• Journal of Life Science
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• v.18 no.1
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• pp.136-142
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• 2008

A recombinant Pichia pastoris carrying double expression cassette of Rhodotorula glutinis epoxide hydrolase(RgEH) gene was developed and used for preparing enantiopure (S)-styrene oxide from racemic mixture of styrene oxide. BglII restriction site of original RgEH gene (pPICZ B/RgEH #2) of previous report was mutated using PCR technique for the construction of double expression cassette containing promoter ($P_{AOX1}$), EH gene and transcription terminator ($TT_{AOX1}$) in pPICZ C vector. Double expression cassette with RgEH was inserted into the chromosomal DNA of P. pastoris. $V_{max}$ ($2.2{\mu}mol\;min^{-1}mg\;dcw^{-1}$) on (R)-styrene oxide of P. pastoris with double expression cassette was about 6-fold higher than that ($0.4{\mu}mol\;min^{-1}mg\;dcw^{-1}$) of P. pastoris with single expression cassette. For the determination of the optimal condition, the effects of detergent and temperature on the enantioselective hydrolytic activity and yield of the enantiomer were investigated. When the reaction was performed at $10^{\circ}C$ for 10 min in the presence of 0.5% Tween 20, enantiopure (S)-styrene oxide with 99.9% ee was obtained as the yield 43.4 % from 20 mM racemic sustrate.

• Bulletin of the Korean Chemical Society
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• v.31 no.4
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• pp.887-890
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• 2010

The family of ClpB protein is a molecular chaperone which protects cellular proteins from being aggregated upon exposure to severe environmental stresses in association with DnaK/DanJ/GrpE in the ATP-dependent manner. In a psychrophilic bacterium which survives at a subzero temperature, any functional role of cold-active ClpB protein can be rather crucial. In order to identify a ClpB encoding gene from a cold-adapted bacterium whose genome sequence has not been fully discovered, we have employed a series of PCR technologies, including a gradient PCR with homologous primers, an inverse PCR and a cassette PCR. The full sequence of PaclpB gene was successfully identified and compared with those of other psychrophilic species. We have further cloned the gene in E.coli expression systems and were able to induce PaClpB protein expression by IPTG, which help us understand a molecular mechanism for survival against extremely cold environments.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.2
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• pp.65-70
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• 2015

Antimicrobial agents or additives have commonly been used in domestic animals for the prevention and treatment of bacterial diseases. Unfortunately, this has resulted in the overgrowth of bacteria that is resistant to antimicrobial agents used by humans, and these might get disseminated to humans via the food. In this study, we investigated the prevalence of integrons, and characterized gene cassette arrays in Proteus mirabilis isolates obtained from chickens in Chungcheong province of Korea. Additionally, the correlation between gene cassette arrays and antimicrobial resistance rate was studied. A total of 26 Proteus mirabilis isolates were recovered from chickens in Chungcheong province in Korea. Antimicrobial susceptibility was determined by disk diffusion method. PCR and DNA sequencing were performed to characterize the gene cassette arrays. In addition, we employed repetitive extragenic palindromic sequence-based PCR (REP-PCR) method for clonality analysis of P. mirabilis strains. Of the 26 P. mirabilis isolates tested, 14 (53.8%) isolates carried class 1 integrons, while class 2 and class 3 integrons were not detected in our study. The class 1 integrons harbored genes encoding resistance to aminoglycosides (aacCA5, aadA2, aadA5 and aadA7), trimethoprim (dfrA17, and dfrA32), lincosamides (linF) and erythromycin (ereA). In particular, the presence of class 1 integron had a significant correlatation to a high resistance rate of aminoglycoside and trimethoprim. We confirmed that class 1 integrons are widely disseminated in P. mirabilis isolates from chickens, contributing to the resistance to diverse antimicrobial agents in Korea. To prevent further spreading of antimicrobial resistant genes among P. mirabilis isolates, constant monitoring and clinical policing will become necessary.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.45-53
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• 2010

We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of a gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic resistance gene to create a "mazF-cassette". A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.835-842
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• 2014

Haloperoxidase (HPO, E.C.1.11.1.7) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics, etc., in the presence of halides and $H_2O_2$. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from Pseudomonas putida ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with P. putida IF-3 sequences. Approximately 600 transformants screened for chimeric genes between P. putida ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with P. putida or Sinorhizobium or Streptomyces strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy. E. coli cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8467-8471
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• 2016

Background: One of the major mechanisms for drug resistance is associated with altered anticancer drug transport, mediated by the human-adenosine triphosphate binding cassette (ABC) transporter superfamily proteins. The overexpression of adenosine triphosphate binding cassette, sub-family B, member 1 (ABCB1) by multidrug-resistant cancer cells is a serious impediment to chemotherapy. In our study we have studied the possibility that structural single-nucleotide polymorphisms (SNP) are the mechanism of ABCB1 overexpression. Materials and Methods: A total of 101 gastric cancer multidrug resistant cases and 100 controls were genotyped with sequence-specific primed PCR (SSP-PCR). Gene expression was evaluated for 70 multidrug resistant cases and 54 controls by real time PCR. The correlation between the two groups was based on secondary structures of RNA predicted by bioinformatics tool. Results: The results of genotyping showed that among 3 studied SNPs, rs28381943 and rs2032586 had significant differences between patient and control groups but there were no differences in the two groups for C3435T. The results of real time PCR showed over-expression of ABCB1 when we compared our data with each of the genotypes in average mode. Prediction of secondary structures in the existence of 2 related SNPs (rs28381943 and rs2032586) showed that the amount of ${\Delta}G$ for original mRNA is higher than the amount of ${\Delta}G$ for the two mentioned SNPs. Conclusions: We have observed that 2 of our studied SNPs (rs283821943 and rs2032586) may elevate the expression of ABCB1 gene, through increase in mRNA stability, while this was not the case for C3435T.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.289-295
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• 2013

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

• Parasites, Hosts and Diseases
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• v.53 no.3
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• pp.335-339
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• 2015

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of $Ca^{2+}$, $Mg^{2+}$, $K^+$, and $HCO_3{^-}$ in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.1008-1013
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• 2014

The use of antimicrobial agents for additives or therapeutics is strongly associated with a prevalence of antimicrobial resistance in commensal Enterobacteriaceae. We aimed to characterize integrons in Enterobacteriaceae isolates obtained from chicken cecums in Korea. Moreover, the correlation between integron gene cassettes and antimicrobial resistance was also investigated. A total of 90 isolates the belonged to Enterobacteriaceae were recovered from chickens grown at Gyeongsang and Chungcheong provinces in Korea. Antimicrobial susceptibility tests were performed by the disk diffusion method. PCR and DNA sequencing were also performed to characterize the gene cassette arrays of the integrons. Of the 90 Enterobacteriaceae isolates tested, 39 (43.3%) and 10 (11.1%) isolates carried class 1 and 2 integrons, respectively. Whereas the class 2 integron did not contain gene cassettes, the class 1 integrons carried seven different gene cassette arrays. The class 1 integrons harbored genes encoding resistant determinants to aminoglycosides (aadA1, aadA2, and aadA5), trimethoprim (dfrA1, dfrA12, dfrA17, and dfrA32), lincosamides (linF), and erythromycin (ereA). Moreover, the presence of a class 1 integron was significantly related to a high resistance rate of antimicrobial agents, such as spectinomycin and trimethoprim. We confirmed that diverse class 1 integrons were widely distributed in Enterobacteriaceae isolates from chickens and directly contributed to the resistance to diverse antimicrobial agents in Korea.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.454-458
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• 2006

HSF1 is the heat shock transcription factor in Saccharomyces cerevisiae. S. cerevisiae KNU5377 can ferment at high temperature such as $40^{\b{o}}C$. We have been the subjects of intense study because Hsf1p mediates gene expression not only to heat shock, but to a variety of cellular and environmental stress challenges. Basing these facts, we firstly tried to construct the hsf1 gene-deleted mutant. PCR-method for fast production of gene disruption cassette was introduced in a thermotolerant yeast S. cerevisiae KNU5377, which allowed the addition of short flanking homology region as short as 45 bp suffice to mediate homologous recombination to kanMX module. Such a cassette is composed of linking genomic DNA of target gene to the selectable marker kanMX4 that confers geneticin (G418) resistance in yeast. That module is extensively used for PCR-based gene replacement of target gene in the laboratory strains. We describe here the generation of hsf1 gene disruption construction using PCR product of selectable marker with primers that provide homology to the hsf1 gene following separation of haploid strain in wild type yeast S. cerevisiae KNU5377. Yeast deletion overview containing replace cassette module, deletion mutant construction and strain confirmation in this study used Saccharomyces Genome Deletion Project (http:://www-sequence.standard.edu/group/yeast_deletion_project). This mutant by genetic manipulation of wild type yeast KNU5377 strain will provide a good system for analyzing the research of the molecular biology underlying their physiology and metabolic process under fermentation and improvement of their fermentative properties.