Two Tor genes in the silkworm Bombyx mori.

Target of rapamycin (TOR), a member of the phosphatidylinositol kinase-related kinase family, plays a critical role in the regulation of growth, metabolism, development and survival, at both the cellular and the organismal levels. Two paralogous Tor genes, BmTor1 and BmTor2, were identified as a pair of inverted repeats in the genome of the silkworm Bombyx mori. The synteny of BmTor1 and CG8360 indicates that BmTor1 is the orthologue while BmTor2 is a duplicate. Analyses of the two BmTor genes at both the nucleotide and amino acid levels reveal that they are evolutionally and structurally conserved. The two BmTor genes had similar expression patterns of tissue distribution with highest levels in the nervous system, and nearly identical developmental change profiles with maximal levels during the 4(th) -larval-moulting and the larval-pupal transition stages. Furthermore, both BmTor genes were up-regulated by either starvation or the moulting hormone 20-hydroxyecdysone (20E), while BmTor2 was more sensitive to both treatments than BmTor1. For the first time, we have identified two copies of the Tor gene in a higher eukaryote, which are induced by starvation and 20E during the larval moulting and the larval-pupal transition stage.

Unusual behavior of the cytoplasmic transcript of hsr omega: an abundant, stress-inducible RNA that is translated but yields no detectable protein product.

Although a major site of transcription in heat shock, the Drosophila hsr omega gene does not encode any known heat shock proteins. Instead, studies of the hsr omega transcripts suggest that the RNA molecules, rather than encoded proteins, are the active products of this gene. The cytoplasmic RNA, omega 3, is spliced and polyadenylated and yet has only very small open reading frames (ORFs), and these are poorly conserved in different Drosophila species. Surprisingly, the work reported here leads us to conclude that one of the tiny ORFs in this RNA is translated. This ORF, designated ORF-omega, is notable in being the only ORF that shows sequence conservation in the three Drosophila species examined. However, translation of this ORF does not lead to detectable accumulation of the protein product. We suggest that ORF-omega may be an example of an unusual type of translated ORF. The act of translation itself may be important rather than the generation of a functional protein product. This nonproductive translation may play a role in regulation of cellular activities.

Multiple inducers of the Drosophila heat shock locus 93D (hsr omega): inducer-specific patterns of the three transcripts.

The Drosophila hsr omega locus produces one of the largest and most active heat shock puffs, yet it does not encode a heat shock protein. Instead, this locus produces a distinctive set of three transcripts, all from the same start site. The largest transcript, omega 1, is limited to the nucleus and appears to have a role there. A second nuclear transcript, omega 2, is produced by alternative termination and contains the sequence found in the 5' 20-25% of omega 1 (depending on the Drosophila species). The cytoplasmic transcript, omega 3, is produced by removal of a 700-bp intron from omega 2. All three hsr omega RNAs are produced constitutively and production is enhanced by heat shock. In addition to being a member of the set of heat shock puffs, the hsr omega puff is induced by agents that do not affect other heat shock loci, suggesting that hsr omega is more sensitive to environmental changes than other loci. We report here that agents that induce puffing of hsr omega loci in polytene nuclei also lead to an increase in hsr omega transcripts in diploid cells. We also show that the relative levels of omega 1 and omega 3 can be modulated independently by several agents. All drugs that inhibit translation, either initiation or elongation, stabilize the omega 3 transcript, which normally turns over within minutes in control cells. Drugs (such as benzamide and colchicine) that induce puffing of hsr omega, but not other heat shock loci, lead to large increases in omega 1. Although the constitutive level of omega 1 is relatively stable, the drug-induced excess is lost rapidly when the drug is withdrawn. The relative levels of hsr omega transcripts may reflect different states in cellular metabolism.

A putative farnesoic acid O-methyltransferase (FAMeT) orthologue in Drosophila melanogaster (CG10527): relationship to juvenile hormone biosynthesis?

Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. Farnesoic acid O-methyltransferase (FAMeT) is thought to be an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF). Previous evidence in other insects suggested that FAMeT is rate limiting and regulated by a neuropeptide family, the allatostatins. A full-length cDNA encoding a 296 amino acid putative FAMeT has been isolated. A recombinant (r)FAMeT was cloned, expressed and a specific antiserum generated. rFAMeT was assayed for enzymatic activity using a radiochemical assay. In this assay, no activity was detected either with rFAMeT alone or when added to a corpus allatum CA extract. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of Drosophila melanogaster ring gland. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on sesquiterpenoid biosynthesis. Drome AST appears to have a select effect on JH bisepoxide biosynthesis and not MF or JHIII. Additional analysis of MF, JHIII and JHB3 release in strains with a deficiency or decrease of FAMeT compared to wild type shows no significant decrease in MF, JHIII or JH bisepoxide synthesis. Deficiency strains that reduce the level of FAMeT showed reduced longevity relative to wildtype but this result may be due to other genetic influences.

Sequence evolution of the Drosophila heat shock locus hsr omega. I. The nonrepeated portion of the gene.

The locus which we now call hsr omega was originally identified as a large heat shock puff in polytene region 93D of Drosophila melanogaster. This puff was subsequently found to have several phenotypic characteristics that distinguished it from other heat shock puffs. These characteristics include induction by a number of agents that do not induce other puffs and the presence of large ribonucleotide particles that are not found elsewhere. Each Drosophila species has one heat shock puff with these phenotypes. In contrast to the strong sequence conservation seen in puffs coding for heat shock proteins, very little cross-hybridization is detected between hsr omega loci in different species, suggesting that the hsr omega loci are diverging rapidly. Comparative analyses of the hsr omega locus from D. melanogaster, D. pseudoobscura, and D. hydei show that, despite the sequence change, the structure of the locus and its transcripts has been conserved, along with a number of short regions of the sequence. The short regions of conservation offer some clues to the function of this unusual locus. In addition, these comparisons offer a view of the evolution of a gene whose primary function does not appear to be protein coding.

Stability of tandem repeats in the Drosophila melanogaster Hsr-omega nuclear RNA.

The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing > 5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is approximately 280 bp. Sequences of 19 1/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than approximately 5 kb nor more than approximately 16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA.

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.

Molecular cloning of the precursor cDNA for schistostatins, locust allatostatin-like peptides with myoinhibiting properties.

The cDNA encoding the precursor polypeptide for schistostatins, allatostatin-like peptides which have been shown to inhibit peristaltic movements of the lateral oviducts of Schistocerca gregaria, has been cloned and sequenced. Translation of this sequence reveals the presence of a pre-proschistostatin consisting of 283 amino acids. It contains ten different peptide sequences which are flanked by dibasic cleavage sites and C-terminal amidation signals. Eight of these peptides were identical to the schistostatins (or Scg-ASTs) that were previously purified from Schistocerca gregaria brain extracts. Two novel peptide sequences were discovered. One of these is the first AST-like peptide which has a C-terminal valine residue. Two peptides contain within their sequence an internal dibasic site which suggests a possible role for alternative processing and/or degradation. The schistostatin precursor differs from cockroach pre-proallatostatins in size, in sequence and in organization. It contains a lower number of peptides (10 versus 13 or 14) which are interrupted only once by an acidic spacer region (versus four in Diploptera punctata and Periplaneta americana). Northern analysis showed the presence of a 2.4 kb mRNA band in the locust central nervous system and midgut. This indicates that schistostatins, like other ASTs, are a good example of insect brain/gut peptides.

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

Characterization of the gene for leucomyosuppressin and its expression in the brain of the cockroach Diploptera punctata.

Using HPLC separation, radioimmunoassay, and subsequent bioassay, we have detected the presence of an active peptide, which co-elutes with the insect myoinhibitory peptide leuco-myosuppressin, in the brain of the cockroach Diploptera punctata. We have isolated a cDNA encoding the precursor for this peptide from cDNA libraries representing D. punctata brain RNA. The cDNA sequence contains an open reading frame that upon translation would result in a prepropolypeptide of 96 amino acids. Proteolytic cleavage of the predicted precursor could result in several peptides, including a 10 amino acid C-terminal peptide that would, upon modification of the NH2 and COOH-terminal amino acids, be identical to the insect FLRFamide, leucomyosuppressin. No other RFamide products are predicted to be processed from the precursor. Southern blot analysis indicates that the gene is present in the D. punctata genome in a single copy. Northern blot analysis shows that the gene is predominantly expressed as a 3.8 kb mRNA in cockroach brain. Study of the expression of the leucomyosuppressin gene in D. punctata brain, using in situ hybridization, indicates that expression occurs primarily in the pars intercerebralis of the protocerebrum, a region showing abundant FMRFamide-like immunoreactive neurosecretory cells. Immunohistochemistry and HPLC coupled to radioimmunoassay indicates that leucomyosuppressin represents a significant proportion of FMRFamide-related peptide production in the brain. However, HPLC analysis also indicates the presence of significant levels of other related peptides, demonstrating the presence of more than one FMRFamide-related gene in this insect.

Molecular characterization of a cDNA from Pseudaletia unipuncta encoding the Manduca sexta allatostatin peptide (Mas-AST).

A 15-residue neuropeptide, Manduca sexta allatostatin (Mas-AST), strongly inhibits juvenile hormone (JH) biosynthesis in vitro by corpora allata (CA) from Manduca fifth-stadium larvae and adult females as well as Helicoverpa zea adult females (Kramer et al., 1991 Proc. Natl. Acad. Sci (USA) 88, 9458-9462). In contrast, this study found that 1.0 microM Mas-AST has no JH biosynthesis inhibitory activity in Pseudaletia unipuncta sixth instar larvae or newly-emerged (day 0) adults but inhibited CA of 5-day-old adult females by 60%. From a P. unipuncta brain cDNA library, was isolated a cDNA that encodes a 125 amino acid polypeptide containing the Mas-AST sequence. Within the precursor, Mas-AST is situated at the carboxy terminus and is flanked by different dibasic proteolytic cleavage signals. The Pseudaletia gene specifying the Mas-AST peptide is present as a single copy per haploid genome. Expression of this gene was low in Pseudaletia sixth instar larvae, prepupae and early pupae but was relatively high in late pupae, and day 1 and 3 adults of both sexes. In day 5 adults, the relative transcript level appears to be maintained in females but declines in males. This pattern of Mas-AST expression does not correlate well with the profile of JH biosynthesis in Pseudaletia, which increases during the first 5 days of adult life, suggesting additional or alternative functions for this peptide.

Expression of allatostatin in the oviducts of the cockroach Diploptera punctata.

The cockroach allatostatins (Y/FXFGL/Ia ASTs) are a ubiquitous family of peptides in the invertebrates. They affect numerous physiological processes including the inhibition of juvenile hormone III (JH) biosynthesis, inhibition of muscle contraction, inhibition of ovarian ecdysteroid biosynthesis and inhibition of vitellogenin (Vg) release from the fat body. We have developed and optimized a sensitive and specific quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Diploptera punctata AST (Dippu-AST) expression. Using this technique we show that tissues of both lateral and common oviducts and the ovary express message for Dippu-AST. Moreover, the pattern of expression observed in the oviducts and ovary is strikingly similar with significant changes occurring during the reproductive cycle. Specifically, expression of AST is drastically reduced during the time of maximal vitellogenin (Vg) uptake, with higher levels measured prior to and following vitellogenesis. Furthermore, using immunocytochemistry, we have shown Dippu-AST-like-immunoreactivity in the terminal abdominal ganglion, as well as in ventral nerve 7, some branches of which innervate the common and lateral oviducts with other branches innervating the bursa copulatrix and brood sac of mated female D. punctata. The pattern of Dippu-AST expression and immunocytochemical staining suggests that ASTs function, in part, to regulate the cycle of vitellogenesis in mated female D. punctata.

Molecular characterization of a cDNA from the true armyworm Pseudaletia unipuncta encoding Manduca sexta allatotropin peptide(1).

Allatotropin (AT) is an insect neuropeptide isolated from the tobacco hornworm, Manduca sexta, stimulates juvenile hormone (JH) biosynthesis by the corpora allata. A cDNA isolated from the true armyworm, Pseudaletia unipuncta, encodes a 135 amino acid AT precursor peptide which contains the AT peptide, with processing sites necessary for its endoproteolytic cleavage and amidation, plus two additional peptides of unknown function. The encoded AT peptide is identical to that isolated from M. sexta and Agrius convolvuli. Southern blot analysis indicated that AT is a single copy gene per haploid genome and is present in two allelic forms. A single transcript of approximately 1.5 kilobases was detected by northern blot analysis. The expression of the AT gene was analyzed during development from sixth instar larvae to five day-old moths. Initial expression was observed in late pupae and this expression was maintained throughout the adult stages in both sexes. In one day-old moths, expression was at its lowest level of the stages that express AT mRNA but levels increased in day 3 and day 5 adults. This pattern of AT expression in adult P. unipuncta moths mirrors that of JH biosynthesis and supports the notion that AT may act in the adult stages. Immunohistochemistry and in situ hybridization revealed that AT expression was localized to numerous structures of the nervous system, suggesting that AT may have functions distinct from regulation of JH biosynthesis.

The regulation of juvenile hormone production in arthropods. Functional and evolutionary perspectives.

Although sesquiterpenoids are probably the ancestral regulators of reproduction and secondarily of metamorphosis in arthropods, our discussion suggests that the neuropeptides that regulate the biosynthesis of these compounds have arisen on several distinct occasions. These peptides probably occurred originally as regulators of other physiological processes and were subsequently co-opted for the regulation of sesquiterpenoid biosynthesis, perhaps first in adult forms and thereafter in larval forms. The evolution of peptides to assume additional physiological functions probably occurred as a result of gene duplication, both at the peptide level and at the receptor level. There are likely to be numerous regulators of sesquiterpenoid biosynthesis in both Insecta and Crustacea, and investigations to date have only begun to reveal the host of peptide families involved in the regulation of juvenile hormone-related biosynthesis across the arthropods.

Allatostatins: a growing family of neuropeptides with structural and functional diversity.

The high degree of conservation of the core sequence of the "cockroach-types" of AST and their widespread distribution suggest that they should be considered a ubiquitous family of peptides within the invertebrates, regulating a range of important physiological processes. These functional processes, by either neural or humoral routes of action, include the inhibition of endocrine function, interneuronal functions, neuromodulatory roles, myotropic and myoendocrine roles, and direct action on biosynthetic pathways. The myomodulatory function appears to be conserved through evolutionary time, whereas the JH inhibitory activity appears to be confined to specific orders. This suggests that the myomodulatory role was the more ancestral of these two particular functions. Certainly, further purification and gene cloning as a means to precursor identification and functional analysis will be a prerequisite to understanding the diverse functions of this peptide family.

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish.

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta.

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.

Molecular characterization of the inhibitory myotropic peptide leucomyosuppressin.

The myoinhibitory peptide leucomyosuppressin (LMS) (pQDVDHVFLRFamide) has been identified and characterized at the molecular level in the cockroach Diploptera punctata through analysis of the organization of both brain cDNA and genomic DNA. Processing of the precursor predicted from DNA sequence would release a single LMS peptide. The organization of the precursor appears to be conserved in other insects and may reflect a functional organization for this subfamily of extended FLRFamides. The expression of the LMS gene appears in numerous cells of the pars-intercerebralis of the cockroach protocerebellum as well as in numerous endocrine cells of the midgut.

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata.

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta.

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.