated five mutants with a 'few polyhedra' (FP) phenotype. Sequence analysis of the 25K gene homologue of the BmNPV FP mutants revealed nucleotide substitutions in the coding region. Rescue experiments indicated that the FP phenotype of the BmNPV mutants resulted from mutations in the 25K coding region. Effects of infection by these FP mutants were analysed following injection of the viruses into silkworm (B. mori) larvae. Compared to infection with wild-type virus, infection with each FP mutant resulted in reduced host degradation (liquefaction). The degree to which liquefaction was blocked corresponded to the degree of functional disruption of the 25K gene product and to the extent to which polyhedron production was reduced. Electron microscopy revealed that (1) polyhedron production was reduced, (2) very few virions were occluded and those that were lacked envelopes, and (3) the basal lamina of fat-body tissue was not destroyed by infection and accumulations of virions occurred along the membrane. Typical NPV-induced liquefaction was observed following infection with a polyhedrin deletion mutant, indicating that host degradation was not related to polyhedron production. These results suggest that (1) the 25K gene product is involved in the host degradation process caused by virus infection and (2) the FP phenotype is an indirect result of disruption of the 25K gene; activation or suppression of a specific host or viral gene related to tissue degradation and polyhedron formation may be involved. (+info)
Studies on silk fibroin of Bombyx mori. I. Fractionation of fibroin prepared from the posterior silk gland.
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1. Fractionation of fibroin prepared from the posterior silk glands of Bombyx mori was carried out. After carboxymethylation of the fibroin, it was fractionated by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-cellulose column chromatography. 2. The fibroin was composed of at least two protein groups of large molecular size and three or four components of small molecular size, and, in addition, a mixture of proteins ranging in size from about 25,000 to more than 100,000 daltons with almost the same amino acid compositions. 3. The latter proteins contained about 48% glycine, 32% alanine, 11% serine, 4.5% tyrosine, 2% valine, and other minor amino acids. The sum of these main five amino acids accounts for more than 97% of the total amino acid residues of the proteins. 4. The present results indicate major heterogeneity in the molecular size of posterior silk gland fibroin, and, in addition, suggest the possibility of repeating sequences with relatively simple amino acid compositions in major peptide chains of fibroin. (+info)
A TATA element is required for tRNA promoter activity and confers TATA-binding protein responsiveness in Drosophila Schneider-2 cells.
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In contrast to yeast and mammalian systems, which depend principally on internal promoter elements for tRNA gene transcription, insect systems require additional upstream sequences. To understand the function of the upstream sequences, we have asked whether the Bombyx mori tRNACAla and tRNASGAla genes, which are absolutely dependent on these sequences in vitro, also require them for transcription in vivo. We introduced wild-type and mutant versions of the Bombyx tRNAAla genes into Drosophila Schneider-2 cells and found that the tRNACAla gene is efficiently transcribed and that its transcription depends strongly on the distal segment of its upstream promoter. In contrast, the tRNASGAla gene is inefficiently transcribed, and this inefficiency results from lack of a specific sequence within the distal tRNACAla upstream promoter. This sequence, 5'-TTTATAT-3', is sufficient to increase the activity of the tRNASGAla promoter to that of the tRNACAla promoter. Moreover, promoters containing the 5'-TTTATAT-3' element are stimulated by increased levels of cellular TATA-binding protein. Together these results indicate that, in insect cells, a TATA-like element is specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of tRNA genes. (+info)