The Mitochondrial Genome of Paramphistomum cervi (Digenea), the First Representative for the Family Paramphistomidae

We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.     

Removal of DNA from RNA by chromatography on acetylated N-[N'-(m-dihydroxylborylphenyl)succinamyl]aminoethyl cellulose.

RNA synthesized in vitro can be separated from contaminating DNA by chromatography on acetylated N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethyl cellulose. The recovery of RNA is greater than 95%, and essentially all the DNA is removed. This procedure is extremely useful in studies in which DNA contamination of RNA interferes with subsequent analysis.     

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.     

Is Thermosensing Property of RNA Thermometers Unique?

A large number of studies have been dedicated to identify the structural and sequence based features of RNA thermometers, mRNAs that regulate their translation initiation rate with temperature. It has been shown that the melting of the ribosome-binding site (RBS) plays a prominent role in this thermosensing process. However, little is known as to how widespread this melting phenomenon is as earlier studies on the subject have worked with a small sample of known RNA thermometers. We have developed a novel method of studying the melting of RNAs with temperature by computationally sampling the distribution of the RNA structures at various temperatures using the RNA folding software Vienna. In this study, we compared the thermosensing property of 100 randomly selected mRNAs and three well known thermometers - rpoH, ibpA and agsA sequences from E. coli. We also compared the rpoH sequences from 81 mesophilic proteobacteria. Although both rpoH and ibpA show a higher rate of melting at their RBS compared with the mean of non-thermometers, contrary to our expectations these higher rates are not significant. Surprisingly, we also do not find any significant differences between rpoH thermometers from other -proteobacteria and E. coli non-thermometers.     

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.     

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.     

Magnesium-binding studies reveal fundamental differences between closely related RNA triphosphatases

The Chlorella virus RNA triphosphatase (cvRTPase) is involved in the formation of the RNA cap structure found at the 5′-end of the viral mRNAs and requires magnesium ions to mediate its catalytic activity. To extend our studies on the role of metal ions in phosphohydrolysis, we have used a combination of fluorescence spectroscopy, circular dichroism, denaturation studies and thermodynamic analyses to monitor the binding of magnesium ions to the cvRTPase. Using these techniques, the thermodynamic forces responsible for the interaction of metal ions with an RNA triphosphatase were also evaluated for the first time. Our thermodynamic analyses indicate that the initial association of magnesium with the cvRTPase is dominated by a favorable entropic effect and is accompanied by the release of eight water molecules from the enzyme. Moreover, both fluorescence spectroscopy and circular dichroism assays indicated that minor conformational changes were occurring upon magnesium binding. Mutational studies were also performed and confirmed the importance of three specific glutamate residues located in the active site of the enzyme for the binding of magnesium ions. Finally, in contrast to the yeast RNA triphosphatase, we demonstrate that the binding of magnesium ions to the cvRTPase does not lead to the stabilization of the ground state binding of the RNA substrate. Based on the results of the present study, we hypothesize that the binding of magnesium ions induces local conformational perturbations in the active site residues that ultimately positions the lateral chains of critical amino acids involved in catalysis. Our results highlight fundamental differences in the role of magnesium ions in the phosphohydrolase reactions catalyzed by the cvRTPase and the closely related yeast RNA triphosphatase.     

Incorporation of a kinin, N, 6-benzyladenine into soluble RNA

Kinin requiring tobacco and soybean tissues incubated on a medium containing N,6-benzyladenine-8-C¹⁴ incorporated C¹⁴ into several RNA components including adenylic and guanylic acids. About 15% of the label taken up by the tissues appeared in RNA while the remainder was distributed among several metabolites in the soluble, nonpolynucleotide fraction. Tissue grown on a kinin labeled in the side chain (N,6-benzyladenine-benzyl-C¹⁴) also incorporated a small, but nevertheless repeatable, amount of radioactivity into minor RNA components.

Ultracentrifugation studies and methylated albumin chromatography indicated that the bulk of the label from benzyladenine-benzyl-C¹⁴ is in soluble RNA. Approximately 50% of the C¹⁴ in soluble RNA is in a component which has chromatographic properties like that of benzyladenine.

It is suggested that the biological action of the kinins may hinge on their providing substituted bases in RNA in tissues which through differentiation no longer synthesize RNA-methylating enzymes. As an alternative it was hypothesized that a small amount of benzyladenine was incorporated into a m-RNA, acting there as a derepressing agent, perhaps by preventing its normal repressing function.

     

The Synthesis of 5s RNA and Its Relationship to 18s and 28s Ribosomal RNA in the Bobbed Mutants of DROSOPHILA MELANOGASTER

Ribosomes contain one molecule each of 5S, 18S and 28S RNA. In Drosophila melanogaster although the genes for 18S+28S are physically separated from the 5S RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various RNA's in ovaries and in adult flies. In ovaries there is a slight excess of 5S RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5S RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18S+28S rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.     

Cloning,expression and cellular localization of Daphnia pulex senescence-associated protein,DpSAP

Daphnia (water fleas) are small crustaceans that undergo an unusual switch from asexual to sexual reproduction that is dependent on environmental conditions. In this study, a senescence-associated protein (SAP) from the common freshwater species Daphnia pulex was cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). Real-time PCR was employed to quantify the expression of D. pulex SAP (DpSAP) in individual organisms. The role of DpSAP in the reproductive transformation was further investigated in both parthenogenetic and sexual females by using digoxin-labeled SAP RNA probes and RNA whole-mount in situ hybridization. DpSAP was more highly expressed in sexual females, indicating a role in growth and reproduction. Cellular localization studies using RNA whole-mount in situ hybridization showed specific expression in the second tentacle joints. These expression patterns suggest an important role for DpSAP in the reproductive transformation of D. pulex.