• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.68-75
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• 1998

The overproduction and high level secretion of Glucose Oxidase (GOD) from A. niger in S. cerevisiae was carried out by cloning GOD gene. For this purpose, using two different strong promoters (ADH1 promoter, GAL10 promoter) and signal sequences (${alpha}$-MF signal sequence of S. cerevisiae and ${alpha}$-amylase signal sequence of A. oryzae) and GAL7- and GOD terminator, four expression vectors were constructed. All the expression vectors were transformed in S. cerevisiae 2805 using auxotroph method. By the flask culture, transformants of pGAL expression vector series containing GAL 10 promotor showed much higher GOD productivity than transformants of pADH expression vector series containing ADH1 promoter Transformants of pGALGO2 containing GAL10 promotor and ${alpha}$-amylase signal sequence has shown the best productivity of GOD ($GOD_{total}$: 10.3 unit/mL, $GOD_{ex}$: 8.7 unit/mL) at 115 hr. This value was three fold higher than that of pGALGO1 containing GAL 10 promotor and ${alpha}$-MF signal sequence, even if the same promotor was involved. Through the ${alpha}$-amylase signal sequence of A. oryzae, GOD was secreted much more than the case of ${alpha}$-MF signal sequence from S. cerevisiae. These results suggest that signal sequence may play a important roles in not only the secretion but also the overproduction of foreign protein. Secretion rate of GOD in pGALGO1 and pGALGO2 was 89% and 84%, respectively, Because of the overglycosylation in S. cerevisiae the molecular weight of recombinant GOD in S. cerevisiae was much larger (250 kDa) than that of nature GOD in A. niger (170 kDa).

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.541-546
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• 1997

There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.181-189
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• 2001

The objective of this study was to evaluate the development of porcine follicular oocytes fertilized by intracytoplasmic sperm injection (ICSI). Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2~7 mm in diameter from a local slaughterhouse. Oocytes were matured in vitro for 40~44 h, and spermatozoa were prepared by swim-up in the presence or absence of 5 mM dithiothreitol (DTT) and then M II stages of the oocyte were either centrifuged or not centrifuged for the following injection of ooplasm. Injected oocytes were cultured in NCSU 23 medium for 6 to 8 days. The results obtained were as follows: 1. The rates of cleavage and development rates into blastocyst by ICSI were not significantly different between the with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively (P＜0.05). 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5 mM DTT treated-sperm were not significantly different (60.4% vs 16.4% and 45.5% vs 22.2%), respectively (P＜0.05). 3. The cleavage and the developmental rates to btastocyst were not significantly different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%) (P＜0.05). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7$\pm$2.9 and 41.9$\pm$4.6).

• Proceedings of the Ginseng society Conference
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• 1984.09a
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• pp.1-11
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• 1984

Ginseng cotyledon calli were cultured on 1/2MS media supplemented with combination of various growth regulators to induce more embryoids and plantlets in a short period. And tissues of ginseng root and calli were also incubated under various factors or conditions to establish methods for the isolation of viable protoplasts in Panax ginseng C.A. Meyer. The calli derived from cotyledon produced numerous embryoids in 1/2MS media containing 0.5mg/$\ell$ 2,4-D and 0.5mg/$\ell$ kinetin after 2 months' culture. But only shoot formation was less frequent. Further development of these embryoids occurred on 1/2MS medium supplemented with the same concentration of BA and GA. Viable protoplasts were isolated from the root tissue and callus of ginseng. The specific conditions for the isolation of viable protoplasts were required of ginseng materials, root tissue and callus, being processed. For the production of viable protoplasts from 1-year old ginseng root tissue, an enzyme mixture of $2\%$ cellulase 'Ono-zuka' and $0.5\%$ macerozyme, an enzyme solution pH of 5.2 to 5.8, a 7- to 8- hour incubation period at $28{\pm}1^{\circ}C$, and 0.9M mannitol as osmoticum in the cell enzyme mixture were optimum, while the treatments with an enzyme mixture of $2\%$ cellulase 'Onozuka', $2\%$ macerozyme and $1\%$ driselase, and 25-hour incubation period at $28{\pm}1^{\circ}C$, were more efficient for the production of viable protoplasts from ginseng callus.

• Tuberculosis and Respiratory Diseases
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• v.42 no.5
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• pp.654-659
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• 1995

Background: Tuberculosis has commonly been associated with various hematologic changes. A difference between the changes found in pulmonary tuberculosis and those found in miliary tuberculosis has been discussed. Up to now some worker were investigated hematological changes associated with pulmonary tuberculosis but was not investigated those associated with miliary tuberculosis in korea. Therefore we examimed the peripheral blood and bone marrow findings in miliary tuberculosis patients to determine hematologic changes. Methods: We performed blood sample at admission and bone marrow biopsy within 7days after admission. For evaluation of the hematologic findings, full blood counts and marrow differential counts were defined by the criteria outlined by Dacie and Lewis. Results: 1) Peripheral blood findings: Pancytopenia in 10% of patients, anemia in all patients, leukocytosis in 10% of patients, leukopenia in 20% of patients, thrombocytopenia in 30% of patients, lymphocytopenia in 90% of patients, monocytosis in 40% of patients and neutrophilia in 10% of patients were found at peripheral blood. 2) Bone marrow findings: Lymphocytopenia in 30% of patients, lymphocytosis in 20% of patients, plasmacytosis in 40% of patients, monocytosis in 100% of patients, and hypocellularity in 30% of patients were found at bone marrow. Erythropoiesis was decreased in 30% of patients. Granulopoiesis was decreased in 20% of patients and increased in 20% of patients. Bone morrow granuloma occured in 25% of patients. Conclusion: Hematologic changes of miliary tuberculosis were seen tendency of cytopenic pattern but monocyte was increased at peripheral blood and bone morrow. This findings would provide additional information for the differential diagnosis of miliary tuberculosis.

• Journal of Ginseng Research
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• v.27 no.2
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• pp.78-85
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• 2003

By the Agrobacterium rhizogenes A$_4$ were induced a transformed callus of ginseng hairy root and examine to find the possibility whether it can produce certain ginsenoside. Investigations for a finding out to optimal culture medium showed that BA application is better than more factorial composition between auxins and cytokinins. For the induction of hairy root callus of ginseng, l/2 MS medium containing 1 to 3 mg of benzyladenine(BA) per liter gave the best result. The growth of ginseng hairy root callus(GHC) cultured with the 1/2MS medium supplemented with 2 mg BA/L was selected for best suspension cultures. The optimum concentration of BA for ginsenosides production was found to be 2 mg/L. Probably the inoculum size of callus plays a role with the ginsenoside production in suspension culture. AS for inoculum size of callus, 50 mg was superior to 150 mg for growth and ginsenoside production. Ginsenoside contents were highest in the suspension culture grown for four weeks under continuous light condition. In fact that continous light treatment promote strongly the synthesis of ginsenoside of the hairy root callus is first result in the world and the numerously induced root hairs of the callus leads a new method for ginsenoside production.

• KSBB Journal
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• v.10 no.5
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• pp.475-481
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• 1995

The effect of metallothionein expression on the metal resistance and removal by recombinant Saccharomyces cerevisiae containing the plasmid pJW9 was investigated. The recombinant strain S. cerevisiae BZ-pJ was constructed by transforming the host strain S. cerevisiae BZ3l-1-7Ba with the gene coding for a metal-binding protein, metallothionein. Introduction of the MT gene yielded an increase in the minimum inhibitory concentration (MIC) of copper more than three times compared with the host strain. The minimum inhibitory concentrations of $Cr^{2+}, Znr^{2+} and Pb^{2+}, $ were not different for the two strains. The recombinant yeast grown in a medium containing 8mM CuSO4 was able to remove copper with a capacity of 18.9mg $Cu^{2+}$/g dry cell. In a mixture of copper and zinc, the presence of copper relieved the toxic effects caused by zinc, resulting in an enhancement of the final cell density and the specific growth rate of the recombinant yeast. The capability to remove copper by the recombinant yeast was linearly proportional to the copper concentrations in the medium. The efficiency of copper removal was rather constant regardless of the initial copper concentrations. The specific removal of zinc was dependent on the zinc concentrations in media, though, and such dependence was not so pronounced as the concentration of copper.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.157-162
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• 2006

Endogenous 84 amino acid parathyroid hormone(PTH) is synthesized as a pre-pro hormone by the chief cells of the parathyroid glands. Physiological actions of PTH include regulation of bone metabolism, renal tubular reabsorption of calcium and phosphate, and intestinal calcium absorption. In addition, PTH stimulates new bone formation by extraordinary stimulation of osteoblastic activity and decreasing calcium excretion by the kidney. In this study, we constructed and tested retrovirus vectors designed to express the human parathyroid hormone(hPTH) gene under the control of the tetracycline-inducible promoters. To increase the hPTH gene expression at turn-on state, woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) sequence was also introduced into retrovirus vector at downstream region of either the hPTH gene or the sequence encoding reverse tetracycline-controlled transactivator(rtTA). Transformed primary culture cells(porcine fetal fibroblast, PFF, chicken embryonic fibroblast, CEF) were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the hPTH gene expression level using two step RT-PCR and ELISA Higher hPTH expression($3{\tims}10^4\;pg/ml,\;5.3{\times}10^4\;pg/ml$) and tighter expression control(up to 8 fold) were observed from the vector in which the WPRE sequence was placed at downstream of the hPTH gene. The resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Korean Journal of Poultry Science
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• v.32 no.4
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• pp.239-244
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• 2005

Uncoupling protein(UCP) is expressed exclusively in brown adipose tissue(BAT). It is blown to uncouple phosphorylation from oxidation and hence to be involved in energy metabolism and heat production, especially under cold exposure. In the present study, we identified single nucleotide polymorphism(SNP) in exon 3 of avUCP gene in Korean native chicken(KNC) population. It was detected a SNP T+1316C in exon 3 of avUCP gene by sequence analysis in KNC population. For PCR-RFLP analysis of the SNP T+1316C, used by AP III restriction enzyme. The result of PCR-RFLP analysis showed that allele T has two fragments of 255 bp and 86 bp, and allele C has only one fragment of 341 bp. The genotype frequencies were TT type, 0.7875; TC type, 0.1875 and CC type, 0.025; and the frequencies of allele T and C were 0.881 and 0.119, respectively in KNC population. Next study was conducted to investigate the effect of the SNP in avUCP gene on economic traits in the KNC population. The TT genotype had a significant higher daily percent lay(84.61) than CC genotype(p<0.05) in KNC population. This study may be useful for genetic studies of avCUP gene and selection on daily percent lay of KNC.