• Journal of Aquaculture
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• v.11 no.4
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• pp.457-463
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• 1998

An expression vector, pUC19N6-luc, containing nuclear matrix attachment region(MAR) isolated from Misgurnus mizolepis liver and control expressino vector, pUC19-luc, were constructed. After these vectors were transferred into CHSE-214 cell line by electroporation, the expression rate of luckferase gens, copy number of vectors and chromosome integration of vectors were analyzed by using assay of luciferase activity, PCR and Southern blotting. While the expression pattern of luciferase gene of pUC19-luc was shown in typicla transient ecpression pattern, that of pUC19N6-luc was highly increased at the 5 days after transfectrion. Although the cope number of pUC19N6-luc vector was higher than that of pUC19-luc vector, these vectors were integrated into chromosome at the same time point in the transfected CHSE-214 cells. In conclusion, the increase of luciferase gene expression of pUC19N6-luc was resulted from not the maintaining of the high copy number but the formation of transcription-favorable structure by MAR effect after chromosomal integration.

• Biomedical Science Letters
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• v.5 no.1
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• pp.17-26
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• 1999

To evaluate the spreading possibilities of the marRAB mutation of E. coli Mar mutant among gram-negative bacilli, chromosomal marRAB mutations of Mar mutants were transduced by $\lambda$placMu9 into pUC19 (Lac$^{+}$, Ap$^{r}$) cloning site in another strains of E. coli or onto the chrmosome of S. typhimurium and P. aeruginosa, selected for transduction by Mar phenotype, Lac$^{-}$, or Ap$^{r}$, and tested for their antimicrobial resistance with or without addition of salicylate (SAL). Compared with wild type strains of JM109, NM522, harboring pUC19 or not, respectively, all strains of JM109 or NM522 carrying pUC19::marRAB mutation showed higher levels of antimicrobial resistance and SAL induction of Mar phenotype than those of wild type. However, in contrast to the original Mar mutants, there were some tendencies of decreased antimicrobial resistance of JM109 or NM522 harboring pUC19::marRAB mutation with SAL induction against chlorarnphenicol (Cm) and tetracycline (Tc), or Tc and ciprofloxacin (Cp), respectively. Almost the same results, as shown as the cases of E. coli JM109 or NM522, were obtained from all transductants of S. typhimurium and P. aeruginosa, except Cp, against which increased antimicrobial resistance with SAL induction was shown. This study, employed the methods of transformation or transduction among intercellular gene transfer methods between gram-negative bacteria, shows the evidences that suggest indirectly the spreading possibilities of marRAB mutation among gram-negative bacilli.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.129-135
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• 1992

A chitinase gene of Serratia marcescens ATCC 27117 was cloned and expressed in Escherichiu di. A genomic library of S, marcescens was constructed with pUC 19 and screened using the swollen chitin agar plate for chitinolytic clones. A positive clone showing chitinclearance contains a recombinant pCHI 89, composed of 8.9 Kb chromosomal DNA fragment and pUC 19. Plasmid pCHI 89 produced 58 KD chitinase in E. coli, which was coincided with one of five extracellular chitinases produced by S. nzarccscens. Restriction endonuclease cleavage sites of the 8.9 Kb insert DNA fragment were mapped. E. coli JM109 harboring pCHI 89 inhibits the growth of a plant pathogenic fungus, Fusarium oxysporum.

• Journal of fish pathology
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• v.6 no.2
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• pp.103-110
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• 1993

Total genomic DNA library of Serratia marcescens was prepared by inserting Sau3AI partial digesting fragments(above 5 kb) into the dephosphorylated BamHl site of pUC19. In primary screening, two colonies were selected by observing the halo around E. coli transformants grown on the swollen colloidal chitin media. Secondary screening was performed by soaking two colonies with a few drops of 4-methylumbelleliferryl N-acetyl-$\beta$-D-glucocosaminide(4-MuNGlcNAc). As 4-MuNGlcNAc is a specific, fluorogenic substrate for chitinase, the positive clones produce light fluorescence by the exposure under the long wave U.V. light(360 nm). From genomic DNA library derived from pUC19, we have isolated two different chitinase clones, pCH1(11.0Kb) and pCH2(7.5Kb), which show completely different restriction map to each other. The cross-hybridization of pCH1EA and pCH2 have not revealed any hybridization signals to each other.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.280-288
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• 1992

Molecular cloning of metalloprotease gene from Serratia marcescens ATCC 21074 into Escherichia coli JM109 was carried out. Chromosomal DNA of S. marcescens was completely digested with Hind111 and southern hybridization with a synthetic oligonucleotide probe revealed that a 50 KD metalloprotease gene was contained in 4.0 Kb chromosomal DNA fragment, 4.0 Kb chromosomal DNA fragments eluted from agarose gel were ligated with pUC19 and transformed into E. coli JM109. Nine positive clones were obtained from about $1\times 10^3$ transformants by colony hybridization. Their recombinant plasmids, pSPl and pSP2 have same chromosomal DNA fragments in pUC19 in opposite-orientations. When cloned metalloprotease gene was expressed in E. coli, about 52 KD precursor protein of metalloprotease was detected by western blot analysis from E. coli harboring a recombinant plasmid pSP2. Plasmid pSP2 showed no protease activities in E. coli but overproduced the active metalloprotease in S. rnarcescens ATCC 27117.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.112-118
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• 2000

A new gene encoding an acidophilic TEX>$\alpha$-amylase of Bacillus cil-culans KCTC3004 was cloned into Eschericlzia coli using pUC19 as a vector. The gene localized in the 5.8 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 40 times greater than that produced by the original B, circulans The optimum pH and temperature of the cloned enzyme were pH 3.6 and 45^{\circ}C.$ respectively. The enzyme hydrolyzed starch to produce maltotriose and maltooligosaccharides. The SDS-PAGE and zymopram of the enzyme produced in E coli(p.4L850) indicated a molecular weight of 55,000.

• KSBB Journal
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• v.11 no.1
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• pp.37-45
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• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• Journal of Life Science
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• v.15 no.5 s.72
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• pp.734-738
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• 2005

A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli $DH5\alpha$ using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with $61\%$ and $59\%$ identical residues, respectively.

• Journal of Plant Biology
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• v.35 no.2
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• pp.165-171
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• 1992

In order to study regulation of rbcL gene expression, rbcL gene of chloroplast DNA (Cp DNA) from maize was cloned. Cp DNA was isolated from intact chloroplast and digested with BamHI. BamHI 9 fragment of Cp DNA containing rbcL gene was ligated to pUC19 and transformed into E. coli DH5a. This recombinant plasmid was named pRLYSl. pRLYSl was hybridized with a part of rbcL gene from rice and digested with restriction enzyme BamHI, HindIIl, and PstI. From these results, it was confirmed that pRLYS1 contains intact rbcL gene and orientation of BamHI 9 fragment of Cp DNA in pRLYS1 was determined.rmined.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.