Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.     

Comparative density functional theory study of the binding of ligands to Cu+ and Cu2+: Influence of the coordination and oxidation state

BP86, B3LYP and MP2 methods, generally used to study large systems containing transition metals, were compared for their ability to accuratly evaluate bond dissociation energies of copper complexes. Various [Cu-L]+ and [Cu-L]2+ complexes in which L are small ligands and the higher coordinated complexes, [Cu(NH3)(4)]+ and [Cu(NH3)4]2+ were studied. For monoligated complexes, the BDEs calculated by the three methods differed by 2 to 60 kcal/mol, the larger differences being obtained for [Cu-L]2+ complexes. The BDEs calculated using the B3LYP functional were in general close to the experimental values whereas the BDEs calculated using the BP86 functional were too high and the BDEs calculated using the MP2 were too low. If we rank the whole ligands according to their increased bond strength, the resulting orders obtained with the three methods are different for the [Cu-L]+ complexes, the B3LYP giving the same order as the experimental one. This result indicates that the BDEs of [Cu-L]+ complexes are better modeled using the B3LYP than using the BP86 and MP2 methods. For [Cu-L]2+, B3LYP also gave the most reliable results whereas BP86 gave too large BDEs and MP2 gave too small BDEs. However, symmetries of ground states can be different using DFT and post-Hartree-Fock methods. For [Cu-N2O]2+ the use of the B1LYP provides a better symmetry of the complex than the B3LYP, as has been recently shown in the literature for [Cu-H2O]2+. MP2 led to an incorrect bent structure for [Cu-N2]2+ in contrast to a linear structure obtained with the other methods, including CCSD(T). However, due to the lack of experimental data for [Cu-L]2+ complexes and to contrasted results for the methods, it is not possible to conclude definitely. For the high coordinated complexes [Cu(NH3)4]+ and [Cu(NH3)4]2+, the PBE calculation method was used in addition to the BP86, B3LYP and MP2. The BDE values were very close to each other when there is no change of the oxidation state during the reaction. On the basis of these calculations, the choice of the method was less crucial for high coordinated complexes [Cu(NH3)4]+ and [Cu(NH3)4]2+ so long as the oxidation state remained the same during the reaction. In contrast, when [Cu(NH3)4]2+ is reduced in [Cu(NH3)3]+ and NH3, the BDE calculated using the four methods were markedly different.     

Theoretical conformational analysis of some hydroxyazoic dyes

The optimum structures of p-hydroxy azobenzene, 4-phenyl azo 1-naphthalene and 4-phenyl azo 1-napthol are determined theoretically using the quantum mechanical MO method PCILO. The conformational minima are obtained by simultaneous optimization of the geometric parameters. The conformers equilibria and their temperature dependence, suggested by experimental dipole moments results, are discussed on the basis of our PCILO calculations.     

Fmoc solid-phase synthesis of peptide thioesters using an intramolecularn,S-acyl shift

[reaction: see text] We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.     

Salt effects on the interaction of an amphiphilic model molecule with immobilized phosphatidylcholine monolayers

The retention of hydrocortisone (used as an amphiphilic model solute) on an immobilized artificial membrane (IAM) column was investigated in relation to the mobile phase concentration of three sodium salts (representing different rankings in the Hofmeister series, i.e. perchlorate, chloride and sulfate) in order to provide insight into the nature of the solute interactions with phospholipid monolayers. The influence of the salt series on solute retention was found to follow the Hofmeister series, emphasizing the role of hydrophobic effect in the solute retention mechanism on phospholipid monolayers. Retention models based on the extended Wyman relations (preferential interaction theory) were developed to analyze more quantitatively the salt effects on the hydrocortisone retention factor. This analysis as well as additional thermodynamic study suggested that the hydrocortisone binding to IAM involved both an insertion into the hydrophobic inside governed by hydrophobic effects and contacts with the interfacial region implying interactions such as van der Waals interactions/hydrogen bonds between the solute hydroxyl groups and the polar headgroups of phospholipidmonolayers.     

Generalite de l'effet structural d'un motif bitertiaire en rmn 13C: effet gem-6 de 6ème substitution methyle

In di-tertiary groups like (Me₃C)₂¹³C, the sixth methyl substituent $\text{inverts}$ or $\text{attenuates}$ the induced ¹³C chemical shift of sp² or sp³ sites (B_σ and y_π effects).     

Structural, electronic, and bonding properties of zeolite Sn-beta: a periodic density functional theory study

The structural, electronic, and the bonding properties of the zeolite Sn-beta (Sn-BEA) have been investigated by using the periodic density functional theory. Each of the nine different T-sites in BEA were substituted by Sn atoms and all the nine geometries were completely optimized by using the plane-wave basis set in conjunction with the ultra-soft pseudopotential. On the basis of the structural and the electronic properties, it has been demonstrated that the substitution of Sn atoms in the BEA framework is an endothermic process and hence the incorporation of Sn in the BEA is limited. The lowest unoccupied molecular orbitals (LUMO) energies have been used to characterize the Lewis acidity of each T-site. On the basis of the relative cohesive energy and the LUMO energy, the T2 site is shown to be the most favorable site for the substitution Sn atoms in the BEA framework.     

Streptavidin chiral stationary phase for the separation of adenosine enantiomers

In this paper, a microbore column packed with streptavidin particles was used, at various temperatures (0-24 degrees C), to separate the adenosine enantiomers by HPLC. Using an aqueous mobile phase, the apparent enantioseparation was high for a small molecule, varying from 11.5 at 0 degrees C to 6.2 at 24 degrees C. From the experiments carried out with a streptavidin-biotin complex stationary phase, it was demonstrated that the blockage of the biotin sites of the immobilized streptavidin was responsible for a strong decrease in the enantioselectivity via a direct and/or an indirect effect. From the analysis of the concentration dependencies of the solute retention factor, it was also shown that a reduction of the D-adenosine specific binding sites occurred at the lowest temperature. The thermodynamic parameters determined from the van't Hoff plots indicated that the D-adenosine binding to the streptavidin specific sites was enthalpically driven.     

SELECTIVE DNA THYMINE DIMERIZATION DURING UVA IRRADIATION IN THE PRESENCE OF A SATURATED PYRIDOPSORALEN

Abstract— It has been recently shown that UVA (320–400 nm) irradiation of DNA in the presence of pyridopsoralens induces the formation of thymine cyclobutane dimers in addition to monoadducts. In this work, we measured the potency of a saturated pyridopsoralen to photosensitize DNA, despite its inability to covalently attach to DNA. First, from spectroscopic fluorescence measurements, we have shown that both analogs, saturated and unsaturated pyridopsoralens, namely 4',5'-dihydro-7-methyl-pyrido[3,4-clpsoralen (DH-MePyPs) and 7-methylpyrido[3,4-c]psoralen, exhibit a similar global affinity for DNA. Secondly, we demonstrated, by footprinting experiments, that exposure of a DNA sequence to 365 nm UV radiation in the presence of DH-MePyPs results in selective cyclobutane thymine dimerization. Thymines located in the immediate proximity of the 5'-TA-3' step are exclusively affected and the frequency of this photoprocess depends on flanking sequences. We thus probe a selective thymine dimer photosensitizer. Results are discussed in terms of drug affinity and physical properties of the helix at the binding site.