• 미생물학회지
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• 제23권3호
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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.

• 미생물학회지
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• 제22권3호
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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.

• 한국미생물·생명공학회지
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• 제16권4호
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• pp.303-309
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• 1988

Cellulose를 이용할 수 있는 Cellulomonas속 균주의 종간 세포의 융합방법을 확립하기 위해 원형질체 재생 및 융합조건에 대해 검토하였다. Cellulomonas속 균주의 원형질체는 osmotic stabilizer를 포함하는 재생배지에서 유동성 한천배지로 중층하여 재생시켰고 재생 확인은 주사전자현미경으로 하였다. 원형질체 융합은 항생물질 내성과 영양요구성 돌연변이주의 유전자 표지에 확인하였다. Lysozyme을 처리하여 형성된 C. flavigena 원형 질체의 세포벽 재생은 osmotic stabilizer로 0.4M sorbitol을 포함하는 재생배지에서 약 15% 수준이었으며 여기에 Polyvinyl Pyrrolidone(PVP) 3% 첨가로 재생율을 약 3배정도 증가시킬 수 있었다. Cellulomonas속 균주의 변이주간의 원형질체 융합은 융합유도제로 PEG 6000을 취하여 최적농도 40% (w/v), 치적 처리시간 15분, Ca농도 25mM에서 약 2.0$\times$$10^{-4}$ - 4.0$\times$$10^{-4}$의 융합빈도를 얻을 수 있었다.

• Mycobiology
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• 제34권2호
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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.

• 미생물학회지
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• 제24권4호
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• pp.364-369
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• 1986

Streptomyces tuberaidicus 의 원형질체 생성, 생성된 원형질체의 정상 균사체료의 환원 그리고 원형질체 융합에 대하여 조 사하였다. 또한 균사체로부터 원형질체가 생성되는 과정을 주사전자현미경을 사용하여 관찰하였다. 원형칠체는 슐샤의 끝부위 에서 뿐만 아니라 균샤 말단의 부풀어오른 부위 그리고 균사의 중간 부위에서도 생성이되는 세 종류의 원형질체 생성양을 보였다. 원형질체의 정상균사체로의 환원률은 tryptone-yeast extract-sodium acetate-$MgCl_2-CaCl_2$-sucrose로 조성된 배 지에서 대략 17%이었으며, 이때 $Ca^{++},\;Mg^{++}$ soccrose의 농도가 각각 50mM, 5mM, 0.4-0.SM일때 최척의 환원률을 보였다. S. tµberaidicµs의 histidine과 adenine을 요구하는 두 균주의 원형질서l간의 융합에서 30% 이상의 융합빈도플 얻

• 미생물학회지
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• 제25권4호
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• pp.274-281
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• 1987

Candida pseudotropicalis의 영양요구성 돌연변이 균주들에 대한 원형질체 형성, 재생 그리고 융합에 있어서 bovine serum albumin (BSA), myoinositol 그리고 ergosterol의 증진효과를 연구하였다. 원형질체 형성율은 영양요구성 균주의 type에 따라 48%-98%였다. O. 5mg/ml의 myoinositol과 0.1mg/ml의 ergosterol을 배양액에 첨가하고 4mg/ ml의 BSA을 원형 질체 형성 완충용액에 첨가했을 때는 50~100%의 원형절체 형성윷을 얻었다. 영양요구성 균주의 생장배지에 myoinositol과 ergosterol을 동시에 첨가하고 원형질체 형성 완충용액에 BSA를 침가하면 2.2-3.0애의 원형질체 재생율 증진을 보여주었다. 원형질체 융합의 최적조건에서 상보적 영양요구성 균주간의 융합율은 $7.0\times 10^{-4}$ ~ $1.5\times 10^{-3}$ 이였으며 상기화합물을 처리하지 않고 얻은 융힐l율과 비교해서 1. 9-2. 3배의 증가를 보였다. 이러힌 결과로 본 연구에서 사용된 균주들에서는 상기화합물 처리 에 의해 원형질체 재생은 물론 세포융합율도 증진됨을 알 수 있었다.

• BMB Reports
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• 제49권6호
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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.

• 미생물학회지
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• 제29권2호
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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.

• 미생물학회지
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• 제21권3호
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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.

• 미생물학회지
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• 제31권4호
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• pp.318-325
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• 1993

아닐린 분해균주 Flavimonas oryzihabitans KU21 의 원형질체 생성, 재생, 그리고 융합 등의 최적 조건을 조사하였다. 세포를 37.deg.C 로 prewarming 시킨 0.2 M Tris-Hcl(pH8.0) 완총액으로 현탁시킹후 0.5% 이상 원형질체로 전환되었으며, 이때 효소처리는 37.deg.C 에서 진탕하지 않고 처리했을 때가 가장 효과적이었다. MgCl/sub 2/ 와 CaCl/sub 2/ 수용액은 원형질체의 안정성을 높였고 완층액에 0.8% BSA 를 첨가함으로써 상온에서 4시간 까지 원형질체의 생존력을 80% 까지 유지할 수 있었다. 원형질체의 재생은 overlaying 방법이 가장 효과적이어서 3.8% 의 재생을 보였다. Rich regeneration medium 에 20 mM CaCl/sub 2/ 를 첨가하였을 때 재생율이 약 3.5배 증가하였고 완충액에 0.8% BSA 를 첨가했을 때는 4.5배의 증가율을 나타내었다. 삼투안정제로 0.5 M sucrose 를 첨가한 rich regeneration medium 에서 F. oryzihabitans 의 원형질체는 top plating 하여 6시나 이후부터 재생되기 시작하여 11 시간까지 80% 의 재생이 이루어졌다. 융합원으로 40.deg. PEC6000 과 CaCl/sub 2/ 를 사용하여 F, oryzihabitans 의 종내 원형질체 융합을 유도하였을때 recombinants 의 생성율은 2.0 * 10/sup -5/-3.6 * oryzihabitans 의 종내 원형질체 융합을 유도하였을때 recombinants 의 셍성율은 2.0 * 10/sup -5/-3.6 * 10/sup -5/ 이었으며 recombinants 들은 여러 세대 후에도 분리되지 않고 안정하였다.