Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas Wittichii RW1.

Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Natural genetic variation of root system architecture from Arabidopsis to Brachypodium: towards adaptive value.

Root system architecture is a trait that displays considerable plasticity because of its sensitivity to environmental stimuli. Nevertheless, to a significant degree it is genetically constrained as suggested by surveys of its natural genetic variation. A few regulators of root system architecture have been isolated as quantitative trait loci through the natural variation approach in the dicotyledon model, Arabidopsis. This provides proof of principle that allelic variation for root system architecture traits exists, is genetically tractable, and might be exploited for crop breeding. Beyond Arabidopsis, Brachypodium could serve as both a credible and experimentally accessible model for root system architecture variation in monocotyledons, as suggested by first glimpses of the different root morphologies of Brachypodium accessions. Whether a direct knowledge transfer gained from molecular model system studies will work in practice remains unclear however, because of a lack of comprehensive understanding of root system physiology in the native context. For instance, apart from a few notable exceptions, the adaptive value of genetic variation in root system modulators is unknown. Future studies should thus aim at comprehensive characterization of the role of genetic players in root system architecture variation by taking into account the native environmental conditions, in particular soil characteristics.

Executive Order Number Twelve, September 1972

Authorize and direct the transfer of powers providing for the maintenance of the State Capital grounds, equipment, etc., as given to the Department of Buildings & Grounds to the General Services Department.

Executive Order Number Six, January 1972

Executive Orders from Governor Ray. Authorize and direct the transfer of powers, duites, etc. as given to the Printing Board of General Services Department

Executive Order Number Ten, June 1972

Authorize and direct the transfer of duties as given to the Administration of State Communications and the State Communications Advisory Council to the General Services Department.

Executive Order Number Eleven, September 1972

Authorize and direct the transfer of powers providing for the purchase of all state-owned vehicles to the General Services Department.

Executive Order Number Thirteen, October 1972

Authorize and direct the transfer from the Executive Council to the Department of General Services the powers, duties, etc. that are contained in Chapter 84 of the Laws of the 64th General Assembly.

Executive Order Number Thirteen, October 1972

Authorize and direct the transfer from the Executive Council to the Department of General Services the powers, duties, etc. that are contained in Chapter 84 of the Laws of the 64th General Assembly.

Executive Order Number Fourteen, December 1972

Authorize and direct the transfer of all powers, etc. contained in Chapter 8A, 1971 Code, specifically including those given to the State Educational Radio and Faciliy Board to the Department of General Services.

The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

Photodamage to Oxygen Evolving Complex - An Initial Event in Photoinhibition of Photosystem II.

Photosystem II (PSII) of oxygenic photosynthesis is susceptible to photoinhibition. Photoinhibition is defined as light induced damage resulting in turnover of the D1 protein subunit of the reaction center of PSII. Both visible and ultraviolet (UV) light cause photoinhibition. Photoinhibition induced by UV light damages the oxygen evolving complex (OEC) via absorption of UV photons by the Mn ion(s) of OEC. Under visible light, most of the earlier hypotheses assume that photoinhibition occurs when the rate of photon absorption by PSII antenna exceeds the use of the absorbed energy in photosynthesis. However, photoinhibition occurs at all light intensities with the same efficiency per photon. The aim of my thesis work was to build a model of photoinhibition that fits the experimental features of photoinhibition. I studied the role of electron transfer reactions of PSII in photoinhibition and found that changing the electron transfer rate had only minor influence on photoinhibition if light intensity was kept constant. Furthermore, quenching of antenna excitations protected less efficiently than it would protect if antenna chlorophylls were the only photoreceptors of photoinhibition. To identify photoreceptors of photoinhibition, I measured the action spectrum of photoinhibition. The action spectrum showed resemblance to the absorption spectra of Mn model compounds suggesting that the Mn cluster of OEC acts as a photoreceptor of photoinhibition under visible light, too. The role of Mn in photoinhibition was further supported by experiments showing that during photoinhibition OEC is damaged before electron transfer activity at the acceptor side of PSII is lost. Mn enzymes were found to be photosensitive under visible and UV light indicating that Mn-containing compounds, including OEC, are capable of functioning as photosensitizers both in visible and UV light. The experimental results above led to the Mn hypothesis of the mechanism of continuous-light-induced photoinhibition. According to the Mn hypothesis, excitation of Mn of OEC results in inhibition of electron donation from OEC to the oxidized primary donor P680+ both under UV and visible light. P680 is oxidized by photons absorbed by chlorophyll, and if not reduced by OEC, P680+ may cause harmful oxidation of other PSII components. Photoinhibition was also induced with intense laser pulses and it was found that the photoinhibitory efficiency increased in proportion to the square of pulse intensity suggesting that laser-pulse-induced photoinhibition is a two-photon reaction. I further developed the Mn hypothesis suggesting that the initial event in photoinhibition under both continuous and pulsed light is the same: Mn excitation that leads to the inhibition of electron donation from OEC to P680+. Under laser-pulse-illumination, another Mn-mediated inhibitory photoreaction occurs within the duration of the same pulse, whereas under continuous light, secondary damage is chlorophyll mediated. A mathematical model based on the Mn hypothesis was found to explain photoinhibition under continuous light, under flash illumination and under the combination of these two.

Molecular genetic approaches to the biology of the moss "Physcomitrella patens"

La mousse haplobiontique Physcomitrella patens est utilisée comme système génétique modèle pour l'étude du développement des plantes. Cependant, l'absence d'un protocole efficace de transformation a constitué jusqu'à présent un gros désavantage méthodologique pour le développement futur de ce système expérimental. Les résultats présentés dans le premier chapitre relatent la mise au point d'un protocole de transformation basé sur la technique de transfert direct de gènes dans des protoplastes par précipitation au PEG. Un essai d'expression transitoire de gènes a été mis au point. Ce protocole a été adapté afin de permettre l'introduction in vivo d'anticorps dans des protoplastes. Le protocole modifié permet d'introduire simultanément du DNA et des IgG dans les cellules, et nous avons démontré que ces anticorps peuvent inactiver spécifiquement le produit d'un gène co-introduit (GUS), ainsi que certaines protéines impliquées dans des processus cellulaires (tubuline). Cet essai, baptisé "essai transitoire d'immuno-inactivation in vivo", devrait être directement applicable à d'autres protoplastes végétaux, et permettre l'élaboration de nouvelles stratégies dans l'étude de processus cellulaires. Le second chapitre est consacré aux expériences de transformation de la mousse avec des gènes conférant une résistance à des antibiotiques. Nos résultats démontrent que l'intégration de gènes de résistance dans le génome de P. patens est possible, mais que cet événement est rare. Il s'agit là néanmoins de la première démonstration d'une transformation génétique réussie de cet organisme. L'introduction de gènes de résistance aux antibiotiques dans les protoplastes de P. patens génère à haute fréquence des clones résistants instables. Deux classes de clones instables ont été identifiés. La caractérisation phénotypique, génétique et moléculaire de ces clones suggère fortement que les séquences transformantes sont concaténées pour former des structures de haut poids moléculaire, et que ces structures sont efficacement répliquées et maintenues dans les cellules résistantes en tant qu'éléments génétiques extrachromosomaux. Ce type de transformation nous permet d'envisager des expériences permettant l'identification des séquences génomiques impliquées dans la replication de l'ADN de mousse. Plusieurs lignées transgéniques ont été retransformées avec des plasmides portant des séquences homologues aux séquences intégrées dans le génome, mais conférant une résistance à un autre antibiotique. Les résultats présentés dans le troisième chapitre montrent que les fréquences de transformation intégrative dans les lignées transgéniques sont 10 fois plus élevées que dans la lignée sauvage, et que cette augmentation est associée à une coségrégation des gènes de résistance dans la plupart des clones testés. Ces résultats génétiques indiquent que l'intégration de séquences d'ADN étranger dans le génome de P. patens a lieu en moyenne 10 fois plus fréquemment par recombinaison homologue que par intégration aléatoire. Ce rapport homologue/aléatoire est 10000 fois supérieur aux rapports obtenus avec d'autres plantes, et fournit l'outil indispensable à la réalisation d'expériences de génétique inverse dans cet organisme à haplophase dominante. THESIS SUMMARY The moss Physcomitrella patens is used as a model genetic system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. But further development of this model system was hampered by the lack of a protocol allowing the genetic transformation of this plant. We have developed a transformation protocol based on PEG-mediated direct gene transfer to protoplasts. Our data demonstrate that this procedure leads to the establishment of an efficient transient gene expression assay. A slightly modified protocol has been developed allowing the in vivo introduction of antibodies in moss protoplasts. Both DNA and IgGs can be loaded simultaneously, and specific antibodies can immunodeplete the product of an expression cassette (GUS) as well as proteins involved in cellular processes (tubulins). This assay, named transient in vivo immunodepletion assay, should be applicable to other plant protoplasts, and offers new approaches to study cellular processes. Transformations have been performed with bacterial plasmids carrying antibiotic resistance expression cassette. Our data demonstrate that integrative transformation occurs, but at low frequencies. This is the first demonstration of a successful genetic transformation of mosses. Resistant unstable colonies are recovered at high frequencies following transformation, and two different classes of unstable clones have been identified. Phenotypical, genetic and molecular characterisation of these clones strongly suggests that bacterial plasmids are concatenated to form high molecular arrays which are efficiently replicated and maintained as extrachromosomal elements in the resistant cells. Replicative transformation in P. patens should allow the design of experiments aimed at the identification of genomic sequences involved in moss DNA replication. Transgenic strains have been retransformed with bacterial plasmids carrying sequences homologous to the integrated transloci, but conferring resistance to another antibiotic. Our results demonstrate an order of magnitude increase of integrative transformation frequencies in transgenic strains as compared to wild-type, associated with cosegregation of the resistance genes in most of these double resistant transgenic strains. These observations provide strong genetic evidence that gene targeting occurs about ten times more often than random integration in the genome of P. patens. Such ratio of targeted to random integration is about 10 000 times higher than previous reports of gene targeting in plants, and provides the essential requirement for the development of efficient reverse genetics in the haplodiplobiontic P. patens.

Differentiated chemical reactivity of nanoparticles toward DTT

RATIONALE: Induction of oxidative stress and impairment of the antioxidant defense are considered important biological responses following nanoparticle (NP) exposure. The acellular in vitro dithiothreitol (DTT) assay is proposed to measure the oxidative potential of NP. In addition, DTT can be considered as a model compound of sulfur containing antioxidants. The objective of this work is to evaluate the surface reactivity in solution of a NP panel toward DTT. METHOD: The NP panel was composed of four carbonaceous particles, six types of metal oxides and silver with primary size ranged from 7 to 300 nm. Suspensions were prepared in surfactant solution with 30 min sonication. DTT was used as reductant to evaluate the oxidative properties of the different NP. The determination of the NP ability to catalyze electron transfer from DTT to oxygen was carried out as described in Sauvain et al., Nanotoxicology, 2008, 2:3, 121−129. RESULTS: All the carbonaceous NP catalyzed the oxidation of DTT by oxygen following the mass based order: carbon black > diesel exhaust particle > nanotubes > fullerene. A contrasting reactivity was observed for the metallic NP. Except for nickel oxide and metallic silver, which reacted similarly to the carbonaceous NP, all other metal oxides hindered the oxidation of DTT by oxygen, with ZnO being the most effective one. CONCLUSIONS : DTT was stabilized against oxidation in the presence of metal oxide NP in the solution. This suggests that different chemical interactions take place compared with carbonaceous NP. To explain these differences, we hypothesize that DTT could form complexes with the metal oxide surface (or dissolved metal ions), rendering it less susceptible to oxidation. By analogy, such a process could be thought to apply in biological systems with sulfur−containing antioxidants, reducing their buffer capacity. Such NP could thus contribute to oxidative stress by an alternative mechanism.

Practical considerations for improving the productivity of mass spectrometry-based proteomics.

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.