Differences in response to the toxin sirodesmin PL produced by Phoma lingam (Tode ex fr.) Desm. on protoplasts,cell aggregates and intact plants of resistant and susceptible Brassica accessions

Summary The selective property of sirodesmin PL, a toxin produced by Phoma lingam, was studied on protoplasts, cell aggregates, leaves and roots. Directly after isolation, protoplasts from all the different Brassica accessions were sensitive when treated with toxin in a concentration higher than 1 M. When more differentiated plant tissue. i.e. cell aggregates, leaves or roots, were investigated, insensitivity to the toxin was found in the plant material resistant to P. lingam, while the plant material susceptible to P. lingam was sensitive. The results reveal that a clear correlation between resistance to P. lingam and insensitivity to sirodesmin PL is present, and that the toxin can be used to distinguish resistant plant material from susceptible both in vivo and in vitro.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Growth and metabolic flexibility in groundwater bacteria

Groundwater bacteria isolated from an oligotrophic-saturated soil showed a mixed strategy of economized metabolism and migration when grown in a continuous-flow column system simulating poor or nutrient-amended growth conditions. The cells were generally <0.5m in diameter in pure groundwater, but doubled in size when the concentration of dissolved organic carbon and phosphate in groundwater was increased 10-fold. The biomass, estimated from analysis of muramic acid (MuAc) in cell wall peptidoglucans, increased at the same time by a factor of 5 when the solid support in the columns was gravel and by a factor of 10 when it was glass beads. Bacteria in pure groundwater stored 10 times more of the energy-rich polysaccharide, poly--hydroxybutyric acid (PHB), than bacteria in enriched groundwater, and those cells that were attached to the gravel stored 10 times as much as cells in the interstitial pore water. Once phosphate was added to groundwater, stored PHB was metabolized. The proportion of free-living to attached bacteria was 2 to 10 times higher in enriched compared with pure groundwater indicating a mass transport of cells as the carrying capacity of their habitat rose.     

Unexpected influence of ionic strength on branched-pathway interactions between beta-lactamases and beta-halogenopenicillanates.

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.     

Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior. Three of the seven hypothetical structures tested here can be rejected as having too many polar side-chain groups in the interior or too few on the protein surface. In addition, a recently designed nutritional protein is identified as being very much unlike globular proteins. These database-derived characteristic quantities are useful in screening designed proteins prior to experiment and may be useful in screening experimentally determined (X-ray, NMR) protein structures for possible errors.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

Analysis of the H+/sugar symport in yeast under conditions of depolarized plasma membrane

H⁺/sugar symport in the obligatory aerobic yeastRhodotorula glutinis was analyzed under conditions where the plasma membrane was selectively depolarized by the lipophilic cation tetraphenylphosphonium (TPP⁺). Control experiments showed that this treatment did not impair the transmembrane pH, the cell energy charge, and the function of plasma membrane H⁺-ATPase. The kinetic data were fitted to elementary functions derived from a model constructed on the basis of some simplifying premises for ordered (either C + H⁺ + S or C + S + H⁺) and random reaction mechanisms. In addition, the comparison of the kinetic parameters in fully energized and depolarized cells provided information about the free carrier charge. It was concluded that the binding sequence of formation of the ternary carrier/H⁺/substrate complex follows a random mechanism and that the carrier bears a negative charge.     

Ultrastructure of the contractile apparatus of rat skeletal muscle embedded in an aqueous medium

The method of tissue embedding in melamine resin was applied to rat skeletal muscle. This method does not require tissue dehydration with organic solvents; only aqueous solutions are used. Electron micrographs of muscles embedded in melamine differ from those embedded in the conventional epoxy resin. In melamine-embedded muscles the actin and myosin filaments appear larger in diameter and subunits can be recognized in cross-sectioned myosin filaments. Within the Z-line, the characteristic patterns described for muscles embedded in epoxy resin are not visible; the spaces between the actin filaments are filled with electron-dense material. This suggests that the Z-line is more compact than could be concluded from epoxy resin-embedded muscle specimens. The M-line appears to be different from what is observed in epoxy-embedded muscle. The membranes appear as several clearly delineated layers. Dehydration rather than the action of the organic solvents per se is the main reason for the differences in the structure of the contractile apparatus between melamine- and epoxy-embedded muscles.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).