Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

The relationship between the fatty acid profiles of the yolk and the embryonic tissue lipids: A comparison between the lesser black backed gull (Larus fuscus) and the pheasant (Phasianus colchicus)

Although substantial information is available regarding the fatty acid composition of lipids of the yolk and of the developing tissues of the chicken embryo, there is little knowledge on this topic for other avian species. The aim of the present study was to compare the yolk and embryonic tissue fatty acid profiles for a species selecting its food in the wild (the lesser black backed gull) with one fed on a standard commercial diet (the commercially reared pheasant). The fatty acid compositions of the yolk lipids were determined, and major differences were observed between the two species. In particular, the phospholipid of the gull yolk was enriched in 20:4n-6 and 22:6n-3 (18.8 and 7.1%, respectively, by weight of total fatty acids) in comparison with the pheasant (4.0 and 4.1%, respectively). The fatty acid compositions of the embryonic tissues were determined using eggs incubated in the laboratory. For the liver and heart, the fatty acid composition of the lipids in the two species reflected the initial yolk composition, with the gull tissue lipids generally containing higher proportions of 20:4n-6 and 22:6n-3 than those of the pheasant. In contrast, the fatty acid profiles of the brain phospholipid were essentially identical in the two species, with 20:4n-6 and 22:6n-3 comprising approximately 9 and 17%, respectively, of total fatty acids in both cases.     

Reevaluation of the role of bicarbonate and formate in the regulation of photosynthetic electron flow in broken chloroplasts

The stimulation of the Hill reaction in CO₂-depleted broken chloroplasts (Pisum sativum L. cv Rondo) by the total amount of dissolved CO₂ and HCO₃⁻ (bicarbonate^*) was measured at several formate concentrations. Formate appears to be a competitive inhibitor of the bicarbonate^* stimulation of electron flow. From these experiments we have obtained a reactivation constant (K_r) of 78 ± 31 micromolar NaHCO₃ and an inhibition constant (K_i) of 2.0 ± 0.7 millimolar HCOONa at pH 6.5. In the absence of formate, significant electron flow was measured at a bicarbonate^* concentration well below K_r, suggesting that electron flow from Q, the primary electron acceptor of photosystem II, to plastoquinone can proceed when no bicarbonate^* is bound to the regulatory site at the Q_B-protein. If so, bicarbonate^* stimulation of electron flow is mainly a diminution of the inhibition of electron flow by formate. In view of the results, it is proposed that regulation of linear electron flow by bicarbonate^* and formate is a mechanism that could link cell metabolism to photosynthetic electron flow.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Redox-Dependent Control of FOXO/DAF-16 by Transportin-1

1. Download : Download high-res image (206KB)
2. Download : Download full-size image

Highlights? FOXO forms redox-sensitive, disulfide-dependent complexes with several proteins ? Transportin-1 binds to FOXO via a disulfide and regulates its nuclear localization ? Redox and insulin signaling govern FOXO nuclear localization via distinct pathways ? Redox control of longevity protein FOXO/DAF-16 is evolutionarily conserved