• Applied Biological Chemistry
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• v.39 no.1
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• pp.25-31
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• 1996

Transgenic tobacco plants expressing calmodulin derivative($lys{\rightarrow}ile$ 115 calmodulin) and hygromycin resistance genes or hygromycin resistance gene alone(control) were generated by Agrobacterium-mediated DNA transfer. Seeds obtained from the transgenic plants($F_o$) were tested for resistance to hygromycin and the expected 3 : 1 ratio was observed. The expression of calmodulin derivative in the tobacco plants was identified by a combined method of Western blot and Chemiluminescence. The effects of surface sterilizers on the germiation of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacoo plants expressing the calmodulin derivative showed no fungi contamination with normal germination by treating with sterilized water alone or sodium hypochlorite(2% effective chlorine). In contrast, seeds from the control transgenic tobacco plants showed severe contamination with fungi by treating with sterilized water alone and showed no contamination with normal germination by treating with sodium hypochlorite(2% chlorine). The effects of calcium concentration on the germination of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacco plants expressing the calmodulin derivative showed better germination frequency than that of the control transgenic tobacco seeds in the medium containing 30 mM $CaCl_2$. The data raise the possibility that the expression of calmodulin derivative gene in tobacco plants could increase adaptability of the seeds to environmental stresses.

• Journal of Genetic Medicine
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• v.5 no.1
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• pp.41-46
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• 2008

Purpose : Fragile X syndrome (FXS) is the most common heritable cause of cognitive impairment. FXS is caused by hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retadation-1(FMR1) gene. Combination of Southern blotting and simple polymerase chain reaction(PCR) amplification of the FMR1 repeat region is commonly used for diagnosis in females. To give a definite diagnosis in a female child suspected of having FXS, we carried out the molecular diagnostic test for FXS using the recently developed Abbott Molecular Fragile X PCR Kit. Methods : The PCR amplification of the FMR1 repeat region was performed using the Abbott Mdecular Fragile X PCR Kit. The amplified products were analyzed by size-separate analysis on 1.5% agarose gels and by DNA fragment analysis using Gene scan. Results : Agarose gel and Gene scan analyses of PCR products of the FMR1 repeat region showed that the patient had two heterozygous alleles with a normal 30 repeats and full mutation of >200 repeats whereas her mother had two heterozygous alleles with the normal 30 repeats and premutation of 108 repeats, suggesting that the premutation of 108 repeats in her mother may have led to the full mutation of >200 repeats in the patient. Conclusion : We diagnosed FXS in a female patient using a simplified molecular diagnostic test. This commercially available diagnostic test for FXS, based on PCR, may be a suitable alternative or complement method to Southern blot analysis and PCR analysis and/or methylation specific(MS)-PCR analysis for the molecular diagnosis of FXS in both males and females.

• Korean Journal of Plant Tissue Culture
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• v.22 no.2
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• pp.95-99
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• 1995

We have previously established a system for plant regeneration through somatic embryogenesis and Agrobacterium-mediated transformation of Korean ginseng. In this study to produce a fungus-resistant plant, we introduced a bean chitinase gene into ginseng using the transformation system. A binary vector pChi/748 was constructed by introducing the bean basic chitinase gene into EcoRI site of pGA748 which carries the CaMV 35S promoter governing the introduced gene and neomycin phosphotransferase II(NPT-II)gene as a positive selection marker. Cotyledonary explants were cocultured with A. tumefaciens strain LBA4404 harboring the binary vertor pChi/748 for 48 h, and transferred to MS medium supplemented with l mg/L2,4-D,0.1mg/L kinetin, 100 mg/L kanamycin, and 500mg/L carbenicillin. Kanamycin-resistant calli were formed on the cut surface of cotyledonary explants after one month of culture, and subsequently they gave rise to somatic embryos. Upon transfer onto medium containing 1 mg/L each of BA and GA$_3$, most of them converted to plantlets after 5 weeks of culture. The genomic DNA of eight kanamycin-resistant regenerants was subjected to polymerase chain reaction (PCR) using two specific 21-mer oligonucleotides derived from the chitinase gene. PCR-Southern blot analysis confirmed that the chitinase gene was incorporated into six out of the eight regenerants..

• Journal of Ginseng Research
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• v.43 no.1
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• pp.38-48
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• 2019

Background: Interspecific ginseng hybrid, Panax ginseng ${\times}$ Panax quenquifolius (Pgq) has vigorous growth and produces larger roots than its parents. However, F1 progenies are complete male sterile. Plant tissue culture technology can circumvent the issue and propagate the hybrid. Methods: Murashige and Skoog (MS) medium with different concentrations (0, 2, 4, and 6 mg/L) of 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and somatic embryogenesis (SE). The embryos, after culturing on $GA_3$ supplemented medium, were transferred to hormone free 1/2 Schenk and Hildebrandt (SH) medium. The developed taproots with dormant buds were treated with $GA_3$ to break the bud dormancy, and transferred to soil. Hybrid Pgq plants were verified by random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses and by LC-IT-TOF-MS. Results: We conducted a comparative study of somatic embryogenesis (SE) in Pgq and its parents, and attempted to establish the soil transfer of in vitro propagated Pgq tap roots. The Pgq explants showed higher rate of embryogenesis (~56% at 2 mg/L 2,4-D concentration) as well as higher number of embryos per explants (~7 at the same 2,4-D concentration) compared to its either parents. The germinated embryos, after culturing on $GA_3$ supplemented medium, were transferred to hormone free 1/2 SH medium to support the continued growth and kept until nutrient depletion induced senescence (NuDIS) of leaf defoliation occurred (4 months). By that time, thickened tap roots with well-developed lateral roots and dormant buds were obtained. All Pgq tap roots pretreated with 20 mg/L $GA_3$ for at least a week produced new shoots after soil transfer. We selected the discriminatory RAPD and ISSR markers to find the interspecific ginseng hybrid among its parents. The $F_1$ hybrid (Pgq) contained species specific 2 ginsenosides (ginsenoside Rf in P. ginseng and pseudoginsenosides $F_{11}$ in P. quinquefolius), and higher amount of other ginsenosides than its parents. Conclusion: Micropropagation of interspecific hybrid ginseng can give an opportunity for continuous production of plants.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.11
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• pp.1385-1390
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• 2007

Anticarcinogenic-active compound was isolated and purified from Angelica gigas Nakai. The compound was identified as decursin ($C_{19}H_{20}O_5$; molecular weight 328) by mass, IR spectrophotometry $^1H-NMR$ and $^{13}C-NMR$. The proliferation decreased in a dose dependant fashion in the MCF-7 cells treated with decursin for 24 hours over the concentration of $20{\mu}g/mL$. The $IC_{50}$ value of the decursin treatment for 24 hours were 31.04, 33.60, 27,24, $20.45{\mu}g/mL$ in the SW480, 293, HepG2 and MCF-7 cells, respectively, The growth inhibitory effect was stronger in the MCF-7 cells compared to other cells including 293 of human normal cells. The chromatin condensation, apoptotic body formation and DNA fragmentation were examined in the cells treated with decursin. These results suggest that decursin from Angelica gigas Nakai inhibited the growth through apoptosis in MCF-7 cells.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.22-29
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• 2011

Although reactive oxygen species (ROS) are inevitable by-products of many redox reactions in eukaryotic cells, they play a crucial role as signaling molecules in many cellular processes for development and defense response to abiotic stresses. The biphasic ROS production which was peaked twice in a first transient phase and a second massive phase was occurred after treatment of abiotic stress such as oxidative stress, high salinity. This biphasic generation of ROS was followed by the biphasic production of stress hormone, ethylene. The mechanism of interactions between ROS and ethylene biosynthesis is studied in tobacco (Nicotiana tabaccum L.) plants under the abiotic stresses. The stress-induced ethylene production was significantly inhibited in RbohD-AS and RbohF-AS, in which antisense expression of NADPH oxidase genes was performed. The accumulation of ROS, which was determined by DAB and DCFH-DA staining, was significantly decreased after abiotic stresses in transgenic plants. The suppression of signaling with ethylene and ROS induced more tolerance in response to abiotic stress. The transgenic plants were more tolerant in MS medium supplemented with salinity stress in contrast with wild-type. Stress-induced cell damage determined by DNA fragmentation was decreased at phase II in those transgenic plants. Therefore, the first burst of ROS is more responsible for making a role as a signaling molecule during stress-induced response. These results suggested that ethylene and ROS act in a positive feedback cycle that results in mutual enhancement of ethylene and ROS production during stress-induced cell death.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.118-122
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• 1995

Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

• Clinical and Experimental Pediatrics
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• v.53 no.3
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• pp.432-436
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• 2010

Transient neonatal diabetes mellitus (TNDM) has been associated with paternal uniparental isodisomy of chromosome 6, paternally inherited duplication of 6q24, or a methylation defect at a CpG island of the ZAC or HYMAI gene. We experienced a case of TNDM in which the patient presented with hyperglycemia, macroglossia, and intrauterine growth retardation, caused by a paternally derived HYMAI. An 18-day-old female infant was admitted to the hospital because of macroglossia and recurrent hyperglycemia. In addition to the macroglossia, she also presented with large fontanelles, micrognathia, and prominent eyes. Serum glucose levels were 200-00 mg/dL and they improved spontaneously 2 days after admission. To identify the presence of a maternal methylated allele, bisulfite-treated genomic DNA from peripheral blood was prepared and digested with BssHII after polymerase chain reaction (PCR) amplification with methylation-specific HYMAI primers. PCR and restriction fragment length polymorphism analysis showed that the patient had only the paternal origin of the HYMA1 gene. TNDM is associated with a methylation defect in chromosome 6, suggesting that an imprinted gene on chromosome 6 is responsible for this phenotype.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.271-279
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• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.