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PubMed1998

Enhanced mitosis in cultured protoplasts: beneficial effects of oxygenated perfluorocarbon and haemoglobin.

Cell suspension-derived protoplasts of Petunia hybrida cv. Comanche were cultured for up to 10 d in (1) aqueous medium, (2) aqueous medium overlaying an oxygenated (10 mbar, 15 min) perfluorochemical liquid (perfluorodecalin; PFC), (3) aqueous medium containing 1:50 (v:v) of the commercial haemoglobin (Hb) solution (Erythrogen), (4) aqueous medium supplemented with 1:50 (v:v) of Erythrogen overlaying oxygenated PFC. Treatments 2, 3, and 4 enhanced mean mitotic division by 111%, 80% and 139% respectively, compared to controls. Both fresh-Hb and stored-Hb significantly (P < 0.05) enhanced mitotic division, although this was 45% greater with fresh-Hb compared to stored-Hb. Both oxygenated PFC and Erythrogen provide approaches for enhancing cellular oxygen supply to eukaryotic cells. Commercially, the recoverability and recycleability of PFCs make these compounds a more attractive option in comparison to Erythrogen, despite a high initial investment cost.

Animals↗
PubMed2006

Growth characteristics of fusants by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis.

Intergeneric fusants were obtained by protoplast fusion between the thermotolerant yeast, Kluyveromyces marxianus, and the starch-assimilating yeast, Schwanniomyces occidentalis. Two thermotolerant fusants growing at 40 degrees C were screened on the medium containing soluble starch. These fusants showed weak growth in a soluble starch medium and the production of a little amylase. The carbon source assimilation and the chromosome composition of the fusants were similar to those of the K. marxianus parent. However, a chromosomal difference from K. marxianus was recognized in the fusants. These results show the possibility that the fusants are amylase-producing strains rearranged from K. marxianus.

Chromosomes, Fungal↗
PubMed1976

Effects of polyene macrolide antibiotics on normal and protoplast type L-form cells of Escherichia coli W1655F+.

The action of the polyene macrolide antibiotics mycotrienin, pimaricin, lucensomycin, tetramycin, rimocidin, nystatin, filipin, lagosin, pentaene antibiotic 2814P, flavomycoin, flavofungin, hexaene antibiotic 5001P, and candicidin, including perhydro derivatives of them, on wall-less stable protoplast type L-form and normal rod form cells of E. coli W1655F+ was studied. No inhibition of the normal rod form cells was detected. In contrast to these results the growth of the L-form cells was inhibited by all of the substances tested, with the exception of pimaricin. Further experiments have shown that the differences in sensitivity of normal and L-form cells cannot be explained by differences in sterol content, the target site of polyene antibiotics in sensitive eukaryotic cells. According to our results it is obvious that the cell wall of the normal cells functions as a penetration barrier to polyene antibiotics.

Anti-Bacterial Agents↗
PubMed1982

Fusion of protoplasts of Streptomyces lavendulae.

Protoplasts of two different auxotrophic mutants of Streptomyces lavendulae were fused with polyethylene glycol (PEG) 1,000, and allowed to regenerate on selective media. Prototrophic colonies overwhelmed other types of recombinants on any selective media. These prototrophic strains were stable after successive isolation. These results suggest that stable diploid cells were formed by cell fusion, which differed from the case of S. coelicolor.

Clone Cells↗
PubMed1982

Genetic and biochemical features of spiramycin biosynthesis in Streptomyces ambofaciens--curing, protoplast regeneration and plasmid transfer.

Spiramycin-producing Streptomyces ambofaciens KA-1028 harboring the pSA1 plasmid gave rise to spiramycin non-producing variants at high frequencies by various curing treatments. However, a number of the spiramycin non-producing progeny obtained by treatment with acridine dyes, still harbored plasmid DNAs which could not be differentiated from plasmid pSA1 by contour length, cleavage patterns and heteroduplex analysis. By treatment with mitomycin C, plasmid pSA1 was cured at high efficiency and spiramycin non-producing strains were obtained. Strain U-1717R obtained by regeneration of protoplasts of plasmid-cured strain U-1717 regained spiramycin production on growth on solid medium only. Furthermore, transconjugants obtained by mating between strain KA-1028 and U-1717R-24 (streptomycin-resistant) regained spiramycin production in both liquid and solid media. We conclude that the genes for the biosynthesis of spiramycin are encoded in a replicon other than plasmid pSA1 but that this plasmid plays a role in the regulation of spiramycin production.

Conjugation, Genetic↗
PubMed1983

Conversion of triostins to quinomycins by protoplasts of Streptomyces echinatus.

Protoplasts of Streptomyces echinatus have been used to investigate the biosynthesis of echinomycin (quinomycin A). It has been shown that this organism has the capacity to convert a series of triostins to the corresponding quinomycins by a mechanism involving methylation. Evidence is presented which suggests that triostin A is the natural precursor of echinomycin. Conversion of tetra-N-demethyl analogues of triostin A to corresponding analogues of echinomycin was not detected.

Anti-Bacterial Agents↗
PubMed1984

Interspecific protoplast fusion among macrolide-producing streptomycetes.

From auxotrophic and idiotrophic mutants of Streptomyces fradiae (tylosin producer) and Streptomyces sp. AM 4900 (pikromycin producer) or Streptomyces narbonensis (narbomycin producer), prototrophic fusants were obtained at a low frequency by the protoplast fusion technique. In the cross of S. fradiae 261-27E (mycaminose-idiotroph, ilv) and Streptomyces sp. AM 4900 N3-4, (pikronolide-idiotroph, arg), an unstable prototrophic fusant, strain No. 14, produced a macrolide antibiotic which was not produced by the wild type, parent strains, and the productivity was lost within a few times transfer. It was concluded that the fusant was not a recombinant, but a heterokaryon. On the other hand, relatively stable fusants were obtained from the cross of S. fradiae TBM (mycaminose-idiotroph, met) and S. narbonensis NA12US3 (narbonolide-idiotroph, his, str) at a frequency of 3.2 X 10(-5). One of the prototrophic fusants produced narbomycin, which is believed to be due to a result of interspecific recombination.

Anti-Bacterial Agents↗
PubMed1988

Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational gene alteration and gene amplification.

Genetic mechanisms involved in the kanamycin (KM)-hyper-resistance of Streptomyces griseus SS-1198PR and NP1-1PR generated by protoplast regeneration were investigated. Southern hybridization of Sph I-digested genomic DNA with a KM-resistance gene (kan) probe revealed that the strain SS-1198PR and its KM-sensitive parent (SS-1198) had the same copy number of a 4.2-kb Sph I fragment hybridizing to the kan probe, while the kan gene of the strain NP1-1PR was located on a highly amplified DNA sequence (100-200 copies/chromosome) consisting of the 6.7 kb amplifiable unit with Sph I site at the junction site region. There was no difference in the restriction endonuclease cleavage map of the kan gene region of the Sph I fragments from the three strains. However, the level (50 micrograms/ml) of KM-resistance conferred by the cloned NP1-1PR kan gene was much lower than that (1,000 micrograms/ml) conferred by the cloned SS-1198PR kan gene in Streptomyces lividans TK21 although the strain TK21 harboring these kan genes produced an aminoglycoside acetyltransferase, AAC(3)-V, with the same substrate specificity. It seemed, therefore, likely that a mutational alteration of the kan gene and a DNA amplification involving the kan region played major roles for the KM-hyper-resistance depending on AAC(3)-V in the strains SS-1198PR and NP1-1PR, respectively.

Base Sequence↗
PubMed1995

Electrofusion of plant cell protoplasts under microgravity--a D-2 spacelab experiment.

Plant cell protoplasts, derived from sexually incompatible plant species, have proved to be a good system for somatic hybridization by electrofusion. Under microgravity, an increase in fusion yield can be expected, especially if the parental cells differ markedly in size or specific density. On the D-2 spacelab mission flown in 1993, electrofusion experiments were performed with three different objects, i.e. tobacco as model system, Helianthus as an important crop, and Digitalis as a plant of pharmacological interest. The resulting fusion products were cultivated (along with parental cells) for 10 days under microgravity, and subsequently regenerated on ground for biochemical analysis. Results are presented on the observation of the fusion process during flight, heterofusion yields, ultrastructural investigation of fusion products immediately after fusion, and characterization of the resulting hybrids. The results are interpreted on the background of earlier microgravity-experiments on sounding rockets or parabolic flights.

Cardenolides↗
PubMed1996

Regeneration of plant cell protoplasts under microgravity: investigation of protein patterns by SDS-PAGE and immunoblotting.

As part of the D-2 Spacelab mission, tobacco (Nicotiana tabacum L.) protoplasts were cultured for 10 days in microgravity and successfully regenerated into microcalli, which, after further cultivation on the ground, gave rise to intact plants. Protein analysis was performed on samples taken during the initial microgravity period and compared to ground controls. Total protein content and protein patterns were monitored, as well as the cytoskeletal proteins tubulin and actin, a key enzyme of secondary metabolism, phenylalanine ammonia lyase, and the pathogenesis-related protein osmotin. None of the investigated proteins showed a gravity-dependent effect. Since relative changes due to culture age were detectable in the immunoblots as well as in the total protein pattern, an adaptation of the cells to microgravity without major modifications of their protein complement may be assumed.

Actins↗
PubMed2001

[Study on intergenera fusion of protoplasts from Rhizobium leguminosorum and Sinorhizobium xinjiangnesis].

Penicillin and chloromycetin were regarded as the sign of resistance to antibodies of R. leguminosorum USDA2370 and S. xinjiangnesis CCBAU110 respectively. Using the protoplast fusion technique, USDA2370 and CCBAU110 were successfully fused. Fusion hybrid can inoculate in the leguminous of parental strains respectively. There were apparent differences between parents and fusion hybrid in cell morphology, colony and pattern of whole-cell protein. The values of DNA homology between fusion hybrid and USDA2370 and CCBAU110 were 56.6% and 10.2% respectively.

Bacterial Proteins↗
PubMed1998

[Observation of female gametophyte development of Ginkgo biloba and its protoplast isolation and culture].

Results of observation showed that the female gametophyte of Ginkgo biloba was at the coenocytic stage from March 30ty to May 30ty. A density of 6-8 x 10(5) protoplasts/ml with a viability of 87.3% was achieved when the female gametopytes collected from May 8th to 15th were treated with 0.5% cellulase Onzuka R-10, 0.5% Pectolyase for 4-5 hours. The thin-layer liquid Murashige and Tuker medium modified by omitting ammonium ions and supplementing with glutamine 1000 mg/L, Vc 5 mg/L, benzyladenine 1.0 mg/L and naphthaleneacetic acid 3.0 mg/L was used for the protoplast culture and multiple cell colonies were obtained.

Cells, Cultured↗
PubMed2002

High performance liquid chromatography and photodiode array detection of ferulic acid in Rubus protoplasts elicited by O-glycans from Fusarium sp. M7-1.

So far only little data have been available concerning the eliciting capacity of well defined glycan molecules isolated from plant pathogens. This study brings new information about changes in plant cells caused by fungal pathogens. Sugar fractions derived from glycoproteins isolated from the fungus Fusarium sp. M7-1 have been tested here as signaling molecules. The ability of three O-glycan fractions (named in this work inducer I, II, III) to trigger responses in Rubus protoplasts has been examined. It was found that inducer III was the most efficient as it elicited changes in the levels of phenylpropanoid pathway intermediates in relation to phenylalanine-ammonia lyase (PAL) activation.

Chromatography, High Pressure Liquid↗
PubMed1999

[Construction of thermotolerant ethanol-producing yeast by protoplast fusion].

Protoplast fusion between Saccharomyces cerevisiae A001 and Kluyveromyces Y034 has been studied. A001 is a high alcohol yield yeast and can ferment maltose into ethanol, but can not grow at 45 degrees C. However, Y034 can grow above 45 degrees C but has a lower alcohol yield and can not ferment maltose. We obtained the fusants which were tested in several aspects such as cell morphology, physiological and biochemical feature, isozyme patterns analysis, alcohol fermentation at high temperature(45 degrees C) and so on. The fusant AY023 with the final ethanol concentration of 7.4% has been selected, which is thermotolerant yeast with the highest alcohol yield at 45 degrees C until now.

Cell Fusion↗
PubMed1992

[Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases].

The culture of Xanthomonas rubrilineans was able to synthesize a number of intracellular aminopeptidases. To study localization of the enzymes in the cells, a protoplasting procedure was developed providing the yield of 99.7 per cent. The following subcellular fractions were isolated: periplasmic, cytoplasmic and membranous. It was shown that alanine aminopeptidase was a cytoplasmic enzyme and glutamate peptidase was a membrane-bound enzyme.

Aminopeptidases↗
PubMed1992

Electrofusion of protoplasts from Porphyra haitanensis and P. yezoensis thalli (Rhodophyta).

In this paper the electrofusion of protoplasts from P. haitanensis and P. yezoensis thalli by using an electrofusion instrument (Shimadzu Company, Japan) under different conditions of AC field, DC pulse, fusion buffer solutions and concentrations of protease are described. The results showed that the fusion rate could reach 21-31% by applying AC field of 30-35V for 25-30s and DC pulse of 300-350 V for 60 microseconds and in the presence of 3 mM Ca2+ and 3 mM Mg2+ in the fusion buffer. The pretreatment with protease had a positive effect on cell fusion. The fusion cells formed cell walls after 7 day's culture and grew into cell aggregates after 41 day's culture.

Buffers↗
PubMed1992

Selection of strains capable of utilizing D-xylose and cellobiose to produce ethanol by electric field-induced protoplast fusion.

The protoplast fusion between Candida guilliermondii S208 (Arg-) and Saccharomyces cerevisiae 314 was induced by three pulses (18kV/cm, 10 microseconds duration) applied at an interval of 1 s with the model GH-401 Electric Induced Gene Transfer/Cell Fusion System. The frequency of appearance of prototrophic hybrids was 3.6 x 10(-3). Stable fusion products were obtained after continual subculture over 20 times on selected and unselected media. Comparative studies showed that the fusion products were real hybrids of both parents. Two fustants, F-106 and F-308, could ferment both D-xylose and cellobiose to ethanol. The ethanol production of F-106 in the media containing 2% D-xylose or 2% cellobiose were 3.1g/l and 1.05g/l, respectively. The ethanol production of F-308 in the above media were 0.2g/l and 2.8g/l, respectively.

Candida↗