• Korean Journal of Clinical Laboratory Science
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• v.43 no.2
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• pp.68-74
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• 2011

Pseudomonas aeruginosa is an aerobic, Gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. Recently, outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. The genes of metallo-${\beta}$-lactamase (MBL), a powerful carbapenemase, are carried as a part of the mobile gene cassettes inserted into integrons playing an important role in rapid dissemination of antibiotic resistance genes among bacterial isolates. In this study, we investigated the prevalence of integron in imipenem resistant P. aeruginosa isolates. A total of 61 consecutive, non-duplicate, and imipenem resistant P. aeruginosa strains were isolated from a university hospital in the Chungcheong province of Korea. We employed repetitive extragenic palindromic sequence-based PCR (rep-PCR) method for the selection of clonally different P. aerusinosa strains. PCR and DNA sequencing were conducted for the detection of integrons. Twenty-one clonally different P. aeruginosa strains were isolated. Only one (P28) of the strains harbored $bla_{VIM-2}$ that was found as gene cassettes in class 1 integrons. Four of 21 carbapenem resistant P. aeruginosa strains harbored class 1 integron containing aminoglycoside resistance determinant. All of the integrons detected in the study contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. To prevent further spreading of the multi-drug resistant P. aeruginosa, conseguent monitoring and clinical polices are required.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.147-152
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• 1998

AL1-gene, necessary for the replication of the genome of a gemini virus TGMV, was inserted in the opposite direction to the promoter CaMV35S resulting in the construction of a plant transformation binary vector pAR35-2. The vector pAR35-2 contains the chimeric gene cassette involving the duplicated promoter CaMV35S, opposite direction of AL1-gene fusioned with hygromycin resistant gene, and the gene cassette of the neomycin phosphotransferase II gene. The plasmid was transferred to tobacco and tomato plants by leaf disk infection via Agrobacterium. The transgenic plants were selected and grown on the MS-agar medium containing kanamycin and hygromycin. The shoots induced from the calli were regenerated to the whole transgenic plants. The antisense AL1-gene was detected in the genomic DNA isolated from the leaves by using the PCR mediated Southern blot analysis. The expression of the antisense AL1-gene was also observed using the RT-PCR mediated Southern blot analysis. The observation of chloroplasts in guard cell pair indicated that the transgenic tomato plants were diploid.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.205-210
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• 1999

16brio cholerne is an important pathogenic organism that causes dimhea in human beings. V ciaoleroe KNIH002 was isolated from patients suffering with dian.heal disease in Korea. From Southern hybridization using the amplified PCR product of 307 bp as a probe. which was obtained from PCR reaction using primer detecting cholera toxin gene, we have found that the c b gene located in 4.5-kb fragmenl double digested with Pstl and BgllI of the chromosome. Therefore, we made mini-libraries of the isolate using PstI and Bgm restriction endonuclease and pBluescript SKU(+) vector. As a result. we cloned 4.5-kb PstI-BglII fragment containing the c a gene encoding a cholera toxin from the constructed mini-libraries of V olzolerae KNlH002 by colony hybridization using the same probes. This recombinant plasmid was named pCTX75. E. coii XL1- Blue harboring pCTX75 showed the cytotoxicity on Chinese Hamster Ovary cells. From the sequencing of he cloned recombinant plasmid, we confinned that it has virulence gene cassette consisting of ace, zot, ctx.4 and cf"~B gene. The ace and zot genes were composed of 291 hp and 1.200 bp with ATG initiation codon and TGA lennination codon, respectively. Nucleotide sequence of the ace gene exhibited 100% identity with that of V cholera E7946 El Tor Ogawa strains. But, nucleolide and amino acid sequence comparison of the zot gene exhibited 99% and 98.8% identity with that of V cholerae 395 Classical Ogawa stram, respectively. Specially. the Ala-100, Ala-272 and Ala-281 sites of Zoi polypeptide presented in V choleme 395 Classical Ogawa strain are replaced by Val in V cholerae KNIH002.

• Journal of Microbiology and Biotechnology
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• v.31 no.12
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• pp.1716-1721
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• 2021

Chikungunya fever is an arboviral disease caused by the Chikungunya virus (CHIKV). The disease has similar clinical manifestations with other acute febrile illnesses which complicates differential diagnosis in low-resource settings. We aimed to develop a rapid test for CHIKV detection based on the nucleic acid lateral flow immunoassay technology. The system consists of a primer set that recognizes the E1 region of the CHIKV genome and test strips in an enclosed cassette which are used to detect amplicons labeled with FITC/biotin. Amplification of the viral genome was done using open-source PCR, a low-cost open-source thermal cycler. Assay performance was evaluated using a panel of RNA isolated from patients' blood with confirmed CHIKV (n = 8) and dengue virus (n = 20) infection. The open-source PCR-NALFIA platform had a limit of detection of 10 RNA copies/ml. The assay had a sensitivity and specificity of 100% (95% CI: 67.56% - 100%) and 100% (95% CI: 83.89% - 100%), respectively, compared to reference standards of any positive virus culture on C6/36 cell lines and/or qRT-PCR. Further evaluation of its performance using a larger sample size may provide important data to extend its usefulness, especially its utilization in the peripheral healthcare facilities with scarce resources and outbreak situations.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.678-682
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• 2010

The deletion of sti from the Streptomyces plasmid pIJ101 made its derivative pHZ1358 an efficient vector for gene disruption and replacement. Here, pHZ1358 was further optimized by the construction of a derivative plasmid pJTU1278, in which a cassette carrying multiple cloning sites and a lacZ selection marker were introduced for convenient plasmid construction in E. coli. In addition, the oriT region of pJTU1278 was also deleted, generating a vector (pJTU1289) that can be used specifically for PCR-targeting. The efficient usage of these vectors was demonstrated by the deletion of the gene involved in avermectin biosynthesis in S. avermitilis.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.379-385
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• 2000

tRNA and 7SL RNA based antisense vehicles were prepared by inserting conserved anti-viral and anti-viroid domains. Anti-PVS coat protein leader sequence (ACPL) and antistructural antihairpin domain of PSTVd (AHII) were inserted in tRNA cassette; anti- zing finger domain of PVS, AHII and anti hop latent viroid ribozyme were inserted in 7SL RNA gene isolated from A. thaliana. These constructs were shown to be transcribed both, in in vitro and in in vivo conditions. However, it followed from our work that closely linked position of PoIII reference genes and PoIIII antisense genes within T-DNA lead to the impairment of RNA expression in transgenic plants. To assay in vivo transcription of antisense genes, hairy root potato cultures were established using h. tumefaciens A4-24 bearing both, Ri plasmid and PoIII-promoterless plant expression vectors with antisense RNA genes. Expression of antisense RNA in transgenic potato tissues was proven by specific RT-PCR reactions.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.43 no.2
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• pp.125-130
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• 2010

A trimethoprim resistant Vibrio parahaemolyticus, which cause acute gastroenteritis in humans, was isolated from farmed fish and seawater. The resistance profiles of isolated V. parahaemolyticus and their correlation with mobile elements were investigated. All of the V. parahaemolyticus were resistance to both rifampin and trimethoprim. The presence of class I integron was confirmed by PCR. PCR-amplified inserted gene cassettes contained aminoglycoside aac6-II, rifampin arr-3 and trimethoprim dfrA27 resistance genes. This study indicated that class I integron mainly contributed to the circulation of trimethoprim resistance determinants in V. parahaemolyticus.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2979-2984
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• 2012

Recent studies have indicated a link between levels of cyclooxygenase-2 (COX-2) and development of the multidrug resistance (MDR) phenotype. The ATP-binding cassette sub-family G member 2 (ABCG2) is a major MDR-related transporter protein that is frequently overexpressed in cancer patients. In this study, we aimed to evaluate any positive correlation between COX-2 and ABCG2 gene expression using the COX-2 inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) in human breast cancer cell lines. ABCG2 mRNA and protein expression was studied using real-time RT-PCR and flow cytometry, respectively. A significant increase of COX-2 mRNA expression (up to 11-fold by 4 h) was induced by TPA in MDA-MB-231 cells, this induction effect being lower in MCF-7 cells. TPA caused a considerable increase up to 9-fold in ABCG2 mRNA expression in parental MCF-7 cells, while it caused a small enhancement in ABCG2 expression up to 67 % by 4 h followed by a time-dependent decrease in ABCG2 mRNA expression in MDA-MB-231 cells. TPA treatment resulted in a slight increase of ABCG2 protein expression in MCF-7 cells, while a time-dependent decrease in ABCG2 protein expression was occurred in MDA-MB-231 cells. In conclusion, based on the observed effects of TPA in MDA-Mb-231 cells, it is proposed that TPA up-regulates ABCG2 expression in the drug sensitive MCF-7 breast cancer cell line through COX-2 unrelated pathways.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.