Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae)

The 60 000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life‐history traits, which range from saprophagy over phytophagy to parasitism and predation. However, the direction of evolutionary change between the different modes remains unclear. Here, we use the Scathophagidae (Diptera) for reconstructing the direction of change in this relatively small family (≈ 250 spp.) whose larval habits mirror the diversity in natural history found in Cyclorrhapha. We subjected a molecular data set for 63 species (22 genera) and DNA sequences from seven genes (12S, 16S, Cytb, COI, 28S, Ef1‐alfa, Pol II) to an extensive sensitivity analysis and compare the performance of three different alignment strategies (manual, Clustal, POY). We find that the default Clustal alignment performs worst as judged by character incongruence, topological congruence and branch support. For this alignment, scoring indels as a fifth character state worsens character incongruence and topological congruence. However, manual alignment and direct optimization perform similarly well and yield near‐identical trees, although branch support is lower for the direct‐optimization trees. All three alignment techniques favor the upweighting of transversion. We furthermore confirm the independence of the concepts “node support” and “node stability” by documenting several cases of poorly supported nodes being very stable and cases of well supported nodes being unstable. We confirm the monophyly of the Scathophagidae, its two constituent subfamilies, and most genera. We demonstrate that phytophagy in the form of leaf mining is the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation has occurred while a second origin of predation is from a saprophagous ancestor. © The Willi Hennig Society 2006.     

Intramolecular interaction between the DEP domain of RGS7 and the Gbeta5 subunit

The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Galpha subunits and exhibits GAP activity. The GGL domain permanently associates with Gbeta5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gbeta5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gbeta5-RGS7, recombinant Gbetagamma complexes, and monomeric Gbeta5 and Gbeta1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gbeta5 and YFP-RGS7, indicating that the DEP-Gbeta5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gbeta5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gbeta5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gbeta5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gbeta5-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gbeta5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation.     

Analysis of rice mitochondrial genome organization using pulsed-field gel electrophoresis

Most of the plant mitochondrial (mt) genomes that have been mapped are believed to be organized as master circle molecules from which sub-genomic molecules arise through homologous recombination. We have evidence to suggest that a major part of the rice mt genome is organized as independent, sub-genomic molecules or mt chromosomes, one of which has already been mapped. This study is aimed at the identification of the other molecular entities that comprise the genome. Pulsed-field gel electrophoresis of the native rice mt DNA and Southern analysis with different mt gene probes have shown that in addition to the 117 kb mt chromosome, at least four more such molecules of sizes 130 kb, 95 kb, 70 kb and 56 kb account for most of the rice mt genome. A majority of the rice mt genes that encode products involved in oxidative phosphorylation are distributed among these five chromosomes. Partial restriction map of the 95 kborf 25/cox 3 chromosome, indicating the sites for the enzymesBglII andHindIII has also been determined.     

Nucleolar localization signals of box H/ACA small nucleolar RNAs.

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.     

Erratum to: Expression of GroES TB antigen in tobacco and potato

Plant Cell, Tissue and Organ Culture (PCTOC) - The lower panel in Fig. 2 of the original publication does not show the gels of the correct experiment. The correct lower panel is printed...     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.