In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

New solution method for equations of reaction and mass transfer in biocatalyst particles

Summary For numerical solution of the reaction-mass transfer equations for immobilised biocatalysts it may be better to start integration at the particle surface and proceed inwards: calculations are targetted on the region to which practically interesting changes are often confined (because concentrations are effectively zero in the interior); and during iterative solution wrong initial estimates may be rejected after detecting anomalies early in the integration.Symbols C_b substrate concentration in bulk (mol m^–3) - c dimensionless substrate concentration (C/C_b) (-) - D_e effective diffusion coefficient (m²s^–1) - Da Damkohler number (V.r_o ²/D_e.K_s) (-) - K_s substrate concentration kinetic coefficient (mol m^–3) - k_e external mass transfer coefficient (ms^–1) - r_o bead radius (m) - Sh Sherwood number (k_e.r_o/D_e) (-) - V maximum rate per unit volume in beads (mol m^–3s^–1) - x dimensionless distance from bead centre (r/r_o) (-) - dimensionless kinetic coefficient (K_s/C_b) (-) - _o effectiveness factor (-)     

Expression of {beta}-galactoside {alpha}2,6 sialyltransferase blocks synthesis of polysialic acid in Xenopus embryos

Polysialic acid is a developmentally regulated carbohydratestructure found on neural cell adhesion molecules (NCAM). Expressionof ß-galactoside 2,6-sialyltransferase in Xenopusembryos, by injection of mRNA, prevents the polysialylationof NCAM, presumably by introducing a different type of sugarlinkage that terminates chain elongation. Abnormalities in neuraldevelopment result from this treatment, but in general the bodyplan of the injected embryos is not severely affected. The resultsprovide evidence that the mis-expression of glycosyltransferasescan be used to interfere with the normal pattern of glycosylationin whole organisms. glycosylation NCAM polysialic acid Xenopus development     

Nucleic acid studies on some heterofermentative lactobacilli: Description of Lactobacillus malefermentans sp.nov. and Lactobacillus parabuchneri sp.nov.

Chemotaxonomic studies were performed on some heterofermentative lactobacilli of uncertain taxonomic position. Two strains from beer and six strains from a variety of habitats were found to be distinct from each other and all other Lactobacillus species examined on the basis of DNA-DNA hybridizations and warrant new species for which the names L. malefermentans and L. parabuchneri , respectively, are proposed.     

The removal of exogenous thiols from proteins by centrifugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.     

Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro

Astrocyte-enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10-90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10-90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.     

The relationship among egg size,density and food level on larval development in the wood frog (Rana sylvatica)

Summary Although inter- and intraspecific variation in egg size among amphibians has been well documented, the relationship between egg size and fitness remains unclear. Recent attempts to correlate egg size intraspecifically with larval developmental patterns have been equivocal. In this study the development of larvae derived from large eggs and small eggs, from a single population in Maryland were compared under a range of food levels and larval population densities. Both food level and density had significant effects on the length of the larval period and size at metamorphosis. However, the response among larvae derived from different egg sizes was not additive. At low densities and high food levels, larvae from small eggs had longer larval periods and a larger size at metamorphosis than larvae derived from large eggs. In contrast, at high densities larvae from small eggs had longer developmental periods but were smaller at metamorphosis than larvae from large eggs. In addition, larvae from small eggs were more sensitive to density irrespective of food level. These results suggest that optimal egg size is correlated with environmental factors, which may explain the maintenance of both geographic and within population variation in egg size commonly observed in amphibians.     

Drought and the organization of tree-hole mosquito communities

Summary In southeastern North America (North Florida, USA), the duration, frequency, and timing of drought differentially affect the survivorship of pre-adult tree-hole mosquitoes. Drought affects survivorship both by the direct action of dehydration on developing larvae and pupae and by the indirect modulation of predation. The drought-susceptible species, Toxorhynchites rutilus, Orthopodomyia signifera, and Anopheles barberi co-occur in more permanent holes that are larger, with larger, more vertical openings, lower down in larger trees, and contain darker water with higher conductivity, pH, and tannin-lignin content than the holes occupied by Aedes triseriatus that has drought-resistant eggs and rapid larval development. Ovipositing mosquitoes cue on physical and chemical attributes of tree holes independently of host tree species. These same attributes differ among drought-prone and drought-resistant holes but mosquitoes track these attributes more faithfully than the attributes predict tree-hole stability.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate