• Korean Journal of Microbiology
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• v.34 no.4
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• pp.236-242
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• 1998

While bacteriophage P2-P4 system is very useful experimental tool for the study of viral capsid assembly, there is no useful plasmid vector for the DNA manipulation in bacteriophage P2-P4 system. In this study, a new vector plasmid, P4 ash8 (sid71) kmr, was constructed by swapping the non-essential region of P4 DNA for kanamycin resistance(kmr) gene cassette of plasmid pUC4-K. P4 ash8 sid71 was starting material for the construction, since it tends to be maintained as a plasmid in the absence of the helper phage. The total size of this chimera was designed to be packaged into P4 or P2 size heads with induction by P2 infection. The conversion of plasmid P4 ash8 (sid71) kmr to bacteriophage was proved by burst size determination experiment and CsCl buoyant equilibrium density gradient experiment. Integrase destructed P4 derivative, P4 ash8 sid71 kmr intS, was able to be constructed easily by in vitro DNA manipulation of P4 ash8 sid71 kmr. The plasmid stability experiment with P4 ash8 sid71 kmr if/tS showed that the integrase of P4 affects the stable maintenance of plasmid P4 state.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.14-21
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologicals using bovine materials have the risk of viral contamination. Therefore viral validation is, essential in ensuring the safety of the products. Bovine herpesvirus type 1 (BHV-1) is the most common bovine pathogen found in bovine blood, cell, tissue, and organ. In order to establish the validation system for the BHV-1 safety of the products, a real-time PCR method was developed for quantitative detection of BHV-1 in raw materials, manufacturing processes, and final products as well as BHV-1 clearance validation. Specific primers for amplification of BHV-1 DNA was selected, and BHV-1 DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $2\;TCID_{50}/ml$. The real-time PCR method was validated to be reproducible and very specific to BHV-1. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BHV-1. BHV-1 DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $10\;TCID_{50}/ml$ of BHV-1 artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BHV-1 contamination during the manufacture of biologics.

• Horticultural Science & Technology
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• v.31 no.2
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• pp.246-254
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• 2013

Five isolates of Tomato yellow leaf curl virus (TYLCV) collected from various regions of Korea were amplified using PCR and determined the sequences of full-length genome, respectively. The PCR-amplified DNA of each TYLCV isolate was introduced into a binary vector to construct infectious clone containing 1.9 copies of the corresponding viral genome. Various cultivars and breeding lines of tomato were inoculated with Agrobacterium tumefaciens harboring infectious clone of each TYLCV isolate to assess resistance against TYLCV. Susceptible cultivar 'Super-sunread' revealed typical yellowing and narrowing of the upper leaves. In contrast, breeding linesTY12, GC9, GC171, and GC173, which contained the TY-1 and/or TY-3 genes that confer resistance against TYLCV in nature, were completely symptomless, suggesting that the lines were resistant to challenging TYLCV isolates. Symptoms of TYLCV in susceptible tomato cultivars are significantly different from those of TYLCV in the resistant tomato cultivars at 30 days after agroinfiltration. Although genomic DNAs of TYLCV were detected from the breeding lines TY12, GC9, GC171, and GC173 using real-time PCR analysis with specific primers, levels of TYLCV DNA accumulation in the resistant breeding lines were much lower than those of TYLCV DNA accumulation in susceptible tomato cultivars. Similar symptom severity and levels of TYLCV DNA accumulation were observed from TYLCV infections mediated by Bemisia tabaci in the resistant and susceptible tomato cultivars. Concentration of agrobacterium did not affect the response of tomato cultivars against TYLCV inoculation. Taken together, these results suggest that TYLCV inoculation via agroinfiltration is as effective as inoculation through Bemisia tabaci and is useful for breeding programs of TYLCV-resistant tomato.

• Journal of Microbiology
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• v.44 no.5
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• pp.530-536
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• 2006

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasm ids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size bead, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

• IMMUNE NETWORK
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• v.2 no.3
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• pp.150-157
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• 2002

Background: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. Methods: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. Results: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. Conclusion: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.114.2-115
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• 2003

Rice stripe virus causes severe damage to rice in Korea, Japan and China. RSV is a type member of the tenuivirus group and transmitted by the small brown planthopper, Laodeiphax striatellus, in a persistant manner. Until now, occurrence of RSV is limited in of southern part of Korea. But recently occurrence of RSV is increasing and spreading in central part of Korea including Chungcheong and Kyonggi province. So we analyzed recent occurrence trend of RSV which is increased and cloned and sequenced coat protein gene for isolating of RSV strain. Infected rice of each species(Ilpumbyeo, Sindongjinbyeo, Keumobyeo-2, Dongjinbyeo, Jongnambyeo, Samcheonbyeo, etc.)is collected, we extracted total RNA from infected leaves and detected RSV viral RNA by reverse transcription(RT)-PCR using specific primer of coat protein gene. The result of RT-PCR, we observed specific band. We already cloned cDNA from the band, is analyzing sequence variety and homology of each species.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.138.2-139
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• 2003

Grapevine leafroll-associated 1 virus (GLRaV-1) and Grapevine leafroll-associated 3 virus (GLRaV-3), member of the genus Ampelovirus, are important viral disease of grapevine in the world. these viruses transmitted only dicotyledonous host by vectors such as mealybugs and there is no suitable herbaceous host for virus. The diseased leaves turn yellowish or reddish depending on cultivars and viruses. Viruses are existed at low concentration and ununiformly distribution in grapevine. Using small-scale double-stranded RNA (dsRNA) extraction method, reverse transcription and polymerase chain reaction (RT-PCR) product of 1Kb long which encoded of coat protein (CP) gene for both viruses was successfully amplified with a specific primers. The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined from selected recombinant cDNA clones. Sequence analysis revealed that the CP of GLRaV-1 consisted of 969 nucleotide, which encoded 323 amino acid residues and CP of GLRaV-3 consisted of 942 nucleotide, which encoded 314 amino acid residues. The CP of GLRaV-1 and GLRaV-3 has 93.8％ and 98.7％ amino acid sequence identities, respectively.

• Korean Journal Plant Pathology
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• v.11 no.3
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• pp.271-277
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• 1995

The RNA nucleotide sequences of the 3/-noncoding regions (3'-NCRs) of two Korean strains of turnip mosaic virus (TuMV), Ca and cqs, have been determined from their cDNA clones that encompassed the 3'-terminal regions of the viral genomic RNAs. The 3'-NCRs of both strains were 209 nucleotides long, terminated with GAC residues and poly (A) tails. The potential polyadenylational signal motif, UAUGU, was located 140 nucleotides upstream from the poly (A) tail in each of the virus. A highly conserved hexanucleotide sequence [A G U G A/U G/C], which was common in the 3'-NCRs of the potyvirus RNAs, was also found at the regions of 119 bases upstream from the 3'-end. Comparison of the 3'-NCRs of the two Korean isolates with those of four strains from Canada, China and Japan showed significantly identical genotypes (94.3∼99.5%). The secondary structure of three loops with long stems was found within the 3'-NCRs by sequence analysis. The substituted bases in the region among the six TuMV strains did not alter their secondary structures. Length of the 3'-NCRs of the know 11 potyviral RNAs and TuMV RNAs was different from one another and their nucleotide sequences showed 55.7% to 24.0% of homology. The 3'-NCR, therefore, is considered to be useful for phylogenetic studies in potyviruses.

• Journal of Oriental Neuropsychiatry
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• v.10 no.2
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• pp.47-57
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• 1999

Recently, essential oils are used for aromatherapy. Most essential oils are said to be anti-bacterial; some may be anti-viral or anti-fungal. I investigated the effects of peppermint pure essential oil on the heat shock-induced apoptosis in human astrocyte cell line CCF-STTGI. In previous studies, heat shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. We studied the heat shock-induced apoptosis through flow cytometry, DNA electrophoresis, and giemsa staining. Interestingly, these events were inhibited by pretreatment of peppermint pure essential oils in CCF-STTGl cells. Peppermint oil also inhibited the heat shock-induced apoptosis in primary cultured rat astrocytes. In addition, this Peppermint essential oil inhibited the heat shock-induced activation of caspase-3. These results suggest that peppermint pure essential oils may modulate the apoptosis through the activation of the interleukin-I -converting enzyme-like protease.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.329-334
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• 2006

Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. In this report, we describe a new synthesized cholesterolic cationic lipid (2-aminoethylcarbamate-cholesterol) and dioleylphosphatidylethanolamine (DOPE) improve the cellular uptake properties of antisense ODNs, as well as plasmid DNA with low toxicity. This formulation of cholesterolic cationic lipid termed Chol-E, efficiently transfects ODNs and plasmids into many cell types in the presense or absence of 10% serum in the medium.