• KSBB Journal
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• 제30권6호
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

• 한국초지조사료학회지
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• 제15권1호
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• pp.1-12
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• 1995

This study was to investigate an effective biological control of forage diseases and provide a basic data and a model in improving variety of antagonistic bacteria, with growth promoting effect on forage, through cell fusion. The results obtained were summarized as follows; 1. The antagonistic himbacterium against soil-borne phathogenic fungi Fusarium oxysporum and Rhizoctonia solani was isolated from continuous cropping himsphere soil of forage, and its biological and physiological characteristics were investigated. This bacterium was identified as Bacillus subrilis and named BS 101. Another strain for cell fusion was Bacillus thur ingiensis ssp. kurstaki HD-I(BT 37669) with insecticidal crystal. 2. The auxotropic mutants of BS 101 and BT 37669 were derived after mutagenesis using N-methyl-N'nitro- Nitrosoguanidine(NTG) to give amino acid requirement marker. n e s e auxotropic mutants of BS 101 and BT 37669 were named BS 1013(his-) and BT 69(asp-), respectively. 3. The best protoplast requirement was obtained using DM 3 medium, containing 5% casamino acid, 1 M $MgCI_2$ and 2% bovine semm albumin, to give Fusant 3, 7 and 8. BT toxin gene was not identified with fusants by Southern blotting. However, SDS-PAGE analysis of strains showed various protein patterns among fusants. 4. From the dark culture experiment, growth of forage in inoculated soil with antagonistic bacteria was delayed than that of non-inoculated soil with antagonistic bacteria in each continuous cropping soil and in each sterilized soil. On the other hand, growth duration of forage was different between continuous cropping soil and sterilized soil. 5. Seed germination of Alfalfa, Italian ryegrass and Orchardgrass were significantly improved by inoculation of antagonistic bacteria(p< 0.05).

• Asian-Australasian Journal of Animal Sciences
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• 제22권7호
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• pp.962-968
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• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.

• BMB Reports
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• 제29권4호
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• pp.359-364
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• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• 한국균학회지
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• 제21권1호
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• pp.9-15
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• 1993

Astaxanthin을 생산하는 효모 Phaffia rhodozyma로 부터 제조된 상보적 돌연변이 균주사이의 융합체에 대한 carotenoid 함량 및 성분을 분석하였으며, 몇가지 화학첨가물에 의한 이들의 색소 증진 효과를 조사하였다. 핵 융합이 확인된 융합체들은 완전배지에서 1년 또는 그 이상 계대 후에도 매우 안정 하였다. 그리고 이들의 astaxanthin 함량은 야생형 모균주의 그것에 비해 약 $2.0{\sim}3.0$배 이었다. 또한 화학첨가물(malt extract, abscisic acid, gibberellic acid, riboflavin)에 의한 색소 증진 효과는 l% malt extract와 1 mM abscisic acid 첨가시 대조구에 비교해 총 caretenoid 함량이 35%와 11%가 각각 증진되었고, 이와 반대로 5 mM gibberellic acid와 0.1 mM riboflavin첨가시 19%와 12%가 각각 감소되었다.

• 한국수정란이식학회지
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• 제10권2호
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 폴리머
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• 제35권6호
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• pp.499-504
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• 2011

본 연구팀은 탈미네랄화 골분(DBP)이라는 천연재료를 졸(sol)화시켜 poly(lactide-co-glycolide) (PLGA)에 함침시킨 지지체를 개발하였다. DBP를 함침시킨 PLGA 지지체 상의 세포증식과 모폴로지를 평가하기 위해 MTT 분석과 SEM을 측정하였다. 또한 sGAG와 콜라겐 함량 측정과 파종된 연골 세포의 표현형 유지에 미치는 영향을 확인하였다. 그 결과 PLGA에 DBP를 함침시킨 지지체가 PLGA 지지체보다 높은 세포 증식률을 보였다. 또한 파종된 연골세포의 표현형 유지에도 긍정적인 영향을 미치는 것을 확인하였다. 이번 연구 결과를 토대로 PLGA에 DBP를 이용한 용액을 함침시킴으로써 DBP내의 성장인자와의 상호작용을 통해 연골세포의 성장에 긍정적 영향을 미쳐 안정되게 연골을 조직화할 수 있는 연골조직공학 지지체로 적합할 것으로 예상된다.

• 대한수의학회지
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• 제35권1호
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• pp.179-185
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• 1995

The fusion rates of nuclear transplant embryos with various DC voltage were 55.6-79.2%. The significantly higher fusion rates of nuclear transplant embryos were achieved at the electric field strenght of DC 1.0-2.0kV/cm(72.0-79.2%) than DC 0.75kV/cm(55.6%, P<0.05). No significant differences in the percentage of embryos that cleaved(48.1, 55.4 and 42.6% respectively) and the percentage of cleaved embryos that developed to morulae/blastocyst(1.9, 5.3 and 1.9% respectively) could be found among the three types of in vitro culture system (Granulosa cell, BOEC co-culture and SOF, P>0.01). The age of the recipient cytoplast(30 vs 40hr) had no effect on the fusion rates and the rates of cleavage development(36.9 vs 44.1%, P>0.01).

• Journal of Plant Biology
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• 제15권4호
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• pp.13-17
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• 1972

Rice callus was treated with 0.56M sucrose solution mixed with 5% pectinase and 10% cellulase, and the protoplasts isolated were transferred to 0.25 M sodium nitrate to induce protoplasmic fusion. Callus tissues were macerated well and degradation of cell walls also proceeded satisfactorily. When the protoplasts were transferred to sodium nitrate solution, many giant roundish protoplasts and some multilobed complex protoplasmic bodies were observed. Most of the fusions took place immediately after the protoplasts were transferred to sodium nitrate. Some multilobed protoplasts which failed to fuse in the initial stage took longer time, about two hours, to get completely fused and rounded-off. Multilobed protoplasmic bodies were invariably multinucleate, while giant round protoplasts had either several nuclei or had one nucleus of large size. Nuclear fusion, also, seemed to occur immediately after the protoplasts were transferred to sodium nitrate.

• 한국자원식물학회지
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• 제19권3호
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• pp.418-421
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• 2006

In this study, we surveyed the influence of IOA (iodoacetamide) and media upon protoplast fusion for the efficient production of the somatic hybrid among S. sisymbriifolium and other Solanum species (S. intergrifolium and S. toxicarium). Regardless of a breed, as the IOA concentration increases, the cell division tends to decrease. As the influence of the media on the colony formation, we could get 5 colonies from the fusion of S. sisymbriifolium and IOA-treated S. integrifolium protoplast, but none was observed in other fusions. As a result of analyzing their IDH isozyme, we found a somatic hybrid in 2 objects.