Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit

Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven beta-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.     

Spatial proximity between two host plant species influences oviposition and larval distribution in a leaf beetle

Everything else being equal, insect herbivores can be expected to oviposit on host plants that provide the qualitatively and quantitatively best food for larvae. However, the selection of a plant for oviposition may be influenced by such ecological factors as natural enemies, host distribution, host patch size or host patch density. We performed a field study to test whether spatial proximity between two host plant species influences the oviposition patterns and larval distribution of the alpine leaf beetle Oreina elongata. In the population studied, O. elongata oviposits and feeds on two host plants, that belong to the same family (Asteraceae): Adenostyles alliariae and Cirsiumspinosissimum. The first species contains pyrrolizidine alkaloids that are sequestered by the beetle as a chemical defence, whereas the second plant does not contain any alkaloids but has hairy and spiny leaves that might give some mechanical protection to beetle larvae.
During two consecutive summers, we quantified oviposition and larval distribution on randomly chosen C. spinosissimum that grew spatially isolated from A. alliariae, on C. spinosissimum that grew in leaf contact with A. alliariae and on A. alliariae that grew in leaf contact with C. spinosissimum (isolated A. alliariae was not considered, because it is rare in the study population). In both years, more eggs were laid on C. spinosissimum than on A. alliariae and more on those C. spinosissimum that were growing close to A. alliariae than on those growing isolated. Large numbers of larvae moved from C. spinosissimum to A. alliariae during the season. Patch size did not influence egg and larval numbers. Eggs survived better on C. spinosissimum than on A. alliariae in the field. The data suggest that C. spinosissimum may provide eggs with better protection against stormy weather. In a separate study of the same population, we found that larval performance was better on A. alliariae than on C. spinosissimum. Our present data suggest that O. elongata preferentially oviposits on plants of the species that maximizes egg survival and that grow in close proximity to plants of the species that provides better food and chemical defence.     

Characterization of the maize xylem sap proteome

The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.     

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.     

Curcumin modulates drug metabolizing enzymes in the female Swiss Webster mouse

Curcumin, the yellow pigment found in turmeric, exhibits potent chemopreventative properties in both in vivo and in vitro cancer models. We hypothesized that this effect may occur via curcumin-mediated changes in enzymes involved in both carcinogen bioactivation and estrogen metabolism. Female Swiss Webster mice were treated with either curcumin (200 mg/kg or 400 mg/kg, p.o.) or vehicle control for 1 or 2 weeks. The results demonstrated that curcumin had no effect on the catalytic activities of ovarian aromatase, hepatic catechol-O-methyltransferase or hepatic UDP-glucuronosyltransferase. However, both doses of curcumin caused a 25% decrease in CYP1A catalytic activity, but not polypeptide levels, following 2 weeks of treatment. Additionally, following 2 weeks of curcumin at 400 mg/kg, there was a 20% decrease in the catalytic activity and a 28% decrease in polypeptide levels of CYP3A. While 2 weeks of curcumin treatment (400 mg/kg) caused a 20% increase in glutathione S-transferase activity, there was no parallel increase in hepatic stores of the co-factor glutathione. In conclusion small changes in CYP1A, CYP3A and GST following long term treatment (2 weeks) suggest that the combination of all three metabolic pathways may play a small role in curcumin's chemopreventative action.