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PubMed2002

Efficient paclitaxel production using protoplasts isolated from cultured cells of Taxus cuspidata.

Efficient isolation of protoplasts from Taxus cuspidata cultured cells, localization of paclitaxel in the cultured cells, and efficient production of paclitaxel by protoplasts were studied. Hemicellulase, potassium citric acid solution, and degassing treatments were effective in increasing the yield of protoplasts isolated from T. cuspidata cultured cells. Protoplasts yields (3.2 - 6.4 x 10(6) number/g-fresh weight cells) were achieved by combining the various treatments with specific culture and cell phases. It was found that about 30% and 35% of paclitaxel in the cells was located in cell walls and/or between the cell wall and cell membrane (CW) of suspension cells in the growth phase and in the stationary phase, respectively. About 30% and 43% of paclitaxel in the cells was located in CW of the cells grown in solid culture in growth phase and in the stationary phase, respectively. In comparison with cell suspension culture, protoplasts in a static culture and the protoplasts immobilized in agarose gel in shaking culture resulted to about 6 times increase in the extracellular paclitaxel accumulation.

Cell Extracts↗
PubMed2003

Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in a moss, Physcomitrella patens.

To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.

Bioreactors↗
PubMed1985

Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes.

Protoplast formation from mycelium and microconidia of Trichophyton mentagrophytes was achieved with Novozym 234. Pretreatment procedures with dithiothreitol or urea mercaptoethanol sodium lauryl sulphate before digestion with Novozym 234 greatly reduced protoplast yield from mycelium. Snail gut enzyme did not protoplasts in good yield. Scanning electron microscopy of mycelium protoplasts showed the acquired spherical shape. The plasma membrane appeared finely granular although remnants of cell wall could sometimes be observed. Transmission electron microscopy showed the cell interior of these protoplasts was plasmolysed. Microconidia treated with Novozym 234 displayed a range of cell wall digestion, with intact protoplasts showing distinct cytoplasmic organelles.

Enzymes↗
PubMed2005

Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI.

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.

Benzofurans↗
PubMed2005

Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light.

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.

14-3-3 Proteins↗
PubMed1990

Expression of resistance to barley stripe mosaic virus in barley and oat protoplasts.

Mesophyll protoplasts from both susceptible and resistant hosts were inoculated with RNA purified from barley stripe mosaic virus (BSMV) strains CV52 and CV42 using the polyethylene glycol (PEG) method. Protoplasts derived from the susceptible Hordeum vulgare L. cv. Black Hulless were susceptible to both BSMV strains, as indicated by fluorescein isothiocyanate staining and ELISA. More than 80% of protoplasts derived from an oat cultivar resistant to CV52, but not to CV42, were readily infected by either CV52 or CV42. Protoplasts from 10 barley lines resistant to CV42 remained resistant to CV42, although a limited number of protoplasts were infected. Functional resistance in the cultured barley protoplasts, but not in those from oat plants, suggests that resistance in these barley lines may be the result of restriction of replication, whereas resistance in oat plants is more likely due to restriction of cell-to-cell movement.

Edible Grain↗
PubMed1993

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA.

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

Base Sequence↗
PubMed1980

Protoplasts from yeast and mycelial forms of Candida albicans.

Protoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of Candida albicans by enzyme digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of C. albicans tested. Treatment with digestive juice of the snail Helix pomatia required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of beta-glucuronidase and chitinase were determined. Electron microscopy of C. albicans showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90% of the protoplasts produced by this model were viable as assessed by vital staining with Janus Green B.

Candida albicans↗
PubMed1986

Polyamine metabolism and osmotic stress. I. Relation to protoplast viability.

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

Avena↗
PubMed1984

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

Base Sequence↗
PubMed1972

Comparison of deoxyribonucleic acid uptake and marker integration in bacilli and protoplasts of Bacillus subtilis.

trp(+)his(-) donor deoxyribonucleic acid (DNA) was added to highly competent trp(-)his(+) recipient bacilli and to protoplasts prepared from these bacilli, and the cell-DNA complexes were incubated for 30 min. The complexes were then washed and lysed, and their DNA was analyzed on a trp(-)his(-) strain for the donor marker trp(+), the resident marker his(+), and for the recombinant trp(+)his(+) combination. The extracts of the bacillary complexes contained a normal percentage of donor markers (0.1-0.2%), and the number of trp(+)his(+) doubles (20% of all trp(+) transformants) indicated that the donor DNA had become integrated into the resident genomes. The protoplast complexes contained 10 to 1,000 times fewer donor markers and almost no recombinants. This indicated that, in protoplasts, marker uptake was minimal and recombination was absent. Uptake was also measured with (3)H-labeled DNA. On the average, protoplasts took up one-fiftieth as much DNA as bacilli. It was concluded that, probably, protoplasts took up no DNA at all, that there were no DNA affinity sites on the surface of the protoplasts, and that the residual marker and radioactivity uptake was due to imperfections in the experimental system. The data and conclusions differed sharply from earlier ones of Hirokawa and Ikeda despite the fact that the techniques of these authors were followed in repeat experiments.

Bacillus subtilis↗
PubMed1975

Characterization of the presumed peptide cross-links in the soluble peptidoglycan fragments synthesized by protoplasts of Streptococcus faecalis.

Protoplasts of Streptococcus faecalis ATCC 9790 were produced with the aid of lysozyme, and the ability of these bodies to synthesize soluble, peptide cross-linked peptidoglycan (PG) fragments was examined. Lysozyme digests of PG isolated using gel filtration from the supernatant medium of protoplasts grown in the presence of [14C]acetate and L-[3H]lysine contained small amounts of PG having KD expected for peptide cross-linked dimers and trimers. Addition of benzyl penicillin (300 mug/ml) to growing protoplast cultures did not affect the net amount of PG fragments synthesized but resulted in inhibition of synthesis of dimer and trimer fractions by 27 and 59%, respectively. Failure of penicillin to completely inhibit the accumulation of the dimer fraction was attributed to the presence of atypical forms of dimer. In fact, the supernatant medium of penicillin-treated cultures did not contain detectable amounts of typical peptide cross-linked dimer. The degree of peptide cross-linkage of protoplast PG was at most only 13% of that found in walls isolated from intact streptococci. The relative amounts of monomers, dimers, and trimers synthesized during early and late stages of protoplast growth was approximately the same. Protoplasts synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci.

Acetates↗
PubMed1978

Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.

Bacillus megaterium↗
PubMed1979

Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts.

Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.

Bacillus subtilis↗
PubMed1979

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).

Bacillus subtilis↗
PubMed1980

Polyethylene glycol-assisted transfection of Streptomyces protoplasts.

In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by deoxyribonucleic acid (DNA) of five temperate phages (phi C31, VP5, R4, phi 448, and S14) belonging to four different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum frequencies of transfection in the ranges of 10(-6)/DNA molecule and 10(-5)/viable protoplast were invariably obtained. It appeared that single DNA molecules initiated transfection events, and that the conformation of the DNA (i.e., circular or linear) was not important. Inhibition of transfection by ethylenediaminetetraacetic acid suggested that divalent cations were also observed. A minor subpopulation of protoplasts appeared to be particularly sensitive to transfection (i.e., "competent") in some DNA-host combinations. In such cases the size of this subpopulation was the major limiting factor in obtaining high transfection frequencies. The same protoplast

Bacteriophages↗
PubMed1981

Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.

Bacteriological Techniques↗
PubMed1964

FORMATION OF "PROTOPLASTS" OF STAPHYLOCOCCUS AUREUS WITH A FUNGAL N-ACETYLHEXOSAMINIDASE.

Hash, John H. (Lederle Laboratories, Pearl River, N.Y.), Marcia Wishnick, and Philip A. Miller. Formation of "protoplasts" of Staphylococcus aureus with a fungal N-acetylhexosaminidase. J. Bacteriol. 87:432-437. 1964.-"Protoplasts" of Staphylococcus aureus were obtained when cells were treated with a fungal N-acetyl-hexosaminidase in hypertonic sucrose solution. The "protoplasts" were sensitive to osmotic shock, and were free of amino sugars and rigid cell walls. They were unable to regenerate cell walls and form colonies. They may be true protoplasts. Osmotic barriers in the "protoplasts" were preserved, and the "protoplasts" incorporated C(14)-l-alanine at about 60% of the rate of intact cells.

Alanine↗