• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.553-559
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• 1996

We have designed nucleotide sequences of hEGF structural gene to eliminate the N-terminal methionine residue incorporated during the translation initiation step, and constructed recombinant human epidermal growth factor (rhEGF) secretion plasmids pYHB101, and pYHB2 in which pelB signal sequence-hEGF gene was expressed under the control of the T7, and tac promoter, respectively. We also constructed pYHB1 vector which contains rhEGF gene controlled by T7 promoter. The transformant with pYHB101 showed relatively slow growth pattern compared to the transformant with pYHB1. However, we observed that the transformant with pYHB101 secreted rhEGF of 13 mg/l significantly after 5 hr induction with 1 mM IPTG and that the T7 promoter was more effective than tac promoter when connected to pelB signal sequence. The amount of rhEGF was 14 mg/l under the sub-optimized condition.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.749-754
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• 2000

PCR primers were designed based on consensus sequences of dTDP-D-glucose 4,6-dehydratase, one of the enzymes involved in the biosynthesis of deoxysugar. The PCR product (360 bp) was obtained from Thermus caldophilus GK24. Colony hybridization was carried out to the cosmid library constructed from T. caldophilus GK24 genomic DNA by the PCR product DNA fragment. We isolated a cosmid clone (pSMTC-1) that was subcloned to call pKCB series plasmid (BamHI fragments), partially sequenced and analyzed. pKCB80 (4.2 kb-BamHI DNA fragment) of them showed ORFs that was orfA, orfB, orfC and orfD. The orfABCD gene cluster is the deosysugar biosynthetic gene ; orfA (glucose-1-phosphate thymidylytransferase), orfB (dTDP-D-glucose 4,6-dehydratase), orfC (dTDP-4-keto-L-rhamnose reductase) and orfD (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase). The gene cluster that was related in biosynthesis of dTDP-L-rhamnose was also identified by computer analysis, and we proposed that the biosynthetic pathway of deoxysugar analyzed from DNA sequencing of pKCB80 is from D-glucose-1-phosphate, dTDP-D-glucose, dTDP-4-keto-6-deoxy-D-glucose via dTDP-4-keto-L-rhamnose to dTDP-L-rhamnose.

• Genomics & Informatics
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• v.12 no.4
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• pp.240-246
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• 2014

Mutation in HNF1B, the hepatocyte nuclear factor-$1{\beta}$ (HNF-$1{\beta}$) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-$1{\beta}$ in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic ${\beta}$-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in ${\beta}$-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.

• YAKHAK HOEJI
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• v.60 no.1
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• pp.1-7
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• 2016

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from retail meat and to characterize the resistant determinants. Determination of minimum inhibitory concentration, the sequence analysis of gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR), the presences of plasmid mediated quinolone resistance (PMQR) and the expression of efflux pump genes were investigated. Of the total 277 retail meat samples, 67 coli form bacteria were isolated. 15 of 67 isolates showed nalidixic acid resistance and 7 of 15 nalidixic acid resistant isolates were also resistant to ciprofloxacin, moxifloxacin and levofloxacin. 11 of 15 nalidixic acid resistant strains were isolated from chicken, 2 of 15 were isolated from beef and 2 of 15 were isolated from pork samples. 11 of 15 nalidixic acid resistant strains have single mutation at codon 87 (D87N or D87G) in gyrA, 2 of 11 gyrA mutants have double mutations at codon 86 and 87 (L86A and L87I) in parC with mutations at codon 434+445+465 or 429 in gyrB. 2 of 15 resistant isolates harbored qnrS, a PMQR determinant. Over expression of the acrB gene, efflux pump gene (3.93~16.53 fold), was observed in 10 of 15 resistant isolates.

• The Journal of Korean Medicine
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• v.21 no.1
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• pp.53-61
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• 2000

Objectives: This study was camed out to examine the effect of five fractions of aqueous extract from Artemisia capillaris THUNB(ACT), on TGF, 1-induced apoptosis, cell viability, cell cycle progression and mRNA expression of apoptosis-related genes in human hepatocyte cell line HepG2. Methods: This study employed Tryphan blue exclusion assay, DNA fragmentation assay, Cpp32 protease activity assay and Quantitative RT-PCR analysis. Results: In the Tryphan blue exclusion assay, the butanol fraction of ACT with $TGF{\beta}$, l showed magnificent (Nice word, ut is it appropriate in a medical abstract\ulcorner) viability and the H2O fraction of ACT with $TGF{\beta}$, l also showed higher viability than only $TGF{\beta}$, l-treated group. DNA fragmentation assay showed that the butanol fraction and the H2O fraction carried inhibitory effects on apoptosis induction, with the butanol fraction displaying greater effects. The Cpp32 protease activity assay showed that the butanol fraction decreased Cpp32 protease activity. The H2O fraction of ACT had no significant effect on the Cpp32 protease activity. Quantitative RT-PCR showed that the butanol fraction suppressed Bax, p 15/INK4B, p21/Waf1, PAI-1 and increased Bcl-2 gene. Conclusions: The data shows that butanol fraction of ACT increases the hepatocyte viability and has the hepatocellular protective effect by the suppression of $TGF{\beta}$, l induced-apoptosis through gene regulation.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.40 no.1
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• pp.52-58
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• 1995

5-methyltryptophan（5MT） resistant mutant plants （MRl） were analyzed for characterization of anthranilate synthetase （AS） and tryptophan synthetase （TS） enzymes. The enzyme was measured in crude extracts from MR1 and control seedlings of Danggin inbred line. There was no significant difference in the level of AS between MR1 and control seedlings when grown on MS medium without 5MT. However, MR1 seedlings grown on MS medium with 25mg/L 5MT showed the level of AS twice higher than that of control seedlings. The activity of AS was inhibited to 50％ in untreated plants when 4mg /L L-tryptophan was added to their extracts. Extracts from MR1 plants required about four times higher concentration of amino acid to cause equal inhibition. In the TS assay, the activity observed in MR1 seedlings was four times higher than that of control seedlings. We have also isolated and sequenced the gene which encoding the tryptophan synthetase B subunit （TSB） from maize. The gene encodes polypeptides with high homology to TSB isolated from other plants, and is expressed in all the developmental stages examined. Northern hybridization analysis indicated that the gene expression in MR1 seedlings grown on MS medium showed a higher level than in control seedlings.

• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.206-209
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• 2002

For the expression of CGTase gene(cgtS) kom Bacillus stearothemophilus NO2 in Saccharomyces cerevisiae, cgtS gene was subcloned into the Eschepichia coll-yeast shuttle vector, pVT103-U. The constructed plasmid, pVT-CGTS was introduced to 5. cemi-siae 2805 cell, and then the cgtS gene under the control of adhl promoter was successfully expressed in the yeast transformant and 87% of the total activity was detected into the fermentation medium. Therefore, the signal peptide of B. stearothemephilus NO2 CeTase showed high secretion efficiency in 5. cerevisiae. Optimal conditions of the recombinant yeast cell f3r expression of CGTase was achieved, when 5. cerevisiae 2805/pv7-CGTS was cultivated on YP medium at 2% dextrose, pH 5.5,$30^{\circ}C$ and the expression level of CGTase was 0.624units/mL for 48 h culture.

• KSBB Journal
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• v.5 no.1
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• pp.49-58
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• 1990

We constructed hybrid plasmids to allow controlled and extracellular production of human alpha-interferon in Escherichia coli. The hybrid plassmids were constructed by transferring alpha-lFN gene from plasmid Hif-2h which has the alpha-lFN gene at PstI restriction site of pBR322, to plasmids pIN -IIIB3 and pIN-IIIC3 at restriction sites between HindIII and BamHI. Plasmids pIN-IIIB3 and pIN-IIIC3 carry E. coli lipoprotein promoter, lac promoter and operator in tandem. The plasmids also have lacl genes which encode for lac repressors, which allows controlled expression of genes cloned to the plasmids by using of inducer IPTG. Lipoprotein signal sequence is located just ahead of cloning sites of the plasmids, which helps cells to excrete or secrete cloned gene products. Plasmid pUC9 was used as a intermediate vector for transferring of alpha-lFN gene from Hif-2h to pIN vectors in order to solve the problem of different restriction sites between Hif-2h and pIN vectors.

• Preventive Nutrition and Food Science
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• v.2 no.3
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• pp.236-240
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• 1997

A crude extract of Smphytum officinale L. (comfrey) was for its ability to inhibit he growth of hepatocellular carcinoma cells and expression of the insulin-like growth factor I (IGF-II) gene. The DNA synthesis of hepatocellular carcinoma cell lines, Hep G2, Hep 3B, and PLC/PRF/5 was inhibited by a crude extract of Smphytum officinale in both a time- and a dose-dependent manners. This plant extract also inhibited expression of the IGF-II gene. Since IGF-II exerts a mitogenic effect on Hep G2 cells, these results suggest that the growth inhibition by Symphytum officinale extract is, in part, mediated through the inhibition of IGF-II gene expression.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.