• Bulletin of the Korean Chemical Society
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• v.26 no.2
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• pp.285-291
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• 2005

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.221-225
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• 2004

Novel cytochrome P450 hydroxylase (CYP) genes were isolated and characterized from P. autotrophica cosmid DNA library using an actinomycete CYP-specific PCR product as a screening probe. The cosmids containing four unique CYP genes (pESK601, 602, 603, 604, 605) were identified, and the four CYP genes were completely sequenced to be homologous to other known Actinomycetes CYP genes involved in various secondary metabolic pathways. Among all novel actinomycete CYP genes found in P. autotrophica, the CYP genes present in pESK601 were revealed to be highly homologous to the CYP genes involved in polyene-type amphotericin and nystatin antibiotic biosynthesis. The nucleotide sequences of the CYP flanking region in pESK601 also revealed the polyene-type biosynthetic genes, implying the presence of a cryptic polyene-type antifungal biosynthetic gene cluster in P. autotrophica.

• IMMUNE NETWORK
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• v.11 no.4
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• pp.203-209
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• 2011

Background: T cell immunoglobulin mucin containing molecule (TIM)-3 is expressed in differentiated Th1 cells and is involved in the suppression of the cytokine production by these cells. However, the regulation of the expression of other T cell genes by TIM-3 is unclear. Herein, we attempted to identify differentially expressed genes in cells abundantly expressing TIM-3 compared to cells with low expression of TIM-3. Methods: TIM-3 overexpressing cell clones were established by transfection of Jurkat T cells with TIM-3 expression vector. For screening of differentially expressed genes, gene fishing technology based on reverse transcription-polymerase chain reaction (RT-PCR) using an annealing control primer system was used. The selected candidate genes were validated by semi quantitative and real-time RT-PCR. Results: The transcription of TIMP-1, IFITM1, PAR3 and CCL1 was different between TIM-3 overexpressing cells and control cells. However, only CCL1 transcription was significantly different in cells transiently transfected with TIM3 expression vector compared with control cells. CCL1 transcription was increased in primary human $CD4^+$ T cells abundantly expressing TIM-3 but not in cells with low expression of TIM-3. Conclusion: CCL1 was identified as a differentially transcribed gene in TIM-3-expressing $CD4^+$ T cells.

• Horticultural Science & Technology
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• v.33 no.5
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• pp.730-736
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• 2015

Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threatens global tomato production. Sw-5b was reported as the resistance gene effective against TSWV. The objective of this research was to develop a single-nucleotide polymorphism (SNP) marker to distinguish tomato cultivars resistant to TSWV from susceptible cultivars for marker-assisted breeding. First, we determined genotypes for TSWV resistance in 32 commercial tomato cultivars using the previously reported Sw-5b gene-based marker. Then, DNA sequences of Sw-5b alleles in tomato cultivars showing resistant or susceptible genotypes were analyzed; a single SNP was found to distinguish tomato cultivars resistant to TSWV from susceptible cultivars. Based on the confirmed SNP, a SNP primer pair was designed. Using this new SNP sequence and high-resolution melting analysis, the same 32 tomato cultivars were screened. The results were perfectly correlated with those from screening with the Sw-5b gene-based marker. These results indicate that the SNP maker developed in this study will be useful for better tracking of resistance to TSWV in tomato breeding.

• Korean Journal of Plant Resources
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• v.14 no.3
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• pp.235-240
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• 2001

Polymerase chain reaction (PCR) is used in a wide array of researches in plant molecular genetics and breeding. However, considerable time and cost are still required for the preparation of DNA suitable for reliable PCR results, especially in plant species containing high amounts of polyphenols. To reduce time and effort for PCR-based analysis, a simplified but reliable method was developed by a combinational employment of a simple and fast DNA extraction procedure and BLOTTO (Bovine Lacto Transfer Technique Optimizer) in reaction mixture. Genomic DNAs prepared by one-step extraction method from recalcitrant plant species such as Rubus coreanus, apple, grape and lettuce were successfully amplified by random primers in the reaction mixture containing 2 to 4% BLOTTO. Successful amplification of ${\gamma}$-TMT transgene in lettuce transformants by the specific primers was also achieved in the same condition, making rapid screening of positive transformants possible. Our results suggest that use of a simple DNA extraction procedure and incorporation of BLOTTO in reaction mixture in combination can reduce time and effort required for the analyses of a large number of germplasms and transformants by PCR-based techniques.

• Journal of Animal Science and Technology
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• v.49 no.3
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• pp.405-414
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• 2007

Pigs have been considered as an ideal source of donor organs because of their plentiful supply and their numerous anatomical and physiological similarities to the human in xenotransplantation. However, for the public health risks associated with the potential for porcine endogenous retrovirus(PERV) infection through xenograft from pig to human, the investigation of methods for elimination and/or control of PERV has been required. In this study we developed the detection and classification methods for PERV based on PCR using specific primers. PERV-A and PERV-B were found in all pigs including Berkshire, Duroc, Landrace, Yorkshire, miniature pig, and Korean native black pig from Jeju by PCR with type-specific primers for PERV. However, PERV-C was detected only from Duroc, miniature pig, and Korean native black pig from Jeju. PERV-A and PERV-B could be distinguished by PCR-RFLP with BamHI. These methods for PERV will be useful in rapid screening of safe organ for xenograft, furthermore, helpful in monitoring of PERV during and after xenotransplantation.

• The Korean Journal of Mycology
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• v.42 no.1
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• pp.50-56
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• 2014

Transposable elements (TEs) are mobile DNA elements that often cause mutations in genes and alterations in the chromosome structure. In order to identify and characterize transposable elements (TEs) in Pleurotus eryngii, a TE-enriched library was constructed using two sets of TE-specific degenerated primers, which target conserved sequences of RT and RVE domains in fungal LTR retrotransposons. A total of 256 clones were randomly chosen from the library and their insert sequences were determined. Comparative investigation of the insert sequences with those in repeat element database, Repbase, revealed that 71 of them were found to be TE-related fragments with significant similarity to LTR retrotransposons from other species. Among the TE sequences, the 70 TEs were Gypsy-type LTR retrotransposons, including 20 of MarY1 from Tricholoma matsutake, 26 of Gypsy-8_SLL from Serpula lacrymans, and 16 of RMER17D_MM from mouse, whereas a single sequence, Copia-48-PTR, was found as only Copia-type LTR retrotransposon. Southern blot analysis of the HindIII-digested P. eryngii genomic DNA showed that the retrotransposon sequences similar to MarY1 and Gypsy-8_SLL were contained as high as 14 and 18 copies per genome, respectively, whereas other retrotransposons were remained low. Moreover, both of the two Gypsy retrotransposons were expressed in full length mRNA as shown by Northern blot analysis, suggesting that they were functionally active retrotransposons.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.19 no.3
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• pp.382-388
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• 2018

In this study, the SELEX method was used to screen for and select aptamers with high selectivity and affinity for Streptococcus mutans, which is the causative agent of dental caries. Aptamers are single stranded oligonucleotides of DNA or RNA with high selectivity and affinity for target molecules because of their specific three-dimensional structures that are used to collect biometric information. When compared to antibodies in vitro, aptamers are more advantageous because of their ease of use in the screening process, low cost, chemical stability, and lack of restrictions on the target molecules. We sorted DNA aptamers, which contain 44 bp or 38 bp primer sequences in 5' and 3', 39 bp random sequences in the middle.We then analyzed the character and affinity to S. mutans. Aptamers of specific oligonucleotides that combine with S. mutans were selected and these results are selectively fused to S. mutans. The results confirmed that DNA aptamers can be used for rapid diagnosis and treatment of dental caries caused by bacteria of the oral cavity, namely S. mutans.

• Journal of Food Hygiene and Safety
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• v.32 no.3
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• pp.199-205
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• 2017

Although many PCR-based assays have been developed, the majority of rapid detection of Toxoplasma gondii in animal and their meat product has been dependent on immunogenic assays. Thus, there is still a need for more reliable PCR based detection method for T. gondii in retail meats. Recently, a 529-bp repeat element that exists in 200-300 copies per genome of T. gondii genome had been spotlighted for its usefulness as potential detection targers. In this study, the 529-bp repeat element was selected for real-time PCR to detect three types of T. gondii (type I, II and III). A primer pair targeting 82-bp of the 529-bp element detected all three types of T. gondii and showed high level of specificity against 14 different food-borne pathogens as well as 3 protozoan parasites such as Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Application of the new real-time PCR assay in meat samples showed improved detection sensitivity compared to the B1-gene targeted method suggesting potential new target for Toxoplasma gondii screening in retail meats.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.117-123
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• 2003

For screening of autoregulator receptor gene from Saccharopolyspora erythraea, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHI site of pUC19 and transformed into the E. coli DH5$\alpha$. The isolated plasmid from transformant contained the fragment of 120 bp, which was detected on 2% gel after BamHI treatment. The insert, 120 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridization using Saccha. erythraea chromosomal DNA were performed with the insert as probe. The plasmid (pEsg) having 3.2 kbp SacI DNA fragment from Saccha. erythraea is obtained. The 3.2 kbp SacI DNA fragment was sequenced by the dye terminator sequencing. The nucleotide sequence data was analyzed with GENETYX-WIN (ver 3.2) computer program and DNA database. frame analyses of the nucleotide sequence revealed a gene encoding autoregulator receptor protein which is a region including KpnI and SalI sites on 3.2 kbp SacI DNA fragment. The autoregulator receptor protein consisting of 205 amino acid was named EsgR by author. In comparison with known autoregulator receptor proteins, homology of EsgR showed above 30%.