• Journal of Korean Society of Forest Science
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• 제104권4호
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• pp.549-557
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• 2015

Novel cpSSR primers were developed based on the sequence information of the Pinus koraiensis chloroplast genome. A total of 30 cpSSR loci were detected in the chloroplast genome, and a total of 30 primer sets flanking those loci were designed. All primer sets were successfully amplified for chloroplast DNA in P. koraiensis. The cross-species transferability of the 30 primer sets was considerably high in P. pumila (100%) and P. paviflora (97%) belonging to the same Subgenus (Strobus) of P. koraiensis. Meanwhile, the transferability was relatively low (73%) in P. densiflora and P. sylvestris belonging to Subgenus Pinus. A total of 13 cpSSR loci out of the 30 loci were polymorphic in the Mt. Jumbong population of P. koraiensis. The mean of haploid diversity(H) was 0.512. The number of haplotypes(N) and the haplotype diversity($H_e$) were 25 and 0.992, respectively. Of the 25 haplotypes, 22 were unique in the analyzed population. The unique haplotypes differentiated 22 individuals (79%) from the total of 28 individuals. In conclusion, the novel cpSSR primers developed in this study would be applicable to other Pinus species, especially the subgenus Strobus, and provide a high level of polymorphism for the study of genetic variation of P. koraiensis.

• Korean Journal of Animal Reproduction
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• 제24권4호
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• pp.419-428
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• 2000

Leptin, the product of obese (ob) gene, is an adipocyte-derived satiety factor that plays a major role in the regulation of food intake, energy homeostasis, body weight, reproductive physiology and neuropeptide secretion. The present study was designed to generate transgenic mice expressing antisense mouse ob (mob) gene. Total RNA was extracted from the adipose tissues of mouse, then reverse transcription was performed. The 303 and 635 bp fragments of anti I and II cDNAs were amplified from mob cDNAs by PCR. The two mob cDNAs were reversely ligated into between adipose tissue specific aP2 promote and SV40 poly(A) site. Transgenic mice carrying two different kinds of antisense mob transgenes were generated by DNA microinjection into pronucleus. Total 14 transgenic mice were born, and the 4 and 5 founder lines of the transgenic mice with anti I and II transgenes were respectively established. Antisense mRNA expression was detected in transgenic F$_1$ mice by RT-PCR analysis. This result suggests that the transgenic mice expressing antisense mob mRNA may be useful as an animal disease model to be obesity caused by decreased amount of leptin secretion.

• Journal of Sericultural and Entomological Science
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• 제52권2호
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• pp.102-109
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• 2014

We constructed the fibroin H-chain expression system to produce enhanced yellow fluorescent proteins (EYFP) in the silk of transgenic silkworm. Fluorescent silk could be made by fusing EYFP cDNA to the heavy chain gene and injecting it into a silkworm. The EYFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EYFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The yellow fluorescence proving that the fusion protein was present in the silk. Accordingly, we suggest that the EYFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Life Science
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• 제18권7호
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• pp.912-917
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• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

• Microbiology and Biotechnology Letters
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• 제33권4호
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• pp.281-287
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• 2005

The endoinulinase gene (inu, 2.733 kb, EC 3.2.1.7) from Paenibacillus polymyxa was subcloned into an Escherichia coli-yeast shuttle vector with GALl promoter for the expression in Saccharomyces cerevisiae. The constructed plasmid, pYGENIU27 (8.6 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil and on the inulin-containing media. The recombinant endoinulinase was predominantly localized in the periplasmic space of the yeast cell. The total activity of the endoinulinase reached 1.81 unit/ml by cultivation of yeast transformant on YPDG medium. The optimized conditions determined for the inulooligosaccharides (IOSs) production from inulin were as follows; pH, 8.0; reaction temperature, $45^{\circ}C$; inulin source, Jerusalem artichoke. Enzyme activity was stably maintained up to the pH of 10.0. Under the optimized condition and with endoinulinase of 36 unit/g-inulin, IOSs started to be produced after 10 min of enzymatic reaction. By the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), and inulotetraose (F4) were produced and F3 was the major product. Consequently, these data would be used as a fundamental parameters for the production of functional sweetener IOSs from inulin by recombinant yeast endoinulinase.

• Journal of the Korea Organic Resources Recycling Association
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• 제11권1호
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• pp.127-128
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• 2003

본 연구는 퇴비화 과정 중 난용성 인산의 가용화와 무기태 인산의 변화를 알아보기 위하여 수행하였다. 비료와 퇴비중의 인산형태는 다른 성분들보다 토양에 흡착 또는 고정되거나 불용화 되는 양이 많아 작물의 흡수량이 적다. 시비된 인산의 흡수율은 낮고, 그 대부분은 난용화되기 때문에 토양에 축적되거나 세탈과 용탈에 의해 수질을 오염화시키는 주원인이 되고 있다. 퇴비화 과정중의 인산형태별 함량변화를 분석조사하여 작물에 시비되는 인산비료와 퇴비의 시용량을 적절하게 조절하여 인산의 과잉 시비량을 저감시키기 위한 연구이다. 돈분을 원료로 한 퇴비화 과정에서 단계별로 퇴비시료를 채취하여 총인산(T-P), 유효인산(Avail. -P)과 무기태인산분획별(Ca-P, Al-P, Fe-P)로 분석한 결과는 다음과 같다. 퇴적더미의 초기부피는 570L였으며, 약 2개월간의 퇴비화를 통해서 시료채취와 미생물등의 분해작용으로 최종부피는 430L정도로 감소하였다. 이는 초기의 부피보다 25% 감소하였다. 퇴적더미의 분해로 인한 용적밀도의 변화를 고려하면, 총인산 함량은 초기 약 17,500mg/kg에서 최종시료는 22,500mg/kg로 증가되었다. 또한 퇴비화가 진행됨에 따라 인산의 가용태가 증가하는 결과를 보였으며, 초기의 유효인산이 4,500mg/kg에서 최종시료에서는 8,900mg/kg으로 증가되었다. 그리고 무기태 인산분획별 인산의 형태별 변화를 조사한 결과, 퇴비화 과정 중 Ca-P의 경우 pH와의 중요한 상관관계를 갖고 있었다. 유기물분해를 통해 유리된 인산과 Ca은 난용태로 전환되는데, 초기의 약 10일 동안 Ca-P의 감소원인은 pH의 감소로 인한 Ca이 유리되는 정도가 낮기 때문인 것으로 해석된다. 초기 Ca-P형태의 인산함량은 11,900mg/kg으로 Fe-P와 Al-P보다 많았다. 또한 퇴비화가 안정화되어 부숙된 최종시료의 무기태 인산분획물 중 Ca-P는 18,000mg/kg로 증가하였으며, Ca-P>Al-P>Fe-P의 순 이었다. 그러나 Al-P와 Fe-P 형태의 무기태인산은 초기의 함량비율보다 다소 감소한 결과를 보였다. 결론적으로, 퇴비화과정 중 단계별 인산함량의 형태전환을 분석한 결과 총인산의 함량은 퇴비화가 안정화될수록 부피감소로 인한 인산함량이 증가하는 경향을 보였지만, 유기물질의 분해로 인한 원료내 인산의 형태가 불용태와 난용태에서 가용태 인산으로 전환되는 것을 도출하였다. 또한 무기태 인산분획물에서는 Ca-P 인산형태가 퇴비화가 진행될수록 증가한다는 결과를 얻었으며, Fe-P와 Al-P는 분해된 유기물의 킬레이트작용으로 감소되었다고 판단되며, 그 존재형태가 경쟁적임을 알 수 있었다. 따라서 화학비료와 퇴비의 시용이 병행될 경우에는 퇴비의 가용태 인산함량뿐만 아니라 무기태 인산의 함량을 분석한 후 인산질비료의 시비량을 조절해야할 것으로 판단된다.

• The Korean Journal of Mycology
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• 제27권6호
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• pp.399-405
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• 1999

Intergeneric hybrids between Saccharomyccopsis fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-) were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic S. cerevisiae mutants and new strains showing an increased starch degrading capability were selected. Maximum production of protoplasts was obtained from the treatment with 0.1 % Novozym 234 at $30^{\circ}C$ for 90 min, and most effective osmotic stabilizer for the isolation of protoplasts was 0.6 M KCl at pH 5.8. The frequency of protoplast regeneration was 14.64% under the conditions. Genectic stability, conidial size, DNA content, and nuclear stain suggested that the fusants were aneuploidy. The specific activity of ${\alpha}-amylase$ was observed to increase about $1.2{\sim}1.9$ folds.

• Microbiology and Biotechnology Letters
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• 제26권6호
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• pp.537-544
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• 1998

The cultivation conditions of transformant Alcaligenes eutrophus AER5 harboring cloned phbC gene for mass production of poly (3-hydroxybutyrate-3-hydroxyvalerate)[P(3HB-3HV)] containing high molar fraction of 3-hydroxyvalerate (3-HV) were investigated. In two-stage batch cultivation, transformant accumulated P(3HB-3HV) containing 52.2 mol% of 3HV compared to 30 mol% of parent strain A. eutrophus H16. The increased 3-HV molar fraction was due to the amplified activity of PHB synthase participating in condensation of 3-HB and 3-HV. To increase efficiency of P(3HB-3HV) accumulation, fructose was added along with precursor compound valerate, and total cell mass and P(3HB-3HV) concentrations remarkably increased, but not 3-HV molar fraction. The effect of magnesium ion showed that P(3HB-3HV) concentration and 3-HV molar fraction were significantly increased upto 6.1 g/L and 71.3 mol% at 0.01 g/L of MgSO$_4$, respectively. The efficiency of several pH adjuster, NaOH, NaOH and (NH$_4$)$_2$SO$_4$, and NH$_4$OH, on total cell mass, p(3HB-3HV) concentration, and 3-HV molar fraction was also compared. To overcome the disadvantage of two-stage cultivation, one-stage intermittent fed-batch cultivation was attempted, such a way 10.0 g/L of fructose was supplied for cell growth at initial 36 hr and then 10.0 g/L of valerate and 5.0 g/L of fructose were applied to induce the accumulation of P(3HB-3HV), consequently, 10.4 g/L of P(3HB-3HV) with 38 mol% of 3-HV fraction could be obtained after 72 hr. These results can be used for elucidating cultivation strategy for mass production of P(3HB-3HV) containing high 3-HV molar fraction using transformant A. eutrophus AER5 harboring cloned phbC gene.

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.203-207
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• 2004

The transgenic mice carrying human Interleukin-10 (hIL-10) gene in conjunction with bovine (3 -casein promoter express hIL-10 in milk during lactation. In this study, stability of germ line transmission and expression of hIL-10 transgene integrated into host chromosome were monitored up to generation F8 of transgenic mice. When male mouse of generation F8 was crossbred with normal females, approximately half of offspring (50.9±5.8%) were identified as transgenic mice. Generation F9 to F15 mice also showed similar transmission rates (66.0±20.1%, 61.5±16.7%, 41.1±8.4%, 40.7±20.3%, 61.3±10.8%, 49.2±18.8% and 43.8±25.9%, respectively), implying that hIL-10 transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human IL-10 from milk of generation F9 to F14 mice were 3.6± 1.2 mg/ml, 4.2±0.9 mg/ml, 5.7±1.5 mg/ml, 6.3±3.5 mg/ml, 6.8±4.5 mg/ml and 6.8±3.1 mg/ml, respectively, which was showed high-level expression compared with that of generation F1 (1.6 mg/ml) mice. In conclusion, our results suggest that transgenic mice can be continuously passed their transgenes to the progeny through the breeding program with the same productivity of human IL-10 protein in their milk.

• Journal of Life Science
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• 제25권10호
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• pp.1164-1168
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• 2015

Previous research showed that chlorphenesin galactoside (CPN-Gal), a preservative in cosmetics, was safer than CPN against human skin cells [9]. To establish a stable and long-term process for CPN-Gal production, we investigated the repeated-batch process. In this process, β-gal-producing recombinant Escherichia coli cells were immobilized in calcium alginate beads, and CPN was converted to CPN-Gal by the transgalactosylation reaction. The process was conducted in a 300 ml flask, which contained E. coli cell-immobilized alginate beads, 33.8 mM of CPN, and 400 g/l of lactose. The pH and temperature were 7.0 and 40℃, respectively. During the repeated-batch operation, four consecutive batch operations were conducted successfully until 192 hr. The conversion yield of CPN to CPN-Gal was 64% during 192 hr, which was higher than the values in previous reports [3, 13]. Thereafter, however, the conversion yield gradually decreased until the operation was finished at 336 hr. Western blotting of immobilized E. coli cells revealed that β-gal gradually decreased after 192 hr. In addition, alginate beads were cracked when the operation was finished. It is probable that, including this loss of E. coli cells by cracks, deactivation, and product inhibition of E. coli β-gal might lead to a gradual decrease in the production of CPN-Gal after 192 hr. However, as the purification of β-gal is not necessary with β-gal-producing recombinant E. coli cells, β-gal-producing E. coli cells might be a practical and cost-effective approach for enzymatically synthesizing CPN-Gal. It is expected that this process will be extended to long-term production process of CPN-Gal for commercialization.