Production of Resveratrol in Recombinant Microorganisms

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).     

Proton NMR detection of porphyrins and cytochrome C in small unilamellar vesicles: role of the dissociation kinetic constant

The molecular tumbling of small unilamellar vesicles is not fast enough to enable the detection of (1)H NMR signals of molecules associated with phospholipids. We show that relatively fast kinetic exchange of the interacting molecules is able to induce a strong decrease of the residual homonuclear dipolar coupling, allowing the acquisition of sharp signals. At low molecule/lipids molecular ratio, this can be lead to signal broadening due to exchange at intermediate rates on the NMR chemical timescale. However, proton resonances can be easily detected when sufficient lipids are added to prevent the occurrence of any free compounds in solution. This is demonstrated, using lipid signal suppression, in the case of paramagnetic porphyrin derivatives as well as diamagnetic hematoporphyrin. Since several peptides and proteins are expected to be associated with lipids having relatively fast dynamics, this study addresses, as a first example, the interaction of cytochrome c.     

Development of selective inhibitors and substrate of matrix metalloproteinase-12

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO2-CH2)-Xaa'-Yaa'-Zaa'-NH2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with Ki values of 0.19 and 4.4 nM toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa'-Zaa' positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr239 and Lys177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm.     

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.     

An amylase gene polymorphism is associated with growth differences in the Pacific cupped oyster Crassostrea gigas

This study investigated the non-neutrality of genetic polymorphism in two alpha-amylase genes (AMYA and AMYB) in the oyster Crassostrea gigas. Bi-parental oyster families, bred to be polymorphic for markers in these genes, were monitored for growth and survival for 1 year under standard culture conditions in two French production sites. Within-family genotype frequencies indicated that the two amylase genes were closely linked (c. 1.7 cM). Within two of three families, significant differences in growth were observed between genotypes at one of the two production sites, suggesting that this polymorphism is not neutral and might be under selection because of its role in digestive function. Estimated daily yields were different between amylase genotypes, indicating the potential value of amylase markers in selective breeding programmes to improve oyster growth.     

Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10 ⁻¹³ M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants.     

The Candida Genome Database: facilitating research on Candida albicans molecular biology

The Candida Genome Database (CGD; http://www.candidagenome.org) is a resource for information about the Candida albicans genomic sequence and the molecular biology of its encoded gene products. CGD collects and organizes data from the biological literature concerning C. albicans, and provides tools for viewing, searching, analysing, and downloading these data. CGD also serves as an organizing centre for the C. albicans research community, providing a gene-name registry, contact information, and research community news. This article describes the information contained in CGD and how to access it, either from the perspective of a bench scientist interested in the function of one or a few genes, or from the perspective of a biologist or bioinformatician interpreting large-scale functional genomic datasets.     

Acetylcholinesterase genes within the Diptera: takeover and loss in true flies

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies.     

Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI), resulting in abortive embryonic development. One Wolbachia host, Culex pipiens complex mosquitoes, displays high levels of variability in both CI crossing types (cytotypes) and DNA markers. We report here an analysis of 14 mosquito strains, containing 13 Wolbachia variants, and with 13 different cytotypes. Cytotypes were Wolbachia-dependent, as antibiotic treatment rendered all strains tested compatible. Cytotype distributions were independent of geographical distance between sampling sites and host subspecies, suggesting that Wolbachia does not promote a reproductive isolation depending on these parameters. Backcross analysis demonstrated a mild restoring effect of the nuclear genome, indicating that CI is mostly cytoplasmically determined for some crosses. No correlation was found between the phenotypic and genotypic variability of 16 WO prophage and transposon markers, except for the WO prophage Gp15 gene, which encodes a protein similar to a bacterial virulence factor. However, Gp15 is partially correlated with CI expression, suggesting that it could be just linked to a CI gene.