Genome Organization And Expression Of Selected Potexviruses

We have used in vitro translation to analyze the protein products produced from the genomic RNAs of members of the potexvirus group, namely, papaya mosaic virus (PMV), clover yellow mosaic virus (CYMV), viola mottle virus (VMV), barrel cactus virus (BCV), and foxtail mosaic virus (FTMV). Polyacrylamide gel electrophoresis of polypeptides made in vitro from the genomic RNAs of these viruses indicated that only PMV and CYMV RNA directed the synthesis of polypeptides which have the same molecular weight and antigenic determinants as their respective coat proteins. All viral RNAs tested in vitro produced high molecular weight polypeptides. Peptide mapping of these high molecular weight polypeptides for PMV and CYMV has indicated that they are related to each other but not to their respective coat proteins. The ability of partially encapsidated PMV and CYMV ribonucleoprotein particles to direct protein synthesis has also been assessed. As the extent of encapsidation increased, the relative synthesis of the high molecular weight products decreased markedly. In contrast, the synthesis of the in vitro coat protein not only persisted but increased relative to the untreated RNA until a substantial fraction of the RNA is encapsidated. In view of the polarity of assembly in vitro this finding indicated that the cistron for PMV and CYMV coat protein is localized towards the 3\u27 end of the RNA. We have also examined the relationship of these viral genomes at the level of nucleotide sequence. Using RNA-cDNA hybridization we found that no homology exists between all members of the potexvirus group tested, not with unrelated members. As a further means of characterizing the genomes of these viruses, we have constructed double-stranded cDNA copies of PMV and CYMV RNA and have subsequently cloned them in E. coli using the plasmid pBR322 as a vector

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

The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

Allatostatins (ASTs) are multifunctional neuropeptides that generally act in an inhibitory fashion. ASTs were identified as inhibitors of juvenile hormone biosynthesis. Juvenile hormone regulates insect metamorphosis, reproduction, food intake, growth, and development. Drosophila melanogaster RNAi lines of PheGlyLeu-amide-ASTs (FGLa/ASTs) and their cognate receptor, Dar-1, were used to characterize roles these neuropeptides and their respective receptor may play in behavior and physiology. Dar-1 and FGLa/AST RNAi lines showed a significant reduction in larval foraging in the presence of food. The larval foraging defect is not observed in the absence of food. These RNAi lines have decreased for transcript levels which encodes cGMP- dependent protein kinase. A reduction in the for transcript is known to be associated with a naturally occuring allelic variation that creates a sitter phenotype in contrast to the rover phenotype which is caused by a for allele associated with increased for activity. The sitting phenotype of FGLa/AST and Dar-1 RNAi lines is similar to the phenotype of a deletion mutant of an AST/galanin-like receptor (NPR-9) in Caenorhabditis elegans. Associated with the foraging defect in C. elegans npr-9 mutants is accumulation of intestinal lipid. Lipid accumulation was not a phenotype associated with the FGLa/AST and Dar-1 RNAi lines

Methyl Farnesoate Plays a Dual Role in Regulating \u3cem\u3eDrosophila\u3c/em\u3e Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce

Methyl Farnesoate Plays a Dual Role in Regulating \u3cem\u3eDrosophila\u3c/em\u3e Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce

Chromosomal-level reference genome of the moth Heortia vitessoides (Lepidoptera: Crambidae), a major pest of agarwood-producing trees

This is the final version. Available on open access from Elsevier via the DOI in this recordAvailability of data and materials: The final chromosome assembly was submitted to NCBI Assembly under accession number JACJUM000000000 in NCBI. The raw reads generated in this study have been deposited to the NCBI database under the BioProject accessions: PRJNA654728, the genome annotation files were deposited in the Figshare (https://doi.org/10.6084/m9.figshare.19633668). The microRNA sequences of known species were obtained from both miRbase [42] and MirGeneDB [21].The moth Heortia vitessoides Moore (Lepidoptera: Crambidae) is a major pest of ecologically, commercially and culturally important agarwood-producing trees in the genus Aquilaria. In particular, H. vitessoides is one of the most destructive defoliating pests of the incense tree Aquilaria sinesis, which produces a valuable fragrant wood used as incense and in traditional Chinese medicine [33]. Nevertheless, a genomic resource for H. vitessoides is lacking. Here, we present a chromosomal-level assembly for H. vitessoides, consisting of a 517 megabase (Mb) genome assembly with high physical contiguity (scaffold N50 of 18.2 Mb) and high completeness (97.9% complete BUSCO score). To aid gene annotation, 8 messenger RNA transcriptomes from different developmental stages were generated, and a total of 16,421 gene models were predicted. Expansion of gene families involved in xenobiotic metabolism and development were detected, including duplications of cytosolic sulfotransferase (SULT) genes shared among lepidopterans. In addition, small RNA sequencing of 5 developmental stages of H. vitessoides facilitated the identification of 85 lepidopteran conserved microRNAs, 94 lineage-specific microRNAs, as well as several microRNA clusters. A large proportion of the H. vitessoides genome consists of repeats, with a 29.12% total genomic contribution from transposable elements, of which long interspersed nuclear elements (LINEs) are the dominant component (17.41%). A sharp decrease in the genome-wide percentage of LINEs with lower levels of genetic distance to family consensus sequences suggests that LINE activity has peaked in H. vitessoides. In contrast, opposing patterns suggest a substantial recent increase in DNA and LTR element activity. Together with annotations of essential sesquiterpenoid hormonal pathways, neuropeptides, microRNAs and transposable elements, the high-quality genomic and transcriptomic resources we provide for the economically important moth H. vitessoides provide a platform for the development of genomic approaches to pest management, and contribute to addressing fundamental research questions in Lepidoptera.Hong Kong Research Grant Council Collaborative Research FundGeneral Research FundChinese University of Hong Kon

Formation of insoluble and colloidally dispersed tannic acid complexes in the midgut fluid of Manduca sexta (Lepidoptera: Sphingidae): An explanation for the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae

Magnesium and calcium ions, in concentrations comparable to those reported in the midgut fluids of lepidopteran larvae, bring about the precipitation of most of the tannic acid present in simple solutions buffered at pH 8.0 and 10.0, but not at pH 6.5. In contrast, when tannic acid is added to Manduca sexta midgut fluid, less than 31% of the tannic acid added to the gut fluid is converted to a form that can be centrifuged into a pellet. The rest remains in the supernatant solution in the form of a colloidal suspension. Very little of the tannic acid, if any, remains in true solution. We suggest that the tannic acid-containing phase that is produced when tannic acid is added to midgut fluid is a complex multi-molecular aggregate of indefinite chemical composition, incorporating varying amounts of tannic acid, surface-active phospholipids, proteins, and polyvalent metal ions. On the basis of this study, we further suggest that the failure of tannins to diffuse across the peritrophic envelopes of lepidopteran larvae is a result of the capacity of the peritrophic envelope to act as a physical barrier to insoluble and colloidally dispersed particles, not the presence of substances in the matrix that strongly adsorb polyphenols or the presence of an extensive network of fixed anionic sites in the matrix that acts as an electrostatic barrier to the passage of polyphenolate anions. Arch. Insect Biochem. Physiol. 39:109–117, 1998.© 1998 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34924/1/3_ftp.pd

Human sat III and Drosophila hsrω transcripts: a common paradigm for regulation of nuclear RNA processing in stressed cells

Exposure of cells to stressful conditions elicits a highly conserved defense mechanism termed the heat shock response, resulting in the production of specialized proteins which protect the cells against the deleterious effects of stress. The heat shock response involves not only a widespread inhibition of the ongoing transcription and activation of heat shock genes, but also important changes in post-transcriptional processing. In particular, a blockade in splicing and other post-transcriptional processing has been described following stress in different organisms, together with an altered spatial distribution of the proteins involved in these activities. However, the specific mechanisms that regulate these activities under conditions of stress are little understood. Non-coding RNA molecules are increasingly known to be involved in the regulation of various activities in the cell, ranging from chromatin structure to splicing and RNA degradation. In this review, we consider two non-coding RNAs, the hsrω transcripts in Drosophila and the sat III transcripts in human cells, that seem to be involved in the dynamics of RNA-processing factors in normal and/or stressed cells, and thus provide new paradigms for understanding transcriptional and post-transcriptional regulations in normal and stressed cells

MicroRNA clusters integrate evolutionary constraints on expression and target affinities : the miR-6/5/4/286/3/309 cluster in Drosophila

This research was supported by the Hong Kong Research Grant Council GRF Grant (14103516), The Chinese University of Hong Kong Direct Grant (4053248), and TUYF Charitable Trust (6903957) (JHLH).A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a microRNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considered together, the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs.PostprintPeer reviewe