Secondary structure of dengue virus type 4 3' untranslated region: impact of deletion and substitution mutations.

Several studies have generated computer-based predictions of secondary structure of the 3' untranslated region (UTR) of Dengue virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 3' UTR with nuclease maps of this region. Maps and predictions were largely consistent. Maps supported the existence of previously predicted pseudoknots and identified putative regions of dynamic folding. Additionally, this study investigated previously identified conserved elements in the flavivirus 3' UTR that differ among viruses with different modes of transmission. Specific regions of mosquito-borne DEN type 4 were either deleted or replaced with homologous sequences from tick-borne Langat virus. All of these mutations caused substantial distortion of secondary structure, yet viruses carrying these mutations were viable.

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid.

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after (13)C(1) glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using (13)C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.

Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.