• Journal of Plant Biology
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• v.35 no.3
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• pp.237-245
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• 1992

To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.14-19
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• 2011

A cyclic undecapeptide-family natural product, cyclosporin A (CyA), which is one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex in a filamentous fungal strain named Tolypocladium niveum. Previously, structural modifications of cyclosporins such as a regionspecific hydroxylation at the $4^{th}$ N-methyl leucine in a rare actinomycetes called Sebekia benihana were reported to lead to dramatic changes in their bioactive spectra. However, the reason behind this change could not be determined since a system to genetically manipulate S. benihana has not yet been developed. To address this limitation, in this study, we utilized the most commonly practiced gene manipulation techniques including conjugation-based foreign gene transfer-and-expression as well as targeted gene disruption to genetically manipulate S. benihana. Using these optimized genetic manipulation systems, a putative cytochrome P450 hydroxylase (CYP) gene named CYP506, which is involved in CyA hydroxylation in S. benihana, was specifically disrupted and genetically complemented. The S. benihana${\Delta}$CYP506 exhibited a significantly reduced CyA hydroxylation yield as well as considerable yield restoration by functional complementation of the S. benihana CYP506 gene, suggesting that the genetically manipulated S. benihana CYP mutant strains may serve as a more efficient bioconversion host for various valuable metabolites including CyA.

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• International Journal of Oral Biology
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• v.44 no.2
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• pp.31-36
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• 2019

Streptococcus mutans is one of the important bacteria that forms dental biofilm and cause dental caries. Virulence genes in S. mutans can be classified into the genes involved in bacterial adhesion, extracellular polysaccharide formation, biofilm formation, sugar uptake and metabolism, acid tolerance, and regulation. The genes involved in bacterial adhesion are gbps (gbpA, gbpB, and gbpC) and spaP. The gbp genes encode glucan-binding protein (GBP) A, GBP B, and GBP C. The spaP gene encodes cell surface antigen, SpaP. The genes involved in extracellular polysaccharide formation are gtfs (gtfB, gtfC, and gtfD) and ftf, which encode glycosyltransferase (GTF) B, GTF C, and GTF D and fructosyltransferase, respectively. The genes involved in biofilm formation are smu630, relA, and comDE. The smu630 gene is important for biofilm formation. The relA and comDE genes contribute to quorumsensing and biofilm formation. The genes involved in sugar uptake and metabolism are eno, ldh, and relA. The eno gene encodes bacterial enolase, which catalyzes the formation of phosphoenolpyruvate. The ldh gene encodes lactic acid dehydrogenase. The relA gene contributes to the regulation of the glucose phosphotransferase system. The genes related to acid tolerance are atpD, aguD, brpA, and relA. The atpD gene encodes $F_1F_0$-ATPase, a proton pump that discharges $H^+$ from within the bacterium to the outside. The aguD gene encodes agmatine deiminase system and produces alkali to overcome acid stress. The genes involved in regulation are vicR, brpA, and relA.

• Archives of Pharmacal Research
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• v.16 no.4
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• pp.271-275
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• 1993

In order to prepare a novel human insulin analogue suhbstituted with homoserine at B$^{30}$ / position, (B$^{30}$ /-homoserine) human insulin, a synthetic gene was designed by linking directly a gene for B chain with that for A chain. This gene was constructed by enzymatic joining of 10 different synthetic oligonucleotides, and then inserted at the polylinker region of pUC19 plasmid. To achieve a high level of gene expression, the gene fusion technique region of pUC19 plasmid. To achieve a high level of gene expression, the gene fusion technique was employed using amino terminal regions of lacZ gene up to Clal or hpal, and either of them has been located under tac promoter. The chemical induction of these fused genes by isopropyl-.betha.-D-thiogalactopyranoside (IPTG) gave a satisfactory level of expression in Escherichia coli harboring the ocnstructed plasmids. It was observed that the fused gene product as a single chain insulin precusor was produced more than 30% of total cell protein of E. coli as a form of inclusion body.

• Genomics & Informatics
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• v.18 no.4
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• pp.38.1-38.9
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• 2020

Chronotype is an important moderator of psychiatric illnesses, which seems to be controlled in some part by genetic factors. Clock genes are the most relevant genes for chronotype. In addition to the roles of individual genes, gene-gene interactions of clock genes substantially contribute to chronotype. We investigated genetic associations and gene-gene interactions of the clock genes BHLHB2, CLOCK, CSNK1E, NR1D1, PER1, PER2, PER3, and TIMELESS for chronotype in 1,293 healthy Korean individuals. Regression analysis was conducted to find associations between single nucleotide polymorphism (SNP) and chronotype. For gene-gene interaction analyses, the quantitative multifactor dimensionality reduction (QMDR) method, a nonparametric model-free method for quantitative phenotypes, were performed. No individual SNP or haplotype showed a significant association with chronotype by both regression analysis and single-locus model of QMDR. QMDR analysis identified NR1D1 rs2314339 and TIMELESS rs4630333 as the best SNP pairs among two-locus interaction models associated with chronotype (cross-validation consistency [CVC] = 8/10, p = 0.041). For the three-locus interaction model, the SNP combination of NR1D1 rs2314339, TIMELESS rs4630333, and PER3 rs228669 showed the best results (CVC = 4/10, p < 0.001). However, because the mean differences between genotype combinations were minor, the clinical roles of clock gene interactions are unlikely to be critical.

• Microbiology and Biotechnology Letters
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• v.15 no.5
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• pp.346-351
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• 1987

A cellulase gene of Clostridium themocellum was transferred to Escherichia coli by molecular cloning with pBR322. The gene was carried in a Hind III digested DNA sequence of about 1.8 kb. This Rind III fragment expressed activities on carboxymethyl cellulose (CMC) and on filter gaper in E. coli. The expression of clostridial cellulase gene in E. coli was studied and compared with the pro-ducts of cellulase genes in C. themocellum.

• BMB Reports
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• v.38 no.1
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• pp.97-103
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• 2005

Under the condition of expression of $\lambda$ P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the $\lambda$ P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the $\lambda$ P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of $\lambda$ P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

• Genomics & Informatics
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• v.1 no.1
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• pp.55-60
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• 2003

The nucleotide sequence of pZMO1, a small cryptic plasmid of Zymomonas mobilis ATCC10988 was determined. Analysis of 1,680 bp of sequence revealed $69\%$ identity with Shigella sonnei plasmid, pKYM and $61\%$ identity with Nostoc sp. ss DNA replicating plasmid. Analysis of a deduced amino acid sequence of an orf of pZMO1 revealed $75\%$ identity and $90\%$ similarity with the repA gene of Synechocystis sp. plasmid pCA2.4. The upstream region of the repA gene of pZMO1 possesses six directed repeat sequences and two inverted repeat sequences at downstream of the IR consensus sequence of nick region of rolling circle replication (RCR) plasmid. A typical terminator hairpin structure was found at the downstream region of repA gene. Degradation of single-stranded plasmid DNA by S1 nuclease was detected by Southern hybridization. It suggests that pZMO1 replicates by a rolling circle mechanism in Z. mobilis ATCC10988 cells.

• Journal of Microbiology
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• v.44 no.3
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• pp.320-326
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• 2006

Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT constributes the major part of the toxigenicity of P, multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture Iysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture Iysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.