Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Fragrep： An Efficient Search Tool for Fragmented Patterns in Genomic Sequences

Many classes of non-coding RNAs （ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum） can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.     

Absence of carbon transfer between Medicago truncatula plants linked by a mycorrhizal network, demonstrated in an experimental microcosm

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Hepatic uptake and metabolism of galactose can be quantified in vivo by 2-[18F]fluoro-2-deoxygalactose positron emission tomography

Metabolism of galactose is a specialized liver function. The purpose of this PET study was to use the galactose analog 2-[(18)F]fluoro-2-deoxygalactose (FDGal) to investigate hepatic uptake and metabolism of galactose in vivo. FDGal kinetics was studied in 10 anesthetized pigs at blood concentrations of nonradioactive galactose yielding approximately first-order kinetics (tracer only; n = 4), intermediate kinetics (0.5-0.6 mmol galactose/l blood; n = 2), and near-saturation kinetics (>3 mmol galactose/l blood; n = 4). All animals underwent liver C15O PET (blood volume) and FDGal PET (galactose kinetics) with arterial and portal venous blood sampling. Flow rates in the hepatic artery and the portal vein were measured by ultrasound transit-time flowmeters. The hepatic uptake and net metabolic clearance of FDGal were quantified by nonlinear and linear regression analyses. The initial extraction fraction of FDGal from blood-to-hepatocyte was unity in all pigs. Hepatic net metabolic clearance of FDGal, K(FDGal), was 332-481 ml blood.min(-1).l(-1) tissue in experiments with approximately first-order kinetics and 15.2-21.8 ml blood.min(-1).l(-1) tissue in experiments with near-saturation kinetics. Maximal hepatic removal rates of galactose were on average 600 micromol.min(-1).l(-1) tissue (range 412-702), which was in agreement with other studies. There was no significant difference between K(FDGal) calculated with use of the dual tracer input (Kdual(FDGal)) or the single arterial input (Karterial(FDGal)). In conclusion, hepatic galactose kinetics can be quantified with the galactose analog FDGal. At near-saturated kinetics, the maximal hepatic removal rate of galactose can be calculated from the net metabolic clearance of FDGal and the blood concentration of galactose.     

Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison

A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.     

Improving genome assemblies by sequencing PCR products with PacBio

Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.