Ozone- and ethylene-induced regulation of a grapevine resveratrol synthase promoter in transgenic tobacco

Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbene, phytoalexins in a small number of unrelated plant species, and are induced by various biotic and abiotic stressors like pathogen attack, UV-irradiation or ozone exposure. To investigate the molecular basis for ozone-induced plant stress responses, we have examined the promoter of the grapevine resveratrol synthase (Vst1). In this report we summarize the influence of ozone on gene regulation. In transgenic tobacco a chimeric gene construct, containing the Vst1 promoter combined with the β-glucuronidase (GUS) reporter gene, is rapidly induced by ozone (0.1 μl·l⁻¹, 12 h). The same construct is also strongly induced by ethylene (20 μl·l⁻¹, 12 h). Promoter deletion analysis of the 5′ flanking sequence identified a positive regulatory element between −430 bp and −280 bp. This region contains ethylene-responsive enhancer elements, as well as an elicitor-responsive sequence in inverse orientation.     

The two-step lysis system of pneumococcal bacteriophage EJ-1 is functional in Gram-negative bacteria: triggering of the major pneumococcal autolysin in Escherichia coli

The holin function Ejh of the pneumococcal bacteriophage EJ-1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli , and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.     

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH₂-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH₂ or the COOH termini of heterologous proteins.

We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

     

UDP-glucose-4-epimerase from Poterioochromonas malhamensis

UDP-glucose-4-epimerase of Poterioochromonas malhamensis, Peterfi has been purified to apparent electrophoretic homogeneity. The enzyme has an apparent MW of 120 000 as determined by gel filtration of the active enzyme. Sodium dodecylsulfate polyacrylamide gel electrophoresis gave a MW of 59 000, thus indicating a dimeric structure. The epimerase does not require external NAD for activity. The apparent K_m values for UDP-glucose and UDP-galactose were calculated to be 1.67 mM and 0.26 mM, respectively. The pH optimum is at pH 8.7 and the isoelectric point is at pH 5.1 ± 0.15.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

The phytotoxin syringomycin elicits Ca2+-dependent callose synthesis in suspension-cultured cells of Catharanthus roseus

Deposition of the 1,3-β-glucan callose onto the cell wall represents one of the defence reactions of plants against pathogens. This process can be induced in suspension cells of Catharanthus roseus by subtoxic concentrations of the bacterial phytotoxin syringomycin and is associated with a slight increase in Ca²⁺ uptake and some K⁺ release. Under these conditions callose formation can be prevented by complexing external Ca²⁺, indicating that some Ca²⁺ uptake is essential as a signal. However, higher syringomycin concentrations elicit increased Ca²⁺ uptake without increasing callose formation, although the potential for callose synthesis is not exhausted – as shown using digitonin as an additional elicitor. These results suggest that, superimposed on Ca²⁺, another, yet unknown signal is also involved in the regulation of callose synthesis.     

Cutin Monomers and Surface Wax Constituents Elicit H2O2 in Conditioned Cucumber Hypocotyl Segments and Enhance the Activity of Other H2O2 Elicitors

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H₂O₂ elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H₂O₂ in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H₂O₂ elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H₂O₂ elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H₂O₂ elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H₂O₂ elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H₂O₂ elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.     

Control of chloroplast ATP synthase (CF0CF1) activity by Δ pH

A biphasic decay of the thiol modulated ATPase activity is observed at fast deenergization of the thylakoids achieved by turning off the light and simultaneous injection of the uncoupler nigericin. Most likely the rapid phase (_1/2 = 5 s) represents an unstable, active E_f-form of the enzyme which decays to a less active, but more stable E_s-form. The two forms have different substrate affinities. Deactivation and reactivation kinetics indicate that the transition from the E_f- to the E_s-form is reversible, requires a low proton gradient (1 to 2 pH units) and most probably involves the release and binding, respectively, of two protons from the thylakoid lumen phase to sites which have an apparent pK of 6.6. The E_s-form decays to the inactive E_i-form with a half time of 90 s. Reactivation of the completely deactivated enzyme is a two-stage process comprising protonation of sites with a pK of 6.8 followed by protonation of sites of pK 4.9. The intermediate E_s'-form has a decay time which is similar to that of the E_s-form, but a different K_m for ATP. Therefore we conclude that activation is not the exact reversal of deactivation. The results are discussed in terms of a model of H+-linked activation/deactivation.