• Korean Journal of Microbiology
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• v.22 no.4
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• pp.249-255
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• 1984

To develop the host-vector system for industrial Coryneform bacteria that seemed to be the most suitable microorganisms for molecular breeding of genes involved in the production of amion acids, nucleotides, and other products of industrial interest, broad host range E. coli plasmid R 1162 DNA was transformed into Brevibacterium ammoniagenes and the plasmids pKU6 isolated from a transformant was physically characterized. All other plasmids from the transformed cells except pKU6 exsisted as multimeric forms in Brevibacterium ammoniagenes. The plasmid DNA was retransformed into Corynebacterium glutamicum with a high frequency ($1.32{\times}10^{-1}$ per cell) and maintained stably both in Brevibacterium ammoniagenes and Corynebacterium glutamicum after 100 generations of cultures with 25-30 copy number per cell. The size of both plasmid pKU6 and plasmid R1162 were the same and restriction maps by EcoR I, Ava I, Pst I, Pvu II and Hinc II were also similar.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.169-173
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• 1990

For the overproduction of L-phenylalanine using Escherichia coli, the authors constructed various recombinant plasmids including pMW 10, pMW 11 and pMW 12. The $aroF{FR}$ and $pheA^{FR}$ genes for the production of L-phenylalanine were isolated from Escherichia coli MWEC 101-5 strains. The productivity and atability of Escherichia coli regulatory mutants containing recombinant plasmids were investigated to evaluate the efficiency of the $aroF^{FR}$ and $pheA^{FR}$ genes. The MWEC 101-5/pMW 11 strain produced 24.3g/l of L-phenylalanine while its stability was 73.8 percent. The specific activity of prephenate dehydratase in the MWEC 101-5/pMW 11 strain increased by 26-fold compared with that of Escherichia coli K-12.

• KSBB Journal
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• v.16 no.6
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• The Journal of the Korean Society for Microbiology
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• v.22 no.3
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• pp.295-300
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• 1987

Salmonella strains isolated from blood in the Dong-san hospital, Taegu during the period from 1971 to 1986 were studied for species distribution, drug resistance, and R plasmids. The number of strains was 2,527 and all of them were classified into S. typhi and S. paratyphi A. Approximately 300 strains were isolated in the period from 1974 to 1976 and 1978, 268 in 1982, and 204 in 1983, but the numbers isolated in the 1980's have a tendency to decrease as compared with those of the 1970's. S. typhi occupied 85% or more of strains isolated until 1976, but the isolation frequency decreased yearly with some variation, and S. paratyphi A increased gradually from 1974. Only 4 strains of S. paratyphi A were resistant to some drugs, and the resistance was not transferred to E. coli by conjugation. S. typhi resistant to drugs were 15 in 1971 through 1973, 24 in 1974, and 13 in 1975, but afterwards only few resistant strains were isolated. These strains were resistant to two or more drugs; chloramphenicol(Cm), tetracycline(Tc), streptomycin(Sm), sulfisomidine(Su), ampicillin(Ap), and kanamycin(Km) and no strain resistant to other drugs tested was found. Strains resistant to 3 or less drugs didn't transfer the resistance to E. coli by conjugation. There were 15 strains resistant to four or more drugs, and were isolated in years from 1972 to 1976. These strains transferred the resistance to E. coli, and the resistance was considered to be mediated by R plasmids. Transfer frequency was higher at $25^{\circ}C$ than at $37^{\circ}C$ and patterns of transferred resistance were Cm, Tc, Sm, Su; Ap, Km; Cm. R plasmids having markers of Cm, Tc, Sm and Su were classified into Inc H1.

• Korean Journal of Veterinary Research
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• v.32 no.2
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• pp.181-194
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• 1992

Two hundred and eighty nine strains of Yersinia enterocolitica isolated from healthy pigs were tested for the presence of 40~50 Megadalton virulence-associated plasmids and plasmidmediated in vitro virulence-associated properties, i.e., congo red uptake, calcium dependency, autoagglutination, CRMOX reaction, crystal violet binding and pyrazinamidase reaction. The correlationships between in vitro virulence-associated properties and the presence of 220 Kdalton outer membrane protein(OMP) were examined in strains with or without virulence-associated plasmids. The correlationships between the presence of plasmids on the production of the OMP and the expression of in vitro virulence-associated properties were studied with $CRMOX^+$ strains and acridine orangecured $CRMOX^-$ mutants. The results were as follows : 1. Of the in vitro virulence-associated tests with 289 strains of Y enterocolitica, 275 strains (95.2%) were positive for pyrazinamidase test, and followed by in order of crystal violet binding test, 226 (79.2% ) ; CRMOX test, 190 (65.7%) ; autoagglutination test, 1.85(64.0%) : calcium dependency test, 86 (29.8%) and congo red uptake test, 47(16.3%). 2. The correlationship between autoagglutination and CRMOX test(r=0.90) was highly significant (p<0.01). 3. In 190 strains(65.7%) bearing the virulence-associated plasmids(MW 40~50 Mdalton), the correlation between the presence of plasmids and their in vitro virulence-associated properties were highest with CRMOX test(r=0.93) and followed by in orders of AAG test(0.81), CV test(0.46), PYZ test(0.37) and CD test(0.18), but no correlationship between the presence of plasmids and CR test(-0.11). 4. The $CRMOX^+$ strains produced the 220 Kdalton OMP when they were cultured at $37^{\circ}C$, but not at $26^{\circ}C$. The presence of 220 Kdalton OMP was correlated significantly with in vitro virulence properties and the presence of virulence-associated plasmid, respectively. 5. In the isogenic $CRMOX^-$ mutant strains, of which plasmid were cured by treatment with acridine orange not only in vitro virulence-associated properties(CR 100%, CD 100%, AAG 82.6%, CV 58.3%) disappeared but also 220 Kdalton OMP(100%) was not produced. These results indicate that the positive CRMOX reaction is plasmid-mediated and the CRMOX test is potential as an in vitro virulence tests with Y enterocolitica.

• Applied Biological Chemistry
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• v.28 no.3
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• pp.149-155
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• 1985

590 strains of Rhizobia were isolated from root nodules of the legumes collected at 223 sites in Korea. According to their host specificities they were classified into R. japonicum(218 strains), R. phaseoli(101 strains), R. trifolii(97 strains), R. meliloti(4 strains), R. leguminosarium(1 strain), Rhizobium species(101 strains), and unidentified species(159 strains). 3 potent strains R-138, R-168, and R-214 of R. japonicum have been selected based on the infectivity to soybean cultivar and effeciency of nitrogen fixation. It was observed that the fast-growing strains of R. japonicum contained 1 to 4 plasmids of M.W. of 35-300 Md. However, plasmids were hardly detected for the slow-growing strains.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.211-220
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• 1986

Nitrogen fixation (nif) genes of Rhizobium leguminosarum were transferred to nif Klebsiella pneumoniae and E. coli by conjugation after partial heat induction of $RP_4$ :: Mu cts in Rhizobium $R^+$ transconjugant, and the hybrid plasmids in the transconjugant strains were isolated and characterized. In order to transfer the nif genes from Rhizobium, the hybrid plasmid $RP_4$ :: Mu cts was transferred by conjugation from E. coil to the symbiotic nitrogen fixer, R. leguminosarum. After stabillity test, the $RP_4$ :: Mu cts in Rhixobium $R^+$ transconjugant was subjected to partial heat induction by culturing it statically at $38^{\circ}C$ for 16 hours, and then conjugated with the nif defective mutant strains of K. pneumoniae or nif mutant strains of E. coli having whole nif gene plasmid. Recombinant strains of K. pneumoniae, which could grow in a N-free medium and exhibit the nitrogenase activity were selected. However, in the case of E. coli, they could grow well in a NA medium containing antibiotices, but hardly frow in a N-free medium. The hybrid plasmids in these transconjugal strains were isolated by gel electrophoresis and compared their molecular sizes.

• Journal of Microbiology
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• v.42 no.2
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• pp.87-93
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• 2004

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide meco-prop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)-and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MPll, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired dif-ferent mecoprop-degradative plasmids in different soils through natural gene transfer.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.46-51
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• 1987

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

• Journal of Sericultural and Entomological Science
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• v.35 no.2
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• pp.120-128
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• 1993

The objective of this study is to identify plasmids of Bacillus thuringiensis var. kurstaki HD-1(B. t k HD-1) toxic to lepidopteran larvae. The results from agarose gel electrophoresis indicated that the bacterium contained 9 plasmids with approximate sizes of 1.4, 4.9, 5.4, 9.3, 10, 29, 44, 52, and 150 megadaltons(Md). By treating the wild type of B. t k HD-1 with either SDS or EtBr as curing agent, 26 cured mutants of the bacterium were obtained, 9 of them were crystallifereous(cry+) and the others acrystallifereous(cry-). Plasmids from B. t k HD-1 were transferred to B. cereus 569 strR cry- recipients(Bc569 M1). Among 13 isolates of Bc569 M1 transcipient, 11 of them were capable of producing the crystal toxic proteins. The plasmid patterns of Bc569 M1 transcipients and partially curved mutants of B. t k HD-1 on agarose gel electrophoresis suggested that the 29 and 44Md plasmids should be involved in the production of crystalline toxic proteins.