• 미생물학회지
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• 제22권1호
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• pp.35-40
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• 1984

Streptomyces속 균주개발의 수단으로서 이용할 목적으로 원형질체 융합방법의 확립을 시도하였다. 특히 융합빈도를 높이고 실험을 간편화하는데 역점을 두었다. 원형질체의 형성 및 재생빈도는 균의 배양시간에 따라 변하였는데 대수기에서 수확한 균체로부터 가장 높은 빈도의 수율을 얻었다. 원형질체의 형성은 다른 용균효소를 사용하지 않고 Lysozyme 단독처리 만으로도 충분히 가능하였고 원형질체의 세포막 재생은 Monolay법 보다는 Overlay법이 훨씬 좋은 결과를 주었다. Monolay법은 1.8%, Overly법은 14%의 재생빈도를 나타냈다. 본 실험에서 PEG1000 (50% W/V)를 사용한 원형질체 융합방법으로 얻은 S. coelicolor의 재조합체의 빈도는 $1.8 {\times} 10^{-2}$이었다.

• 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.

• 미생물학회지
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• 제22권2호
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• pp.103-110
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• 1984

The conditions for the protoplast fusion of auxotrophic mutants of Trichoderma koningii were determined. A preparation of commercial enzyme Driselase was used successfully to isolate protoplasts from the 18 hr old mycelium of T. koningii. The yields of protoplasts production were ranged from $0.3{\times}10^8$ to $2.5{\times}10^8$ protoplasts per mg of damp mycelium of various auxotrophic mutant strains. The regeneration frequencies from $9.3{\times}10^{-3}\;to\;2.0{\times}10^{-1}$ were obtained when the protoplasts from auxotrophic mutants were plated on the malt extract medium containing 0.6M $MgSO_4$, and 2% agar, and the optimal concentration of PEG for protoplst fusion was 30%. Exposure of protoplasts to PEG for 10 min was found to be sufficient to induce high frequency heterokaryon formation. Optimal pH of fusion mixture was determined as 5.5, and 1 mM of calcium chloride in fusion mixture was found to be sufficient to enhance protoplast fusion frequency. Under optimal condition, the fusion frequency of the cross between protoplasts from various auxotrophic mutants were $1.6{\times}10^{-2}\;and\;4.1{\times}10^{-2}$.

• Applied Microscopy
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• 제13권1호
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• pp.31-40
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• 1983

Ultrathin sections of intact mycelia, released protoplast and fused protoplast of Micromonospora rosaria were observed by electron microscopy Intact mycelia showed a typical gram-positive bacterial cell wall structure and mesosomes. Released protoplasts had no cell wall components and fibrous nuclear region was distinguished from cytoplsmic region clearly. Protoplasts which treated with sucrose supplemented buffer were stable. But those treated with buffer without sucrose were extensively damaged, forming mom braneous vesicles. It was surmised that those vesicles originated from the damaged cytoplasmic membrane. High frequency of fusion was achieved by 50%(w/v). polyethylene-glycol 1,000 Fusion bodies in different stage of fusion were observed. Cell membrane barrier was stepwise relieved.

• 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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• 제31권3호
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• KSBB Journal
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• 제13권4호
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• pp.399-403
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• 1998

This study was carried out to optimize the concentration of Ca2+ and pH of fusion medium which affected electrofusion frequency of protoplasts isolated from Nicotiana tabacum L. (cv. BY4) mesophyll cells and callus. The protoplasts were electrofused in the fusion media containing two different Ca2+ concentrations and three different pH regions. Fusion frequency was lower in the fusion medium containing only 13% mannitol as osmotic stabilizer. However, higher degree of fusion frequency (47.3%) was observed in the fusion medium containing 50mM CaCl2 at pH 10.5 than any other conditions. Cell viability was decreased by Ca2+ and high pH treatment in the fusion media, while fusion frequency was increased. It is concluded that Ca2+ is involved in electrofusion of protoplasts.

• 한국미생물·생명공학회지
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• 제18권5호
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• pp.460-465
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• 1990

Asp.wentii와 Asp.nidulans의 원형질체 재생은 2-DG가 30$\mu g$/ml 첨가된 포자현탁액을 4시간 전배양 할 때 적당하였고 ergosterol, myoniositol, casamino acid, BSA가 함유된 CBE 재생용 배지에서 효과적이었으며, 30 이상 재생률을 나타내었다. 원형질체 융합은 10mM $CaCl_2$가 함유된 pH7.5의 30PEG 4000으로$37^{\circ}C$ 에서 10분간 처리했을 때 가장 양호하였으며, 융합빈도는 $8.2\times 10^{-4}$을 나타내었다. 가장 우수한 융합주인 FWN-56은 CMCase, avicelase, $\beta$-glucosidase 및 xylanase를 동시에 분비하였으며 친주에 비하여 활성이 2.3배, 1.5배, 1.8배, 2.5배 각각 증가하였고, 또한 MM에 4중 이상 보관 후의 segregant율이 1 이내였으므로 유전적 안정성은 높았으며, 분생포자 DNA함량은 1.4-1.6배였다. 또한 핵의 크기도 친주에 비하여 큰 것으로 보아 융합주임을 확인하였다.

• 한국식품과학회지
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• 제25권4호
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• pp.366-372
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• 1993

본 연구는 미림 제조시 미림의 품질을 높이기 위한 방법의 하나로 세포 원형질체의 융합체 의해 ACPase 활성이 높은 균주를 얻으려고 시도하였다. 돌연변이 방법으로 acid carboxypeptidase(ACPase) 활성이 높은 A. oryzae 9-12와 A. shirousamii IFO 6082-60을 선별하여 사용하였다. A. oryzae 9-12와 A. shirousamii IFO 608260의 원형질체의 형성조건은 chitinase (10mg/ml), cellulase(10mg/ml) 및 zymolase(20T(5mg/ml)등의 효소 혼합용액에서 $30^{\circ}C$에서 4시간 반응시켰을 때 가장 높았으며, pH6.0일 때, 안정제로서는 0.6M KCl 및 무기 염류로서는 0.01M $CaCl_2\;2H_2O$가 가장 높았고, 30% PEG 6,000이 가장 좋았다. 이때의 융합빈도는 0.71%였다. 융합주의 ACPase의 활성은 F-50이 20,800unit/g으로 변이주보다 1.4배 높았다.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.265-270
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• 1986

L. casei세포의 유전연구를 위한 도구로서 세포융합 기술을 연구하였으며 융합세포(recombinant)를 선발하고 확인하기 위한 유전자 선발표지 인자로서 항생제 저항성이 이용되었고, 항생제 저항성 돌연변이 균주는 nitrosoguanidine을 처리하여 분리하였다. 선발 배지에서 항생제의 적절한 최종 농도는 streptomycin 25 $\mu\textrm{g}$/$m{\ell}$, hostacillin 0.5 I. U./$m{\ell}$, lincomycin 0.5$\mu\textrm{g}$/$m{\ell}$ 그리고 methicillin 5 $\mu\textrm{g}$/$m{\ell}$로 확인되었다. L. casei균주에서 높은 세포융합은 PEG 분자량 4,000에서 40%농도, 중성 부근의 pH, 3$0^{\circ}C$에서 약 1분간 처리하였을 때 얻어 졌다. 항생제 저항성의 자연돌연변이주의 출현빈도는 세포융합 출현빈도 보다 $10^2$-$10^3$ 정도 낮은 수준으로 나타났다. 세포융합 빈도는 모균에 대해 약 $10^{-4}$ 비율로 나타났다.