Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Photosynthesis: Molecular biology and bioenergetics

Book Review

Photosynthesis: Molecular biology and bioenergeticsG.S. Singhal, J. Barber, R.A. Dilley, Govindjee, R. Haselkorn and P. Mohanty (Eds), Proceedings of the International Workshop on Application of Molecular Biology and Bioenergetics of Photosynthesis. Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong and Narosa: Springer-Verlag, 1989. xiv+441 pages. DM 148,00. ISBN 3-540-50451-6     

Towards an explanation of carboxylation/oxygenation bifunctionality in Rubisco. Transition structure for the carboxylation reaction of 2,3,4-pentanetriol

We construct a theoretical model of the transition structure for the carboxylation reaction of ribulose-1,5-biphosphate catalyzed by Rubisco. This is a first-order saddle point on the energy hypersurface for the nucleophilic attack of carbon dioxide on CH₃-(CHOH)₃-CH₃ at the C2 center.Ab initio analytical gradients methods at a 4-31G basis set level are used.The carbon framework and oxygens of the stationary structure superpose with the corresponding atoms of 2-carboxyarabinitol-1,5-biphosphate, which is a transition state analog that has recently been highly refined with X-ray methods. The hydroxyl group in C3 iscis to the C2 oxygen. The C3 center is somewhat pyramidized, the dienol O2-C2-C3-O3 is not planar.The geometry of the transition state allows for simple explanations of both the enolization of Rubisco's substrate ribulose-1,5-biphosphate, O₃PO-CH2-CO-(CHOH)₂-CH₂-OPO₃ and oxygenation reaction. The former is due to the pyramidal deformation at C3 and out of plane of O2-C2-C3-O3 framework: the enoliation is intramolecular and is probably enhanced by proton tunnelling. The latter is related with the fact that a rotation around an ethylene-like bond brings the triplet state down in energy. The reactive skeleton has a stationary geometry in the triplet state not very different from the one obtained in the global transition structure. There, the triplet is only 9 kcal/mol above the singlet. The spin densities at C2 and C3 centers clearly indicate the place where oxygenation will take place.     

Hidden smectic properties of a chiral side-chain co-oligomer

We recently showed that a side-chain industrial co-oligosiloxane presents a quenchable enlarged blue phase behaviour at the cholesteric-isotropic phase transition. In this paper, we present the results of a structural study based on X-ray diffraction, differential scanning calorimetry and optical measurements. In particular, the smectic A organisation is demonstrated in the lower temperature domain, which was hitherto understood as a cholesteric phase. A structural model for this phase is proposed on the basis of the analysis of the anisotropic scattering of stretched fibers. Our results also suggest that the observed glass transition is indeed a rather complex phenomenon, which seems to involve not only the freezing of the main chains, but also smectic correlations at the side-chain level. Moreover, the calorimetric study indicates that, notwithstanding the conservation of the processed film's optical properties, low kinetic reorganisations occur at room temperature.     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Nucleosomal organization of chromatin in sperm nuclei of the bivalve mollusc Aulacomya ater

The sperm nuclei of Aulacomya ater, family Mitylidae, contain three proteins (X, Aa5 and Aa6) which are specific to this cell type coexisting with a set of five somatic-type histones. Information about the chromatin structure resulting from this kind of association is scarce. Therefore, we have probed the structure of this sperm chromatin through digestion with micrococcal nuclease in combination with salt fractionation. The data obtained have allowed us to propose a nucleosomal arrangement for this chromatin. However, two types of nucleosomes would be present in agreement with their protein components.     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Membrane Operational Impedance Spectra in Chara corallina Estimated by Laplace Transforms Analysis

The membrane operational impedance spectrum of Chara corallina Klein ex Willd. (R. Brown) cells is investigated using Laplace transform analysis. The spectrum changes with both amplitude and sign of the electrical stimulation when time- and voltage-dependent K⁺ channels contribute to the membrane conductance. We compare the advantages and disadvantage of this technique for studying membrane impedance with those of the alternating current method and the white noise method.