The effect of cycloheximide (actidione) on cell wall synthesis in yeast protoplasts (original) (raw)
World Journal of Microbiology & Biotechnology, 1992
The optimal conditions for protoplast formation of Candida apicola were by using an enzyme from Arthrobacter sp. in combination with 2-mercaptoethanol. The kinetic data support the two-layered structure model of cell wall for this yeast but the structure of the cell wall depended on the age of cells and culture conditions. To regenerate the protoplasts, the type of osmotic stabilizer was important: sorbitol gave 16 to 30% regeneration. Electron
Inhibitory effect of 2-deoxy-d-glucose on the formation of the cell wall in yeast protoplasts
Journal of bacteriology, 1969
2-Deoxy-d-glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall components in Saccharomyces cerevisiae protoplasts. The synthesis of fibrillar glucan cell wall component was inhibited at a glucose to 2DG ratio of 4:1 in the cultivating medium. The completion of the formation of cell wall by the synthesis of the amorphous mannan-protein cell wall component was inhibited at a glucose to 2DG ratio of about 20:1. The inhibition could be reversed by increasing the glucose to 2DG ratio in the nutrient medium. No incorporation of 2DG into fibrillar glucan cell wall component was observed.
Plant Physiology, 1977
The effects of two protein synthesis hinbitors, cydoheximide and chloramphenicol, on the synthesis of nitochondrial proteins in maize (Zea mays) have been studied. The results of these investigations suggest that while most of the mitochondrial proteins are synthesized in the cytoplasm and are subsequently asociated wth the nitochondrion, several proteins of the mitochondrial inner membrane are synthesized within the mitochondrion. These resut are consistent with those observed in several other systems, but not previously reported for higher p-ts. of CH or CAP. The sets were then mixed and homogenized. The 3H incorporation thus served as an internal standard, and the 3H/ 14C ratio served as an indicator of the influence of the protein synthesis inhibitors. SUBCELLULAR FRACTIONS Various subcellular fractions were obtained as described by Longo and Longo (10). Three fractions were studied, namely the soluble fraction (25,000g supernatant), the mitochondrial fraction (which banded at an isopycnic density of 1.22 g/cm3 in a 35-65% sucrose gradient), and the dense particulate fraction which passed through the 65% sucrose in the gradients. This fraction contained mitochondrial inner membranes and glyoxysomes. In higher organisms, protein synthesis occurs on both 80S (cytoplasmic) and 70S (mitochondrial) ribosomes. In yeast, Neurospora, and mammals (2, 13, 14, 16), less than 10% of the
Correlative Studies of Cell Wall Enzymes and Growth
Plant Physiology, 1975
If cell wall hydrolytic enzymes are involved in extension growth, a correlation may be expected between hydrolytic activity of the cell walls and growth rate of the tissue from which the walls are prepared. Epicotyl sections from 0 to 5 mm, 6 to 10 mm, and 11 to 13 mm below the apical hook of pea seedlings (Pisum sativum var. Alaska) have relative growth rates of 100:15:2, respectivelv. The relative fl-glucosidase activities (units/mg wall) of cell walls from these sections are respectively, 100:24:23, for walls prepared in glycerol and 100:42:23 for walls prepared in aqueous solution. Thus, there is a correlation between growth rate of the tissue and specific activity of the wall-associated ,B-glucosidase. Similar correlations were found for other cell wall-associated hydrolases. Relative cell numbers for the above sections, as determined by counting, were 100:25:16, and with these data it could be calculated that the amount of cell wall ,3-glucosidase activity per cell is essentially a constant. Thus, for epicotyl sections the amount of enzyme per cell does not change during the process of cell elongation but the specific activity declines as the result of deposition of new wall material. Data from several laboratories suggest that glycosidases play a role in wall plasticization, thus permitting cell elongation during extension growth (9). There is evidence that oligosaccharidehydro
Archives of Microbiology, 1996
Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a β-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.
CELL-WALL POLYMER MAPPING IN THE COENOCYTIC MACROALGA CODIUM VERMILARA (BRYOPSIDALES, CHLOROPHYTA)1
Journal of Phycology, 2010
Cell walls in the coenocytic green seaweed Codium vermilara (Olivi) Chiaje (Bryopsidales, Chlorophyta) are composed of 32% (w ⁄ w) b-(1 fi 4)-D-mannans, 12% sulfated polysaccharides (SPs), and small amounts of hydroxyproline-rich glycoprotein-like (HRGP-L) compounds of the arabinogalactan proteins (AGPs) and arabinosides (extensins). Similar quantities of mannans and SPs were reported previously in the related seaweed C. fragile (Suringar) Hariot. Overall, both seaweed cell walls comprise 40%-44% of their dry weights. Within the SP group, a variety of polysaccharide structures from pyruvylated arabinogalactan sulfate and pyruvylated galactan sulfate to pyranosic arabinan sulfate are present in Codium cell walls. In this paper, the in situ distribution of the main cell-wall polymers in the green seaweed C. vermilara was studied, comparing their arrangements with those observed in cell walls from C. fragile. The utricle cell wall in C. vermilara showed by TEM a sandwich structure of two fibrillar-like layers of similar width delimiting a middle amorphous-like zone. By immuno-and chemical imaging, the in situ distribution of b-(1 fi 4)-Dmannans and HRGP-like epitopes was shown to consist of two distinct cell-wall layers, whereas SPs are distributed in the middle area of the wall. The overall cell-wall polymer arrangement of the SPs, HRGP-like epitopes, and mannans in the utricles of C. vermilara is different from the ubiquitous green algae C. fragile, in spite of both being phylogenetically very close. In addition, a preliminary cell-wall model of the utricle moiety is proposed for both seaweeds, C. fragile and C. vermilara.
Reversion of Yeast Protoplasts in Media containing Polyethylene Glycol
Microbiology, 1983
Liquid medium supplemented with 35% (w/v) PEG supported regeneration of the cell wall in protoplasts of Saccharomyces cerevisiae. The regeneration was followed by reversion to normal cells. Both these morphogenetic processes were similar to those described previously in gelatin media. The frequency of reversion was estimated to be 20 to 30%. The regeneration medium with PEG also induced protoplast fusion, When complementary auxotrophs were used for fusion only the fused products proliferated, giving first regenerated protoplasts and then hybrid cells. METHODS Strains. The following strains of Saccharomyces cereuisiae were used : diploid wild-type CCY 2 1-4-59, haploid strain 9 hisa and 55R5-3C uraa, kindly provided by Dr M. Opekarovi and Dr J. h b i k. Culture media. All the strains were maintained on wort-agar slants. Cells were grown in malt extract medium, protoplasts were cultured in either minimal glucose medium (minimal medium; Leupold, 1955) or in Difco Nitrogen Base-glucose medium (YNBG medium). Both media were supplemented with 0.6 M-KCl (as an osmotic stabilizer) and/or PEG. In some experiments, medium containing 3% (w/v) yeast extract (Difco) and glucose was used (YEG medium). When required PEG 4000 or 6000 was included at concentrations ranging from 10 to 40% (w/v). Protoplast preparation and cultiwtion. A modified method of Eddy & Williamson (1959), using lyophilized snail enzymes and 0-6 M-KCl, was employed. Freshly prepared protoplasts
Polyamines and cell wall organization inSaccharomyces cerevisiae
Yeast, 1992
Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.