• Bulletin of the Korean Chemical Society
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• v.27 no.11
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• pp.1891-1893
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• 2006

• Journal of the Korean Society of Tobacco Science
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• v.19 no.2
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• pp.117-123
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• 1997

Viral coat protein (CP) encoding cDNA with artificial start and stop codons was synthesized by reverse-transcriptase polymerase chain reaction (RT-PCR) from the Korean isolate of potato virus Y-vein nectrosis strain (pVY-VN). To make PVY CP cDNA to untranslatable form, three stop codons were inserted near the start codon by "megaprimer-PCR" method. The untranslatable CP cDNA was subcloned to plant expression vector and transferred to N. tabacum cv. NC82 by Agrobacterium-mediated transformation. Highly resistant plants to PVY infection were screened, based on symptom development after mechanical virus inoculation. By genomic PCR and Southern blot analysis, one or more copies of the untranslatable CP gene were found in all transformants. From northern blot analysis, highly resistant transgenic lines had very low level of CP transcript but susceptible lines had high level, suggesting resistance to PVY infection should be related to RNA-mediated mechanism.mechanism.

• BMB Reports
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• v.39 no.6
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• pp.743-748
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• 2006

The complement (C) regulatory proteins decay accelerating factor (DAF, CD55) and CD59 could protect host cells using different mechanisms from C-mediated damage at two distinct levels within the C pathway. Co-expression of DAF and CD59 would be an effective strategy to help overcome host C-induced xenograft hyperacute rejection. In this study, we made a construct of recombinant expression vector containing DAF and CD59 cDNA and the stable cell lines were obtained by G418 selection. Extraneous genes integration and co-expression were identified by PCR, RT-PCR and Western blot analysis. Human c-mediated cytolysis assays showed that NIH/3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-DAF, and pcDNA3-CD59DAF-DP were protected from C-mediated damage and that synchronously expressed human CD59 and DAF provided the most excellent protection for host cells as compared with either human CD59 or DAF expressed alone. Therefore, the construct represents an effective and efficacy strategy to overcome C-mediated damage in cells and, ultimately, in animals.

• BMB Reports
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• v.35 no.3
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• pp.297-301
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• 2002

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress. Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the oxyR deletion mutant in regard to growth kinetics, viability, and DNA damage. The deletion of the oxyR gene in E. coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage. Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.

• Journal of Microbiology
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• v.34 no.1
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• pp.37-37
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• 1996

The internal regions of nuclear small subunit rRNA from 6 plaeurotus species and 5 Pleurotus ostreatus strains were amplified by PCR and sequenced. The DNA sequences of 8 Pleurotus strains (P. ostreatus NFFA2, NFFA4501, NFFA4001, KFFA4001, KFCC11635, P florida, P. florida, P. sajor-cuju, P. pulmonarius, and P. spodoleucus) were idential, but P. cornucopiae differed from them in two bases out of 605 bases. However, p[hylogenetic analysis of the sequences by DNA-distance matrix and UPGMA methods showed that P. ostreatus NFFA2m1 and NFFA2m2, known as mutants of P. ostreatus NFFA2, belonged to anther group of Basidiomycotina, which is close to the genus Auricularia. The difference of the SSU rDNA sequences of P. cornucopiae from other Pleurotus species tested corresponds to the difference of mitochondrial plasmid type present in Pleurotus species as observed by Kim et al. (1993, Korean J. Microbiol. 31, 141-147).ishement of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with .alpha.-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de nono protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 /.mu.g/ml) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.15-23
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• 2000

The subcellular localization of the SopB protein, which is encoded by the Escherichia coli F plasmid and is involved in the partition of the single-copy plasmid, was directly visualized through the expression of the protein fused to the jellyfish green fluorescent protein (GFP). The fusion protein was found to localize to positions close but not at the poles of exponentially growing cells. Examination of derivatives of the fusion protein lacking various regions of SopB suggests that the signal for the cellular localization of SopB resides in a region close to its N terminus. Overexpression of SopB led to silencing of genes linked to, but well-separated from, a cluster of SopB-binding sites termed sopC. In this SopB-mediated repression of sopC-linked genes, all but the N-terminal 82 amino acids of SopB can be replaced by the DNA-binding domain of a sequence-specific DNA -binding protein, provided that the sopC locus is also replaced by the recognition sequence of the DNA-binding domain. These results suggest a mechanism of gene silencing: patches of closely packed DNA-binding protein is localized to specific cellular sites; such a patch can capture a DNA carrying the recognition site of the DNA -binding domain and sequestrate genes adjacent to the recognition site through nonspecific binding of DNA.

• YAKHAK HOEJI
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• v.39 no.6
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• pp.661-665
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• 1995

Ginsenoside-Rg$_{1}$(G-Rg$_{1}$) has been shown to stimulate DNA synthetic activity in SK-HEP-1 cells. This study was therefore designed to determine in SK-HEP-1 cells whether the stimulatory effect of G-Rg$_{1}$ may be mediated by protein kinase C (PKC) which is known to play a key role in the signal transduction pathway leading to the cell proliferation. Using the tn situ PKC assay method, the PKC enzyme activity was determined in SK-HEP-1 cell cultures in response to G-Rg$_{1}$ at 3*10$^{-5}$ M or phorbol 12-myristate 13-acetate(PMA) at 10$^{-6}$ M which in the enzyme activity by 1.5- and 7-fold, respectively. Furthermore, G-Rg$_{1}$, was also able to synergistically increase the enzyme activity by 11-fold m the cell cultures in the presence of PMA. These stimulatory effects of G-Rg$_{1}$ or PMA on the DNA synthetic activity and the PKC activity were ablished by a specific PKC inhibitor, GF109203X. These results suggest that the stimulatory effect of G-Rg$_{1}$ on the DNA synthetic activity may be partly due to stimulation of PKC-mediated signal transduction pathway leading to the proliferation of SK-HEP-1 cells.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.111-116
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• 2010

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

• YAKHAK HOEJI
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• v.50 no.1
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• BMB Reports
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• v.47 no.11
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• pp.609-618
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• 2014

DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development.