• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.535-538
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• 2000

Streptomyces lavendulae FRI-5 produces the ${\gamma}$-butyrolactone autoregulator IM-2, which is required for nucleoside antibiotic producetion. We have developed a system for introducing DNA into S. lavendule FRI-5 via conjugal transfer from Esherichia cole. Conditions were established for conjugation of the oriT-and attP-containing plasmid pSET152 from E. coli ET12567 (pUZ8002) to FRI-5. Conjugation resulted in integration of the plasmid at the chromosomal C31 attB site. The frequency of intergeneric conjugation varied with the medium used. The highest frequency ($1.6\times10-5$ per recipient) was obtained on ISP medium 2 containing 10mM MgCl2. Southern blot and phenotypic analyses of exconjugants revealed that S. lavendulae FRI-5 contains a unique C31 attB site, and that integration of heterologous DNA into the attB site did not interfere with morphological differentiation or IM-2-dependent signal transduction, including the production of a blue pigment. This system will now enable detailed genetic analysis of the regulation of antibiotic production in S. lavendulae FRI-5.

• Journal of Life Science
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• v.27 no.8
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• pp.857-863
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• 2017

The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.

• Journal of Pharmaceutical Investigation
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• v.37 no.1
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• pp.39-43
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• 2007

Previously, we have reported that PLGA nanoparticles were prepared for sustained release of water-soluble blue dextran and the particle size, in vitro release pattern and encapsulation were modulated by varying polymers. This study was designed to encapsulate plasmid DNA in PLGA nanoparticles and to investigate the effect of Polymers and temperatures. PLGA nanoparticles were fabricated with poloxamer 188 (P188) or poloxamer 407 (P407) by using spontaneous emulsification solvent diffusion method. As a model plasmid DNA, pCMV-Taq2B/1L-18 was encapsulated in PLGA nanoparticles. Then, the particle size, zeta potential and encapsulation efficiency of nanoparticles containing plasmid DNA were investigated. Particle sizes of PLGA nanoparticles prepared with P188 and P407 were in the range of 200-330 nm and 250-290 nm, respectively. Zeta potentials of nanoparticles were negative regardless of nanoparticle compositions. Encapsulation efficiency of P407 nanoparticles prepared at $30^{\circ}C$ was higher than those at other preparation condition. From the results, the PLGA nanoparticles prepared with poloxamers at different temperature, could modulate the particles size of nanoparticles, and encapsulation efficiency of plasmid DNA.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.312-320
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• 2000

Abstract The full sequence of the plasmid pKJ36, which was derived from Bifidobacterium longum KJ, was determined and analyzed to construct shuttle vectors between E. coli and Bifidobacterium. The plasmid pKJ36 was composed of 3,625 base pairs with a 65.1% G+C content. The structural organization of pKJ36 was highly similar to that of pKJ50, and the three major ORFs on pKJ36 showed high amino acid sequence homologies with those of pKJ50. The putative proteins coded by these three ORFs were designated as RepB (32.0 kDa, pI=9.25), MembB (29.0 kDa, pI=12.25), and MobB (39.0 kDa, pI=IO.66), respectively. The amino acid sequence of RepB showed a 57% identity and 70% similarity with that of the RepA protein of pKJ50. Upstream of the repB gene, the so-called iteron sequence was directly repeated four-and-ahalf times and a conserved dnaA box was identified. An amino acid sequence comparison between the MobB and MobA of pKJ50 revealed a 48% identity and 61 % similarity. A conserved oriT sequence with an inverted repeat identical to that of pKJ50 was also found upstream of the mobB gene. A hydropathy analysis of MembB revealed four possible transmembrane regions. The expressions of the repB and membB genes were confirmed by RT-PCR. The in vitro translation reaction of pKJ36 showed protein bands with anticipated sizes with respect to each putative gene product. S 1 endonuclease treatment and Southern hybridization suggested that pKJ36 replicates by a rolling circle mechanism via a single-stranded DNA (ssDNA) intermediate. A shuttle vector between E. coli and Bifidobacterium sp. was constructed using the pKJ36, pBR322, and staphylococcal chloramphenicol acetyl transferase (CAT) gene. The successful transformation of the Bifidobacterium strains was shown by Southern hybridization and PCR. The transformation efficiency differed from strain to strain and, depending on the electroporation conditions, with a range between $1.2{\times}10^1-2.6{\times}10^2{\;}cfu/\mu\textrm{g}$ DNA.X> DNA.

• Fisheries and Aquatic Sciences
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• v.12 no.1
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• pp.24-28
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• 2009

A tetracycline resistant Vibrio parahaemolyticus, capable of growing on TCBS medium containing tetracycline, was isolated from cultivated fishes. A gene responsible for the tetracycline resistance was cloned from chromosomal DNA of the V. parahaemolyticus strain using Escherichia coli KAM3, which lacks major multi-drug efflux pumps (${\Delta}acrB$) as host cells. The nucleotide sequence and homology analysis revealed an open reading frame (ORF) for tetracycline resistance protein (TetB). In order to characterize the antibiotic resistance of TetB originated from the V. parahaemolyticus strain, the gene was sub cloned into plasmid pSTV28. The resulting plasmid was designated as pSTVTetB and transformated into E. coli KAM3. E. coli KAM3 cells harboring the recombinant plasmid pSTVTetB are able to grow on plates containing tetracycline and oxytetracycline but not doxycycline, indicating that the tetB gene confers the tetracycline- and oxytetracycline-resistance to the host cell.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.79-84
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• 2001

An aniline-utilizing microorganism identified as a species of Pseudomonas was isolated from soil contaminated highly with aniline and urea-herbicide. This strain was able to utilize aniline as the sole source of carbon and energy, and was shown to harbor a single large plasmid mediating the aniline assimilation. Subsequent plasmid-curing of this bacterium resulted in the abolishment of the aniline utilizing phenotype and the loss of catechol-C2,3O-oxygenase. The reestablishment of the plasmid, denoted pB10, in cured Pseudomonas sp. via filter surface mating, resulted in restoration of the aniline assimilation abilities and enzyme activity.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.31-36
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• 1991

A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

• Journal of Pharmaceutical Investigation
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• v.35 no.5
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• pp.337-341
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• 2005

The purpose of the study was to prepare the pegylated liposome carrying plasmid DNA with optimal encapsulation efficiency. Plasmid DNA (pCEP4 clone 790, 10.6 kb) was entrapped in the pegylated liposome composed of neutral lipid, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), cationic lipid, DDAB (dimethyl dioctadecyl ammonium bromide) and anionic lipids, DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide by freezing/thawing method. Free plasmid DNA was separated from the encapsulated one by Sepharose CL-4B column chromatography. The DNA amount encapsulated into the pegylated liposome was increased as cationic lipid concentration, initial amount of plasmid DNA and total lipid amount were increased.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.551-553
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• 2000

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the endoxylanase gene in B. subtilis. The expression plasmid, pHJKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter ($P_B$), and introduced into B. subtilis DB104. Through batch fermentation of the trasformant cell on a maltose medium, endoxylanase was produced in a growth-associated manner as the predominant protein. The total activity reached about 600 unit/ml at the end of the cultivation, which corresponded to 698 mg endoxylanase protein/l with a specific activity of 860 unit/mg protein. It was also found that the segregational plasmid instability was less than 30% and most of the endoxylanase activity was detected in the culture medium. This result suggests that the secretory production of endoxylanase can be significantly enhanced with the use of the $P_{JH}$ promoter and high-cell density culture techniques, quantitatively as well as qualitatively.

• Microbiology and Biotechnology Letters
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• v.11 no.1
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• pp.75-79
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• 1983

To exploit a suitable vector and recipient strain for molecular cloning in Bacillus subtilis, oxytetracycline-resistant plasmic DNA has been prepared from Streptomyces rimosus by phenol-buffer extraction of lysozyme-lysed cells and introduced into B. subtilis KPM 60 [St $r^{R}$-mutant of RM 125 (leu A8, arg 15, hsm $M^{-10}$ , hsr $M^{-10}$ )] by transformation. Oxitetracycline-resistant plasmid was well transferred into B. subtilis KPM 60 with average frequency of 10$^{-4}$ per $\mu\textrm{g}$ of DNA. The highest frequency of plasmid transformation was obtained after 3 hours incubation of recipient cells in the growth medium and 30 to 60 minutes incubation in the competence medium, and then 20 minutes contact of DNA and host cells. Optimum pH for competence was 7.5, and optimum temperature for transformation was 2$0^{\circ}C$.>.