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At least 469 records · Page 26Linked to original sources
PubMed1968

Composition of the protoplast membrane from Saccharomyces cerevisiae.

1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0.8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7.0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13-20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C(16:0), C(16:1) and C(18:1) acids. Whole cells contained a slightly greater proportion of C(16:0) and a somewhat smaller proportion of C(18:1) acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas-liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Delta(5,7,22,24(28))-ergostatetraen-3beta-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C(29) di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.

Amino Acids↗
PubMed1992

Instability of the protoplast membrane of facultative alkaliphilic Bacillus sp. C-125 at alkaline pH values below the pH optimum for growth.

Cell walls of facultative alkaliphilic Bacillus sp. C-125 consist of three polymers (peptidoglycan, teichuronopeptide and teichuronic acid). Protoplasts prepared from the strain with egg-white lysozyme regenerated cell walls at neutral pH, but not at pH above 8.5. The protoplasts were susceptible to lysis at alkaline pH. The protoplasts exposed to alkaline pH rapidly burst and lost ability to regenerate their cell walls. The alkali-instability was similar to that of protoplasts from neutrophilic Bacillus subtilis 168. The membrane vesicles were also labile at alkaline pH. The acidic wall components of strain C-125 may contribute to stabilization of the cytoplasmic membrane of cells growing at alkaline pH, probably by shielding the membrane from direct exposure to an alkaline environment.

Bacillus↗
PubMed1997

T-DNA tagging reveals a novel cDNA triggering cytokinin- and auxin-independent protoplast division.

Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.

Amino Acid Sequence↗
PubMed1998

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.

Arabidopsis↗
PubMed2000

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis.

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.

Animals↗
PubMed2001

The auxin signal for protoplast swelling is perceived by extracellular ABP1.

Protoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C-terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti-AtERabp1 antibodies on Arabidopsis protoplasts or anti-ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.

Amino Acid Sequence↗
PubMed2001

Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina.

Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta-mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta-mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.

Animals↗
PubMed1969

Infection of tobacco mesophyll protoplasts by tobacco mosaic virus.

It was proved that the protoplasts prepared from mesophyll of Nicotiana tabacum are infected by tobacco mosaic virus. The infection occurred when purified tobacco mosaic virus particles were added to a protoplast suspension in the presence of poly-L-ornithine. The virus multiplied in these protoplasts to a level of 10(6) virus particles per infected protoplast during 24 hours of incubation. The efficiency of infection was remarkably high, exceeding that by mechanical inoculation of tobacco leaves.

Chloramphenicol↗
PubMed2001

Cytochalasin D attenuates the desensitisation of pressure-stimulated vesicle fusion in guard cell protoplasts.

Fusion of vesicular membranes with the plasma membrane during pressure-driven swelling of guard cell protoplasts was studied using patch clamp capacitance measurements. Hydrostatic pressure pulses were applied via the patch pipette and resulted in an immediate and linear increase in membrane capacitance, a parameter proportional to the surface area. In any given protoplast, pressure-stimulated increases in membrane capacitance could be provoked repetitively. However, the rate of rise in capacitance upon the same strength of stimulation decreased exponentially with time (tau = 4 min) for subsequent pressure stimuli. This process was the result of a desensitisation of the plasma membrane to mechanical forces. Incubation of guard cell protoplasts in cytochalasin D, which depolymerises actin filaments, nearly abolished this desensitisation process. These results suggest that membrane stretch initiates a reactive process that may fortify or stabilise the plasma membrane of guard cell protoplasts.

Actin Cytoskeleton↗
PubMed1994

Production and regeneration of protoplasts from the Y-phase of the human pathogenic fungus Paracoccidioides brasiliensis.

Protoplasts of Y-cells and partially converted M-cells from several human isolates of Paracoccidioides brasiliensis were obtained with a combined enzyme system containing Novozym 234 and a chitinase. A laboratory made extract from Trichoderma harzianum CBS-345-33 supplemented with chitinase induced the release of protoplasts from partially converted M-cells but not from established Y-cells. A similar yield of protoplasts (1-2 x 10(5) ml-1 after 16 h) was produced by using either enzymatic system. Protoplasts regenerated on nutrient gelatin at 23 degrees C at a frequency of 0.1%.

Cell Wall↗
PubMed2001

Haemoglobin (Erythrogen) enhanced microcallus formation from protoplasts of Indica rice (Oryza sativa L.).

The beneficial effects have been studied of supplementing culture medium with 1:100-1:500 (v:v) of a commercial haemoglobin solution (Erythrogen) on the mitotic division of cell suspension-derived protoplasts of Indica rices (Oryza sativa L.). Protoplasts were cultured in liquid medium, at densities of 1.5 x 10(6) or 2.5 x 10(6) ml(-1), on nitrocellulose membranes overlaying a semi-solidified medium layer that was supplemented with both Erythrogen and nurse cells of Lolium multiflorum. The mean final plating efficiencies (FPEs) of rice cv. BR26 protoplasts cultured with 1:200 (v:v) Erythrogen, at 1.5 x 10(6) ml(-1) (0.018+/-0.003%; n = 8) and 2.5 x 10(6) ml(-1) (0.016+/-0.002%; n = 8), were both significantly (P < 0.05) greater than controls lacking Erythrogen (0.0058+/-0.002%; n = 8 and 0.0041+/-0.001%; n = 8, respectively). Similarly, the mean FPEs of cv. Bini protoplasts cultured with 1:200 (v:v) Erythrogen at 1.5 x 10(6) ml(-1) (0.012+/-0.003%; n = 6) and 2.5 x 10(6) ml(-1) (0.017+/-0.001%; n = 6) were also significantly (P < 0.05) greater than their respective controls (0.003+/-0.001%, n = 6 and 0.002+/-0.001%, n = 6). In contrast, supplementation with 1:100 or 1:500 (v:v) Erythrogen did not lead to sustained mitotic division and microcallus formation in both rice cultivars.

Cell Culture Techniques↗
PubMed1958

Cytological and chemical studies of the growth of protoplasts of Bacillus megaterium.

Aeration of protoplasts of Bacillus megaterium in a succinate buffered nutrient broth led to marked growth similar to that already described by McQuillen, and the degree of chromatin synthesis in these growing forms prompted a combined cytological and chemical study. Growth was followed by phase contrast and by Feulgen stains, as well as by lipide phosphorus, nucleic acid, and protein analyses. In slide cultures, growth and compression led to monstrous flattened forms with readily visible, but coalescent nuclear structures. In fluid cultures, the protoplasts grew as phase dense spheres. Orderly reproduction of apparently discrete nuclear bodies was observed during the initial hours of spherical growth, but in older cultures, the chromatin arrangement tended to be more haphazard and was influenced by the concentration of Mg ions. In the same medium, protoplasts free of lysis showed a linear rise in optical density, while vegetative cells exhibited an exponential increase. However, protoplasts were able to synthesize DNA at the same rate as vegetative cells, but their increase of RNA was always less. Thus, as they grew, the ratio RNA/DNA fell. The lipide P increased in proportion to the expanding surface. With growth and lysis, large amounts of water-insoluble slime accumulated. Analyses indicate it to be a phospholipoprotein material containing some RNA.

Bacillus megaterium↗
PubMed1993

Characterisation of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon.

The tobacco Tnt1 retrotransposon is the only plant retrotransposon that has been shown to be transcriptionally active, and its transcription is strongly induced when preparing leaf-derived protoplasts. We have analysed in this paper the LTR sequences important for Tnt1 expression in tobacco protoplasts. We show that LTR sequences upstream of the TATA box are sufficient to confer protoplast-dependent induction to a heterologous promoter. We also show that this region contains two short activator elements, and that one of these sequences, BII, interacts with protoplast-specific nuclear factors.

Base Sequence↗
PubMed2004

Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level.

Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.

Gene Expression Regulation, Plant↗
PubMed2002

Hyperosmotic stress induces formation of tubulin macrotubules in root-tip cells of Triticum turgidum: their probable involvement in protoplast volume control.

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.

Adaptation, Physiological↗
PubMed1975

Inhibition of multiplication of tobacco mosaic virus in protoplasts by antibiotics and its prevention by divalent metals.

At concentrations that inhibit bacterial growth, some antibiotics including gentamicin completely inhibited virus multiplication in protoplasts, and other antibiotics partially inhibited virus multiplication. The inhibition caused by each antibiotic was largely prevented by adding a divalent metal; MnC1(2) was more effective than CaC1(2) and other salts of divalent metals when added at 10 mM to the incubation medium. When added immediately after infection, 1 mug/ml of gentamicin halved the final virus concentration and 3 mug/ml completely inhibited virus multiplication, although 10 mug/ml was required to stop bacterial growth. Gentamicin inhibited virus multiplication even when added 24 h after virus inoculation. Also, when protoplasts were exposed to gentamicin for only 1 or 2 h, either immediately after inoculation or 2 h later, the virus concentration was considerably decreased. Gentamicin seemed not to affect virus multiplication in whole plants. Sap from Dianthus barbatus also strongly inhibited virus multiplication in protoplasts but, unlike gentamicin, it acted in the presence of MnC1(2). By contrast, chelating agents such as 1 mM-EDTA or 5 mM-potassium citrate was strong inhibitors of virus multiplication that were inactive in the presence of MnC1(2). It is suggested that gentamicin and other antibiotics may chelate metals from the protoplast membranes, thus disorganizing their function and affecting virus multiplication.

Anti-Bacterial Agents↗
PubMed1998

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts.

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.

Closterovirus↗
PubMed1978

Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration.

Conditions for highly efficient genetic recombination in Streptomyces by protoplast fusion are described. Protoplasts of S. fradiae and S. griseofuscus were formed by a modification of the glycine-lysozyme-lytic enzyme method (Okanishi, Suzuki & Umezawa, 1974). Regeneration of cells from protoplasts was monitored throughout the growth cycle and was most efficient when cells of either S. fradiae or S. griseofuscus were taken from the transition phase between the exponential and stationary growth phases. Fusion of protoplasts carrying different auxotrophic or chromosomal drug-resistance markers was achieved by treatment with polyethylene glycol, and high frequencies of stable genetic recombinants were obtained.

Polyethylene Glycols↗