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PubMedDate not supplied

[Isolation of Staphylococcus aureus protoplasts using lysoamidase].

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.

Hydrolysis↗
PubMed1986

[Isolation and regeneration of the protoplasts of the streptomycete producers of actinomycins C and X].

Protoplasts of S. michiganensis, S. chrysomallus and Streptomyces sp. 26-115, organisms producing actinomycins C and X form in hypertonic salt solution under the action of 3-4,5 mg/ml of lysozyme on the mycelium suspension. For protoplasting, the streptomycetes were grown on the soybean medium in the presence of 0.2-0.8 per cent of glycine. The mycelium of the streptomycete exponential growth phase was more favourable for protoplast formation. Protoplast regeneration was studied on the medium described by Okanishi et al. The quantitative composition of this medium was not optimal for regeneration of protoplasts of the above streptomycetes. The level of their regeneration depended to various extents on concentration of phosphate, magnesium and calcium ions and sucrose in the regeneration medium.

Culture Media↗
PubMed1989

Plant regeneration of mesophyll protoplasts from axenic potato (Solanum tuberosum L.) shoot.

Mesophyll protoplasts from commercial potatoes were cultured in shallow liquid media. The regenerated cells divided and formed calli. Regenerant plants of two potato lines (Ke Xin 4; 68-62) were obtained from the regenerated calli after transfer to solid medium. The division frequency of the regenerated cells was influenced by culture protoplast density. Growth of the protoplast-derived colonies was sensitive to sucrose concentration in the liquid medium. The protoplast culture medium obviously affected differentiation frequency for the protoplast-derived calli (P-calli). The P-callus differentiation frequency was raised by supplementing the medium with 3% mannitol, which also shortened the P-callus differentiation.

Cells, Cultured↗
PubMed1989

Cell wall formation in regenerating protoplasts of Schizosaccharomyces pombe: study by high resolution, low voltage scanning electron microscopy.

The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.

Antifungal Agents↗
PubMed1986

Transfer of functional adenovirus E1A transcription activator proteins into mammalian cells by protoplast fusion.

Human adenovirus 2/5 E1A proteins were used to evaluate protoplast fusion as a method of transferring functional proteins into mammalian cells. Both the E1A 13 and 12 S mRNA products expressed in Escherichia coli are shown to activate in trans adenovirus gene expression following transfer into monkey kidney cells by protoplast fusion. Approximately 20% of the recipient mammalian cells exhibited positive nuclear E1A-specific immunofluorescence following fusion with protoplasts containing E1A protein. E. coli-expressed E1A protein was modified post-translationally in Vero cells following protoplast fusion, as evidenced by its shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. These results establish protoplast fusion as a simple rapid method for examining the functional activity, intracellular distribution, and post-translational modification of E. coli-expressed proteins in intact mammalian cells.

Adenovirus Early Proteins↗
PubMed1988

[Cytological study of early stages of regeneration of protoplasts of fungi and Streptomyces].

Early stages of Penicillium chrysogenum 51 and Streptomyces lividans 66 protoplast regeneration on solid media were studied microscopically under conditions of microcompartments. It was shown that at the early regeneration stages there were both rapid reversion into the mycelial form and a retarded one. In P. chrysogenum retarded regeneration resulted in formation of hypha-like structures or protoplast breaking into fragments of various sizes. Some of the fragments restored the cell walls and mycelial organization whereas the others lysed. As a result of the breaking and compartmentalization of the viable areas one protoplasts formed several centers of P. chrysogenum colony reversion. Retarded regeneration of protoplasts in S. lividans 66 resulted in their growth and multiplication in the protoplast-like L-form. On media with penicillin, glycine and horse serum there were isolated colonies of S. lividans L-forms subject to passages or reversion depending on the medium composition.

Cell Wall↗
PubMedDate not supplied

[Development of protoplasts of Streptomycetes on media favoring the regeneration and formation of L-forms].

The protoplasts of three Streptomyces species and their regenerative ability were studied using light microscopy. When Streptomyces lividans and S. erythraeus protoplasts are cultivated on regeneration media, their regeneration is not synchronous during the first day; some protoplasts revert to yield the mycelial form and also L-forms of these cultures are produced. If the protoplasts are transferred to a medium inducing L-forms, they grow and multiply for a long time with the production of L-form colonies. This process is maintained if S. lividans L-form cells are passaged on the medium inducing L-forms, but the protoplasts revert to yield the mycelial form on the regeneration medium.

Culture Media↗
PubMed1988

[Isolation and properties of the protoplasts of Streptomyces erythraeus].

The paper is concerned with development of conditions for cultivation of the erythromycin-producing organism and preparation and maintenance of its stable viable protoplasts. Optimal conditions for the culture growth and protoplasting were developed. Two-stage cultivation of the organism on media PB and S provided dense diffuse or diffuse local growth characterized by low differentiation and higher homogenicity. The incubation time at stage I was 66 hours and that at stage II was not more than 24 hours at respective temperatures. The culture was incubated on a shaker in the presence of glycine at the minimum concentration. The presence of glycine in the medium altered the culture cell walls which was evident from changing of staining by Gram from + to +/- . Treatment of such a culture with lysozyme for 30 minutes provided formation of up to 2.10(9) protoplasts per 1. ml. It is possible to maintain the protoplasts in frozen state at -20 degrees C in medium P for 1 month. Under such storage conditions the titer of the viable protoplasts as compared to the initial one decreased only 2-fold after the one-month storage.

Culture Media↗
PubMed1979

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.

Aspergillus nidulans↗
PubMed1984

Protoplast fusion in Saccharomyces cerevisiae.

The ability of different Saccharomyces cerevisiae yeast strains to form protoplasts and protoplast fusion were studied. The protoplast formation depended mainly on strains used and the time of snail gut enzyme action. The percentages of the regenerating protoplasts varied, depending on strain, from 3 to 33 per cent. From the fusion experiments one can establish that kariogamy is prerequisite for stable for stable diploid formation. The yields of protoplast fusion were higher when both strains were rho+ as compared with rho+ and rho 0 combinations.

Cell Fusion↗
PubMed1980

The effect of calcium on protoplast release from species of Aspergillus.

The effect of various osmotica and lytic enzyme treatments, on the release of stable protoplasts from Aspergillus niger and two strains of Aspergillus fumigatus, has been investigated. Most osmotica supported protoplast release at a concentration of 0.6 M except for CaCl2 and Ca(NO3)2. Supplementation of 0.6 M KCl with 0.2 M CaCl2 enhanced release from both species, but depended upon strain of organism, concentration of osmoticum and enzyme system employed. More protoplasts were released at low concentrations of KCl and/or when chitinase was present in the digestion medium. This enhancement effect was concerned with the Ca2+ ion since addition of Ca(NO3)2 also gave a higher yield and EDTA severely depressed protoplast release. Further investigations suggested that the effect of Ca2+ was not primarily acting as a supplementary osmoticum or concerned with protoplast membrane stabilization. Experiments using 45CaCl2 showed that the enhancement effect was probably due to Ca2+ affecting chitinase activity.

Aspergillus fumigatus↗
PubMedDate not supplied

[Preparation of protoplasts and localization of ribonuclease, beta-1,3-glucanase and glucosoisomerase in the fungal mycelium of Penicillium brevi-compactum].

Optimal conditions for the formation and isolation of protoplasts from the fungus Penicillium brevi-compactum were investigated. Localization of ribonuclease, glucosoisomerase and beta-1,3-glucanase in the mycelium was examined. To produce protoplasts, the mycelium was treated for 3-4 hours at 40 degrees C with the incubation mixture, containing chitinase from Actinomyces kurssanovii, lytic enzymes from Act. cellulose, lysozyme and 0.8 M mannitol as an osmotic stabilizer. The levels of activities of RNase, beta-1,3-glucanase, and glucosoisomerase were measured in the fungal mycelium before preparation of protoplasts, in the incubation mixture after their preparation, and in the protoplast lysate. The protoplast formation facilitated the release of RNase, beta-1,3-glucanase and glucosoisomerase from the fungal mycelium into the incubation mixture.

Actinomyces↗
PubMed1993

[Isolation and characterization of genetic recombinants in protoplast fusion in Saccharopolyspora erythraea].

Formation of genetic recombinants after conjugation and protoplast fusion in Saccharopolyspora erythraea, an organism producing erythromycin, was studied comparatively. After the protoplast fusion the frequency of all the classes of the haploid recombinants increased 10 to 460 times by comparison with the conjugation. The protoplast fusion was characterized by higher diversity of the recombinant classes, up to 45.5 per cent of the recombinants being formed at the account of multiple crossing overs. It was shown that unlike conjugation of the S. erythraea strains the protoplast fusion had no gradient of inheritance of the parent genetic markers by the recombinants. The results indicated that in S. erythraea protoplast fusion the recombination involved more genes of the parent strains. This makes promising the procedure for genetic analysis and design of erythromycin-producing strains.

Conjugation, Genetic↗
PubMed1987

Protoplast-mediated gene transfer into human leukemia (K562) cells.

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.

Acetyltransferases↗
PubMed1992

Factors controlling variability in measurements of the transient gene expression in electroporated protoplasts of wheat callus.

Factors controlling variability in enzyme transient expression assays have been investigated in electroporated protoplasts isolated from wheat embryogenic and nonembryogenic calli. The level of variation was reduced to a minimum through the optimization of the beta-glucuronidase measurements in the pellet and the supernatant of the homogenized protoplasts, by expressing the data on the basis of the number of protoplasts found to be viable immediately before the assay and on the amount of protein in the pellet and supernatant. Protoplast separation on the basis of size was also useful in eliminating some of the variation resulting from the heterogeneity of the callus used. Efforts to partially synchronize the callus tissue by auxin starvation have not resulted in a significant decrease of this variation. Our results indicate that the level of variation in enzyme transient activity in protoplasts resulting from calli can be reduced by implementation of the experimental techniques presented here.

Cell Membrane Permeability↗
PubMed1993

Protoplast fusion: a tool for intergeneric gene transfer in bacteria.

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.

Journal Article↗
PubMed2001

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica.

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. 'Lovely Tokyo' and P. obconica cv. 'Aalsmeer Giant White'. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.

Journal Article↗
PubMed1993

Characterization of Paraquat Transport in Protoplasts from Maize (Zea mays L.) Suspension Cells.

Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat. [14C]Paraquat uptake was measured in 400-[mu]L centrifuge tubes using silicon oil centrifugation techniques. Approximately 50% of accumulation from a 100 [mu]M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min. Membrane binding accounted for about 30% of apparent accumulation. Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component. The Km of the saturable component was 132 [mu]M, and the Vmax was 0.512 nmol [mu]L of protoplasts-1 min-1. In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent. Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response. This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface. Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive. Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake. These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings.

Journal Article↗