Evidence for the acid-growth theory of fusicoccin action

Three predictions of the acid-growth theory of fusicoccin (FC) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue shows that these FC-induced processes are strictly correlated in time and respond coordinately to cations present in the medium. ii) Fusicoccin (1 mol l^-1) induces a rapid acidification of the cell-wall solution, reaching a final level of pH 3.8–4.0. Exogenous protons are able to substitute quantitatively for FC in causing segment elongation at pH 3.8–4.0. At pH 4, FC has no additional effect on cell elongation. iii) Neutral buffers (pH 7) completely abolish the FC-mediated growth response. iv) Cycloheximide (10 mg l^-1) inhibits both FC-induced and acid-buffer(pH 4)-induced elongation after a lag of 40–45 min, and FC-induced H⁺ excretion after a lag of 2 h. Under the same conditions, indole-3-acetic acid-induced elongation and H⁺ excretion are inhibited without detectable lag. It is concluded that these results are fully compatible with the acid-growth theory of FC action.Abbreviations IAA indole-3-acetic acid - CHI cycloheximide - FC fusicoccin     

Ethylene synthesis and growth of tomato hypocotyls: Induction by auxin and fusicoccin and inhibition by vanadate

The effects of fusicoccin (FC) on growth and ethylene synthesis of tomato (Lycopersicon esculentum Mill.) hypocotyls were compared to those of indole-3-acetic acid (IAA). Fusicoccin promoted both growth and ethylene production maximally at <2M. Growth was stimulated to a slightly greater extent by FC as compared to IAA, while ethylene synthesis rates in response to FC were about 50% less than those induced by IAA. Cycloheximide (0.5 M) inhibited auxin-induced growth by 80% but had no effect on FC-induced growth; ethylene production was inhibited to the same extent (58%) when induced by either IAA or FC. Both IAA and FC caused tissue contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC to increase, indicating that like IAA, FC induces ethylene synthesis by stimulating the formation of ACC. Orthovanadate, a potent inhibitor of proton-translocating plasma membrane ATPases, reduced both IAA- and FC-induced growth and ethylene synthesis at concentrations less than 1 mM, with ethylene synthesis being approximately 10 times more sensitive to inhibition than growth. Vanadate did not affect tissue ACC levels, slightly reduced total ACC production, and inhibited conversion of ACC to ethylene. However, significant inhibition of in vivo ethylene-forming enzyme activity required high concentrations of vanadate (1 mM) and was less effective than inhibition by cobaltous ion. The site of action of vanadate in inhibiting ethylene synthesis remains unclear, but the ion did not prevent the elevation of tissue ACC levels in response to IAA or FC. It is unlikely, therefore, that stimulation of plasma membrane H⁺-ATPase activity is required for the induction of ACC synthase by IAA and FC.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell

Fusicoccin (FC) is commonly thought to promote electrogenic H⁺ extrusion through its action on the H⁺-ATPase of the plant plasma membrane. Nonetheless, essential support from rigorous electrophysiological analysis has remained largely absent. The present investigation surveys the effects of FC on the charge transport properties at the membrane of a higher-plant cell — stomatal guard cells of Vicia faba L. — for which the electrical geometry is defined, and from which the voltage-dependent kinetic characteristic for the pump has been identified. Current-voltage (I-V) relations of the guard cells were determined before and during treatments with FC, and during brief exposures to NaCN plus salicylhydroxamic acid. Responses of the pump and of the ensemble of secondary transport processes were identified in the whole-membrane conductance-voltage relations and in the difference-current-voltage (dI-V) characteristic for the pump. In 0.1 mM K⁺, exposure to 10 M FC shifted guard-cell potentials negative by 29–61 mV. Current-and conductance-voltage profiles indicated limited changes in the pump I-V characteristic, an observation which was confirmed through explicit kinetic analysis of pump dI-V relations. However, the voltage response was accompanied by a 1.5-to 2.6-fold fall in membrane conductance. These results challenge conventional views of fusicoccin action by ascribing the electrical responses to reduced current passage through secondary transport pathways as well as to enhanced electrogenic ion pumping.Abbreviations and symbols Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SHAM salicylhydroxamic acid - FC fusicoccin - V _m free-running membrane potential - G _m membrane slope conductance at V _m - (d)I-V (difference) current-voltage (relation) - G-V slope conductance-voltage (relation)     

Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region

The expression of a stress- and salicylic acidinducible protein gene from tobacco, PR1a protein gene, was determined after its Introduction to lettuce (Lactuca sativa L.) plants. The 5 flanking 2.4 Kb fragment from PR1a gene was joined to the bacterial -glucuronidase (GUS) gene (PR-GUS) and introduced into lettuce cotyledons by Agrobacterium-mediated gene transfer using a binary vector containing a kanamycin-resistance gene as a selectable marker. As a control with constitutive expression, the chimeric gene consisting of CaMV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-resistant regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the lettuce genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These results showed that the promoter of the stress-inducible tobacco PR1a protein gene was introduced into lettuce plants, and the introduced chimeric gene was expressed normally under the regulated control of the PRla promoter.Abbreviations BA N6-benzyladenine - GUS -glucuronidase - NAA -naphthaleneacetic acid - Km kanamycin - Km^s kanamycin resistant - Km⁰ kanamycin sensitive - NPT- II neomycin phosphotransferase II - PR pathogenesis-related - SA salicylic acid - MS Murashige and Skoog medium - NOS nopaline synthase     

Antifouling activity of seaweed extracts on the green alga Enteromorpha prolifera and the mussel Mytilus edulis

Twenty-seven species of common seaweeds from the coast of Korea havebeen screened for antifouling activity. The seaweed extracts were tested inlaboratory assays against the marine fouling green alga Enteromorphaprolifera and the blue mussel Mytilus edulis. Tissue growth, sporesettlement, zygote formation and germlings of the E. prolifera wereinhibited by methanol extracts of the seaweed Ishige sinicola (= I. foliacea) and Sargassum horneri. Spore settlement was stronglyinhibited by using extract concentrations as low as 30 g mL^-1with I. sinicola and 120 g mL^-1 with S. horneri. The repulsive activity of the foot of the mussel was completely inhibited bymethanol extracts of I. sinicola and Scytosiphon lomentaria atconcentrations of 40 g per 10 L drop supplied to eachmussel. These extracts also showed strong antifouling activities onlarval settlement with, respectively, no or only 6% of the spat settlingwhen a test concentration of 0.8 mg mL^-1 was used. This work isthe first stage towards the development of novel antifouling agents frommarine macroalgae.     

Efficient micropropagation of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> applying meristem wounding: a three-step protocol

A highly efficient three-step protocol for in vitro propagation of Ensete ventricosum (enset) was developed that consisted of initiation, bud proliferation, and shoot elongation and rooting stages. At the initiation stage, it was crucial to use shoot tips (5–8 mm) with subtending corm tissues as explants to obtain growth. The addition of 0.5–1% (w/v) activated charcoal to the medium was essential to prevent phenol exudation which otherwise leads to the loss of cultures. During the bud proliferation stage, modified MS macronutrients and micronutrients together with a combination of cytokinins (1.6 M naphthaleneacetic acid, 4.4 M 6-benzylaminopurine, 23.2 M kinetin, 22.6 M N⁶ 2-isopentyladenine) was used. This novel composition of macronutrients was based on the analysis of leaf nutrient content of glasshouse-grown enset sprouts. Multiple bud formation on the enlarged corm tissue was induced only when the meristem region was wounded before transfer to the bud proliferation medium. Up to 75 healthy shoots per explant were produced, whereas unwounded explants produced, only one to two shoots per explant. A third stage with a low concentration of cytokinin enabled shoot elongation as well as root development. The plantlets were acclimatized with 100% success and they showed no apparent phenotypical deviation.Abbreviations BAP 6-Benzylaminopurine - IBA Indole-3-butyric acid - DW Dry weight - EV Ensete ventricosum medium - 2-iP N⁶ 2-Isopentyl adenine - NAA -Naphthaleneacetic acid     

Molecular organization of the human CD3 gene family on chromosome 11q23

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 , , and chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 and chain genes in vectors designed for rapid and efficient chromosome walking. The human CD3 gene was located in the region immediately downstream of the CD3 and genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 gene located 26 kb downstream of the CD3 and genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11g23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.     

Inhibition of elongation and H+ and K+ transport by the phosphodiesterase inhibitor aminophylline in plant tissue

Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H⁺ and K⁺ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.Abbreviations cAMP adenosine 3:5-cyclic monophosphate - FC fusicoccin - IAA indole-3-acetic acid - MES 2-N-morpholinoethanesulfonic acid - PDE cyclic nucleotide phosphodiesterase     

Carbon dioxide and water vapor exchange over a <Emphasis Type="Italic">Miscanthus</Emphasis>-type grassland: Effects of development of the canopy

The eddy correlation technique was employed to measure net ecosystem carbon dioxide (CO₂) (NEE) and water vapor exchange (LE) over a C3/C4 co-occurring wet temperate Miscanthus-type grassland in the Kanto plain of Japan in the 1999 growing season. The maximal mean canopy height and maximal leaf area index were 1.0m and 5.5, respectively. The daily maximal LE was approximately 540Wm^–2. The maximum value of daily accumulative LE was 16.3MJday^–1. Daily variation of the decoupling factor () suggests that in the morning LE decoupled with the atmosphere, and the available energy was the major driving force for LE, whereas in the afternoon LE coupled strongly with the atmosphere, and the atmospheric evaporative demand played a critical role in LE. The decline in (from 0.8 to 0.5) with the growing season demonstrates that LE decoupled from the atmosphere in the later growth season. The peak NEE value was 57.4µmolCO₂m^–2s^–1 (the positive value signifies the canopy carbon gain was from the air). The maximal daily integrated NEE was 1.06molCO₂m^–2day^–1 observed during the peak growth stage. A rectangular hyperbolic model was used to describe the relation between daytime NEE and incident photosynthetic photon flux density (PPFD). The net ecosystem CO₂ was not light-saturated up to a PPFD level of 2000µmol m^–2s^–1. The initial slope estimated with the NEE–PPFD response model was approximately 0.042molCO₂mol^–1photon on average. The canopy light compensation point ranged from 210 to 430µmolm^–2s^–1 with an average of approximately 310µmolm^–2s^–1. Both the initial slope and the canopy light compensation point decreased as the canopy senesced. The switch in dominance from C3 to C4 plants played an important role in the canopy fluxes.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

The slow wound-response of<Emphasis Type="Italic"> γVPE</Emphasis> is regulated by endogenous salicylic acid in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Vacuolar processing enzyme (VPE) is a cysteine protease responsible for the maturation of various vacuolar proteins in higher plants. The Arabidopsis thaliana (L.) Heynh. VPE gene, encoding a VPE homologue, is slowly up-regulated in both local and systemic leaves in response to wounding. To clarify the activation mechanism of VPE, we examined the accumulation of VPE mRNA after hormone treatments or after wounding in wild-type and various mutant plants of Arabidopsis. Both ethylene and jasmonic acid (JA) are known as signal molecules that activate the wound-responsive genes. However, treatment with exogenous JA had little effect on the VPE response, although JA activated the vegetative storage protein (VSP) gene, a typical wound-responsive gene. Wounding activated VPE even in two ethylene-insensitive plants (etr1-1 and ein2-1). Thus, the wound-induced expression of VPE was independent of ethylene and JA. We found that the wound-induced expression of VPE was reduced in two SA-deficient plants (pad4-1 and NahG), while the wound-induced expression of VSP increased in these mutants. Appreciable accumulation of SA was not observed in either the local or systemic leaves after wounding. These results suggest that endogenous SA enhances the wound-induced expression of VPE and attenuates the wound-induced expression of VSP, although SA is not a wound-signal that directly activates these genes.Abbreviations ABA abscisic acid - GST glutathione S-transferase - INA 2,6-dichloroisonicotinic acid - JA jasmonic acid - MeJA methyl jasmonate - PR pathogenesis-related - RBCS Rubisco small subunit - SA salicylic acid - VPE vacuolar processing enzyme - VSP vegetative storage protein     

Stable isotope evidence of benthic microalgae-based growth and secondary production in the suspension feeder Cerastoderma edule (Mollusca,Bivalvia) in the Marennes-Oléron Bay

The contribution of natural food sources to the growth and secondary production of the suspension feeding bivalve Cerastoderma edule (L.) was estimated under field conditions in the Marennes-Oléron Bay (Atlantic coast, France). Monthly estimates of abundance, biomass and cockle growth were combined with seasonal analyses of ¹³C and ¹⁵N ratios of juvenile and adult cockles, together with their potential food sources [i.e. suspended particulate organic matter (POM), microphytobenthos, macroalgae and seagrass] sampled at mid-tide level in a muddy sandflat. Adult cockles grew mainly in spring, whereas juveniles grew in summer and autumn, following spat recruitment in early summer. Total annual production and elimination of cockles were estimated to be 32.5 and 34.7 g AFDW m^–2 yr^–1. Relative contributions of each year class to production were ca 40, 41, 11 and 6% for 0-group, 1-, 3- and 4-yr-old cockles in 1995, respectively. Quantitative assessment of proportions of food sources to the annual secondary production of cockles was obtained by using a simple carbon isotope-mixing model with microphytobenthos (¹³C = –16.0±0.6) and POM (¹³C = –22.2±1.1) as end-members. On average, more than 70% of the total annual cockle production originated from microphytobenthos, with a much higher contribution for the 0-group (88%) than for adult cockles (60%). The between-age difference was induced mainly by changes in the availability of food resources (benthic versus planktonic) during the non-synchronous growing seasons of juvenile and adult cockles.     

Expression of<Emphasis Type="Italic"> ENOD40</Emphasis> during tomato plant development

In legumes, ENOD40 expression is increased upon interaction of plants with rhizobia. Little is known of the expression pattern of ENOD40 during other stages of the plant life cycle. Studies of ENOD40 expression in non-legume development may give an indication of the function of the gene. To investigate the ENOD40 expression pattern during plant development, a fusion between the -glucuronidase (GUS) reporter gene and 150 bp of the 5 untranslated region plus 3,000 bp of 5 untranscribed tomato ENOD40 sequence was constructed and introduced into Lycopersicon esculentum Miller. Based on the observed GUS expression patterns in transgenic tomato we speculate that ENOD40 in tomato has a role in counteracting ethylene-provoked responses.Abbreviations GUS -glucuronidase - FISH fluorescence in situ hybridisation - RACE rapid amplification of cDNA ends - RFLC restriction fragment length polymorphism     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

Chorismate mutase of<Emphasis Type="Italic"> Thermus thermophilus</Emphasis> is a monofunctional AroH class enzyme inhibited by tyrosine

aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-/ barrel enzyme that is organized as a trimer. Comparison of the enzymes structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the proteins surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a K _m of 290 M, and a k _cat/K _m value of 180 s^–1 mM^–1. TtCM was inhibited by tyrosine with a K _i =34 M, possibly in a competitive manner.     

The role of acidification in gibberellic acid- and fusicoccin-induced elongation growth of lettuce hypocotyl sections

The roles of gibberellic acid (GA₃) and fusicoccin (FC) in the elongation growth and acidification of the medium by excised hypocotyl sections of lettuce (Lactuca sativa L.) were investigated. Hypocotyl sections incubated in buffer without GA₃ elongate optimally at pH 4.0–4.25 while sections incubated with GA₃ show the same growth between pH 4.25 and 6.0. Preincubation of sections at pH 6.0 for 6 h does not affect the subsequent elongation response to acidic medium (pH 4.25); however, the sections become refractory to further acid treatment after their initial burst of growth in response to pH 4.25. Sections made refractory to acid are responsive to GA₃ application, however, and the rate of growth in response to GA₃ of sections pretreated for 6 h at pH 4.25 is 85% of that of sections pretreated at pH 6.0. Although preincubation of sections for 48 h in medium at pH 6.0 abolishes the GA₃ response, it does not affect the response to buffer at pH 4.25. FC stimulates elongation growth in letuce hypocotyls at an optimal concentration of 1 M, and pretreatment of sections at pH 4.25 does not affect this elongation response. Although both GA₃ and FC increase elongation of the section, neither causes appreciable acidification of the medium. Addition of KCl or NaCl to FC-treated sections causes rapid medium acidification but addition of salts to GA₃-treated tissue does not cause acidification. Abrasion of the hypocotyl to remove the cuticle does not enhance acidification of the medium by the sections nor deos it affect elongation of the sections in response to GA₃ or FC. Medium acidification by the sections is not a passive process since it is abolished both by low temperature (2° C) and metabolic inhibitors (carbonyl cyanide-m-chlorophenyl-hydrazone, azide). The acidification of the medium by barley (Hordeum vulgare L.) roots in response to FC is also dependent on the presence of KCl. We conclude that the acid-growth hypothesis does not explain GA₃- or FC-induced elongation in lettuce hypocotyls.Abbreviations FC tusicoccin - GA₃ gibberellic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - MES 2-(N-morpholino)ethanesulphonic acid - Tris tris-(hydroxymethyl)aminomethane     

Plant regeneration and genetic transformation of Lotus angustissimus

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N⁶ – ² or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - 1AA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP N⁶ – ² - PA proanthocyanidins - NOS nopaline synthase - NI TII neomycin phosphotransferase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Genetic transformation of selected mature cork oak (Quercus suber L.) trees

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and -glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.Abbreviations BA N⁶-Benzyladenine - GUS -Glucuronidase - MSSH Expression-proliferation medium - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase gene - uidA -Glucuronidase gene     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride