• Korean Journal of Microbiology
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• v.23 no.3
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• pp.190-196
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• 1985

For frequency improvement of protoplast fusion in Brevibacterium flavum, Brevibacterium lactofermentum lactofermentum and Corynebacterium glutamicum, the effect of plasma expanders on fusion and cell wall regeneration, compatison between direct and two-step selection method, tendency of fusion frequency according to pH of fusion fluid and polyethylene glycol concentration were examined. By addition of 3% polyvinyl pyrrolidone to cell wall regeneration medium, regeneration frequencies were expressed 23 (Brevibacterium lactofermentum), 10.4 (Brevibacterium flavum) and 2.7 (Corynebacterium glutamicum) times higher than those of none polyvinyl pyrrolidone medium respectively.

• Korean Journal of Microbiology
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• v.22 no.3
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• pp.175-181
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• 1984

In order to develope a protoplast fusion system for industrial coryneform bacteria, the optimum conditions for the formation and regeneration of progoplast were examined for Brevibacterium flavum and Corynebacterium glutamicum and the protoplast fusion was performed. For the formation of the protoplast of B. flavum and C. glutamicum, the optimum time for penicillin G. treatment to obtain protoplast was mid-exponential growth phase ($O.D_{580}=0.6-0.8,\;8.0{\times}10^7-1.0{\times}10^8cell/ml$). At the optimum conditions (0.3units/ml penicillin G and $400{\mu}g/ml$ lysoyme for treatement), frequencies of protoplast formation and protoplast regeneration were 99% and 25%, respectively. Protoplast regeneration frequency was highest under the optimum conditions for the protoplast formation. Addition of 25mM $Mg^{2+}\;and\;50mM\;Ca^{2+}$ to the regeneration medium further increased the regeneration frequencies. The protoplast fusion frequencies of B. flavum and C. glutamicum in intraspecies fusion were $1.0{\times}10^{-8}\;and\;7.8{\times}10^{-4}$, of the regenerated protoplast respectively, when 33% of PEG (polythylene glycol) 6,000 was used as the fusing agent.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.303-309
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• 1988

In order to establish the process of interspecific protoplast fusion of the genus Cellulomonas capable of utilizing of cellulose, C. flavigena NCIB 12901 and Cellulomonas sp. CSI-1, the optimum conditions for the regeneration and fusion were examined. The condition of suitable osmotic stabilizer for the protoplast regeneration of C. flavigena was established by using 0.4M sorbitol. And then, by addition of 3% po]yvinyl pyrrolidone (PVP) to cell wall regeneration medium, regeneration frequency was increased 3 times higher than that without PVP addition. The optimum conditions for the interspecific protoplast fusion between auxotrophic and antibiotics resistant mutants were obtained with 40%(W/V) of PEG (polyethylene glycol) 6000 as the fusogenic agent and 25mM of CaCl$_2$on treating time for 15 min. The fusion frequency between mutants was from 2.0$\times$10$^{-4}$ to 4.0$\times$10$^{-4}$ under the optimum conditions. The fusants were confirmed to revert from protoplast to cells of rod type during regeneration process and the aggregation of protoplast by PEG was observed. Also the progress of fusion was observed by scanning electron microscopy, Many isolated fusants were shown to be complement clones of both parents which occured at a high frequency among the isolated clones.

• Mycobiology
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• v.34 no.2
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• pp.73-78
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• 2006

Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulted in the maximum release of protoplasts from 3-day-old mycelia of P. florida ($5.3{\sim}5.75{\times}10^{7}$ protoplasts/g) and P. pulmonarius ($5.6{\sim}6{\times}10^{7}$ protoplasts/g). The isolated protoplasts of P. florida regenerated mycelium with 3.3% regeneration efficiency while P. pulmonarius showed 4.1% efficiency of regeneration. Polyethyleneglycol (PEG)-induced fusion of protoplasts of these two species resulted in 0.28% fusion frequency. The fusant produced fruiting bodies on paddy straw but required a lower temperature of crop running ($24{\pm}2^{\circ}C$) than its parents which could fruit at $28{\pm}2^{\circ}C$. The stable fusant strain was selected by testing for the selected biochemical markers i.e. Carbendazim tolerance and utilization of the lignin degradation product, vanillin.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.364-369
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• 1986

A procedure for the preparation, regeneration and fusion of protoplasts of Streptomyces tubercidicus was confirmed. Also, protoplast releasingprocesses from mycelia were observed by scanning electron microscope. Three types of protoplasts releasing processes-from the hyphal tip, hyphal end regions and lateral regions of the hyphae-were observed. More than 17% regeneration efficiency was obtained by regeneration medium that is composed of tryptone-yeast extract-sodium acetate-$MgCl_2-CaCl_2$-sucrose. Optimal concentrations of $Ca^{++},\;Mg^{++}$ and sucrose in the regeneration medium were 50mM, 0.4-0.5M respectively. Above 30% of fusion frequency of the protoplasts derived from two auxotrophic strains of S. tubercidicus was induced by polyethylene glycol 4000(60% w/v).

• Korean Journal of Microbiology
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• v.25 no.4
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• pp.274-281
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• 1987

The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

• BMB Reports
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• v.49 no.6
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• pp.303-304
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• 2016

Myoblast fusion is important for skeletal muscle formation. Even though the knowledge of myoblast fusion mechanism has accumulated over the years, the initial signal of fusion is yet to be elucidated. Our study reveals the novel function of a phosphatidylserine (PS) receptor, stabilin-2 (Stab2), in the modulation of myoblast fusion, through the recognition of PS exposed on myoblasts. During differentiation of myoblasts, Stab2 expression is higher than other PS receptors and is controlled by calcineurin/NFAT signaling on myoblasts. The forced expression of Stab2 results in an increase in myoblast fusion; genetic ablation of Stab2 in mice causes a reduction in muscle size, as a result of impaired myoblast fusion. After muscle injury, muscle regeneration is impaired in Stab2-deficient mice, resulting in small myofibers with fewer nuclei, which is due to reduction of fusion rather than defection of myoblast differentiation. The fusion-promoting role of Stab2 is dependent on its PS-binding motif, and the blocking of PS-Stab2 binding impairs cell-cell fusion on myoblasts. Given our previous finding that Stab2 recognizes PS exposed on apoptotic cells for sensing as an "eat-me" signal, we propose that PS-Stab2 binding is required for sensing of a "fuse-me" signal as the initial signal of myoblast fusion.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.123-129
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• 1991

In order to develop the protoplast fusion method of the strains of Fusarium, the interspecific protoplast fusion was attempted between Fusarium poae and F. sporotrichioides. Various auxotrophic mutants were isolated by the treatment of N-Methyl-N'-Nitro-N-Nitrosoguanidine. The optimal conditions for the formation and regeneration of protoplasts were examined and the characteristics of a fusant were studied. As a results, protoplasts were readily obtained from 18 hours cultured mycelia by the treatment of driselase for 3 hours and 0.6 M KCl as a best osmotic stabilizer at pH 6.0 for the formation of protoplast. Sucrose was the most suitable for the regeneration. Polyetylene glycol (M.W. 8,000) in $CaCl_{2}$-glycine solution was used to induce the protoplast fusion. The interspecific fusion frequency between protoplasts among the auxotrophic mutants of the two strains ranged from $2.7*10^{-2}$ to $5.7*10^{-3}$ . DNA content and cellulase activity were rather increased in the interspecific fusant. The lag phase of growth curve was slightly elongated in the fusant.

• Korean Journal of Microbiology
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• v.21 no.3
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• pp.156-162
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• 1983

Conditions for effcient formation and regeneration of protoplasts of Micromonospora rosaria and Micromonospora purpurea were investigated. The state of inoculm, culture stage and growth in a medium containing partially growth-inhibiting concentration of glycing have significant effects on portoplasting. A high frequency of regeneration (up to 30%) was accomplished with a hypertonic regeneration agar medium defined by Okanishi for Strptomyces. Using the optimal conditions for protroplasting and regeneration, protoplast fusion of auxotrophic M.rosaria was carried out. Polyethylene glycol 1,000 was chosen for fusogenic agent. When signgle auxotrophs were used, the recombinant frequency of auxortrophic markers varied from 1.3 to 3.2%. Using two double auxotrophs, the recombinant frequencies of 0.7-4.3% were obtained. Much lower frequencies(three or more orders of magnitude) were observed by the conventional matings.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.318-325
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• 1993

The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at $37^{\circ}C$ for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were $2.0{\times}10^{-5}~3.6{\times}10^{-5}$. All tested recombinant clones were very stable on further propagation.