• The Korean Journal of Physiology
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• 제28권2호
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• pp.233-240
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• 1994

In this study, it was investigated whether immortalized proximal tubule cells transformed with pRSVT could survive through the numerous passages. Results were as follows: 1. The cells transfected with pRSVT formed rapidly growing, multilayered colonies within 2 weeks in a hormone defined medium. Domes were also observed in some of the cultures. 2. r-glutamyl transpeptidase activity was equivalent to that observed in primary renal proximal tubule cell cultures. 3. Transformed cells with pRSVT form tubules in matrigel following 20 passages. 4. Genomic DNA of transformants was digested with either the restriction enzyme Xba or BamH1. A band of approximately 7.5kb was detected with Xba. Three BamH1 bands were detected at approximately 15 kb, 6.5 kb, and 3 kb.

• 아시안잔디학회지
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• 제18권4호
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• pp.211-219
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• 2004

Transgenic zoysiagrass (Zoysia japonica cv. Zenith) plants have been obtained by particle bombardment of embryogenic callus with the plasmid pSMABuba, which contains hygromycin resistance (hpt) and bialaphos resistance (bar) genes. Parameters on DNA delivery efficiency of the particle bombardment were partially optimized using transient expression assay of a chimeric $\beta-glucuronidase$(gusA) gene driven by the CaMV 35S promoter. Stably transfarmed zoysiagrass plants were recovered with a selection scheme using hygromycin. Transgenic zoysiagrass plants were confirmed by PCR analysis with specific primer for bar gene. Expression of the transgene in transformed zoysiagrass plants was demonstrated by Reverse transcriptase (RT)-PCR analysis. All the tested transgenic plants showed herbicide BastaR resistance at the field application rate of $0.1\%-0.3\%$.

• Archives of Pharmacal Research
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• 제16권4호
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• pp.300-304
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• 1993

Genetic transformation of streptomyces gaespitosus by plasmid plJ 702 was camied out. Optimal conditions for the protoplast preparation of streptomyces casepitosus, its regeneration, and its transformation by plJ 702 were evaluated. Addition of 2% glycine to the culture broth was optimal for protoplast yield. Formation and regeneration of protoplasts were most efficient when the mycelium were harvested at between late log and stationary growth phase. The regeneration frequency of the protoplasts was 15% when the protoplats were regenerated on R2YE agar media containing 0.5M sucrose. Under the best condition for protoplats (M.W. 4,000) treatment for 2 minutes.

• 디지털융복합연구
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• 제13권12호
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• pp.313-318
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• 2015

본 연구에서는 세균의 항균제 내성기전을 연구하기 위해 일개의 대학병원에서 분리된 Enterobacter cloacae를 대상으로 extended spectrum ${\beta}$-lactamase (ESBL) 및 16S rRNA methyltransferase 유전자를 검출하고 항균제 감수성 양상을 조사하였다. 대상균주 중 총 8 균주가 CTX-M-15형 ESBL을 생성하는 것으로 확인되었으며 이 균주들 중 3 균주는 16S rRNA methyltransferase의 한 종류인 armA 유전자도 동시에 가지고 있는 것으로 나타났다. CTX-M-15형 ESBL 유전자와 armA 유전자를 동시에 가지고 있는 E. cloacae는 3세대 cephalosporin 계열 및 aminoglycoside 계열의 항균제 뿐 만 아니라 fluoroquinolone 계열의 항균제에도 내성을 보였다. 더구나 이러한 항균제 내성 유전자들은 플라스미드를 통해 다른 세균으로 전달 될 수 있어 다제내성 세균의 출현 및 확산을 촉진 할 수 있다. 따라서 E. cloacae를 대상으로 지속적인 항균제 내성 유전자를 모니터링 하는 것은 항균제 내성 확산방지를 위해 중요할 것으로 사료된다.

• 대한의생명과학회지
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• 제9권4호
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• pp.195-201
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• 2003

Aromatic hydrocarbons are of major concern among genotoxic chemicals due to their toxicity and persistence. Some microorganisms can utilize aromatic hydrocarbons as carbon and energy sources by inducing expression of catabolic operon(s). The XylR regulatory protein activates transcription of the catabolic enzymes to degrade BTEX (benzene, toluene, ethylbenzene, and xylene) from its cognate promoters, Pu and Ps upon exposure of the cells to the aromatic hydrocarbons. The activity of XylR on the promoters was previously monitored using luciferase luc reporter system. The xylR, its promoter Pr and the promoter Po for the phenolic compound catabolic operon were introduced upstream of firefly luciferase luc in the pGL3b vector to generate about 7.1 kb of pXRBTEX. Here E. coli harboring the plasmid was freeze-dried under various conditions to fin,d optimal conditions for storage and transport. The cell viability and luciferase activity were maintained better, when the cells were freeze-dried at -7$0^{\circ}C$ in the addition of the 10% skim milk or 12% sucrose. However, coaddition of protectants such as 10% skim milk plus 10% glucose or 12% sucrose plus 10% glucose, resulted in much better viability and bioluminescence activity compared with the effect of single addition of each protectant. In addition, it was shown that the freeze-dried cells maintained almost intact bioluminescent activities and cell viability for at least 1 week after freeze-drying. This work demonstrated that the properly freeze-dried recombinant bacterial cells could be utilized as a whole-cell biosensor for simple and rapid monitoring of BTEX in the environment.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.431-438
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• 2009

The alkaline serine protease asp, which was shown to be a virulence factor of Vibrio alginolyticus as a purified protein, was cloned from V. alginolyticus EPGS, a strain recently isolated from moribund Epinephelus coioides in an outbreak of vibriosis in a mariculture farm of Shenzhen. The asp null mutant was constructed by homologous recombination with suicide plasmid pNQ705-1. Compared with the wild-type strain, the asp null mutant exhibited a significant decrease of total extracellular protease activity, and caused a IS-fold decrease in virulence of V. alginolyticus. In our previous study, the luxO and $luxR_{val}$ genes from V. alginolyticus MVP01 were cloned and identified, and the luxO-$luxR_{val}$ regulatory couple was shown to regulate various genes expression, suggesting that it played a central role in the quorum sensing system of V. alginolyticus. In this study, the regulation of the asp gene was analyzed by using RT-PCR and quantitative real-time PCR methods; we proved that its transcription was greatly induced at the late stage of growth and was regulated by a luxO-$luxR_{val}$ regulatory system.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.637-645
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• 1999

Two putative regulator genes, mocR and mocS, of the moc (mannityl opine catabolism) operons in pTi15955 of the octopine-/mannityl opine-type Agrobacterium tumefaciens strain 15955, were tested for their possible roles as repressors in the moc operons. The regions upstream of macC and mocD, the first structural genes in the two divergently oriented moc operons, were transcriptionally fused into the promoterless lacZ reporter gene. Each of the lacZ-fusions was introduced into Agrobacterium strain UIA5, a Ti plasmid-cured derivative, harboring either a mocR or a mocS clone. The resulting strains were grown in media containing various sugar sources, and the $\beta$-galactosidase activities were quantitatively measured. The results suggested that MocR repressed the expression of macC and macD. The expression of the fused $\beta$-galactosidase was not induced by mannopine (MOP) or possible catabolic intermediates of the opine, e.g. santhopine (SOP), glucose, mannose, or glutamine. However, the repression was significantly relieved by the supplementation of MOP and the concomitant introduction of the agcA gene encoding MOP cyclase that catalyzes the lactonization of MOP to agropine (AGR). These results suggested that AGR, rather than MOP or the other catabolic intermediates, is the inducer for the expression of the operon. On the contrary to previous report showing that the induction levels of macC and macD were lowered by the supplementation of inorganic nitrogen in media, the expression of these genes was not affected by the level of nitrogen in our reporter system. MocS did not strongly repress the expressions of macC and mocD. It is possible that MocS may be involved in the regulation of the operons present downstream of the moc operon, which are responsible for the utilization of mannopinic acid and agropinic acid.

• Molecules and Cells
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• 제38권12호
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• pp.1079-1085
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• 2015

Originally, activins were identified as stimulators of FSH release in reproduction. Other activities, including secondary axis formation in development, have since been revealed. Here, we investigated the influence of activin ${\beta}_A$ on the body, including the gastro-intestinal (GI) tract. Initially, the activin ${\beta}_A$ protein was detected in the serum proportional to the amount of pCMV-rAct plasmid injected. The induced level of activin ${\beta}_A$ in muscle was higher in female than male mice. Subsequent results revealed that stomach and intestine were severely damaged in pCMV-rAct-injected mice. At the cellular level, loss of parietal cells was observed, resulting in increased pH within the stomach. This phenomenon was more severe in male than female mice. Consistent with damage of the stomach and intestine, activin ${\beta}_A$ often led to necrosis in the tip of the tail or foot, and loss of body weight was observed in pCMV-rAct-injected male but not female mice. Finally, in pCMV-rAct-injected mice, circulating activin ${\beta}_A$ led to death at supraphysiological doses, and this was dependent on the strain of mice used. Taken together, these results indicate that activin ${\beta}_A$ has an important role outside of reproduction and development, specifically in digestion. These data also indicate that activin ${\beta}_A$ must be controlled within a narrow range because of latent lethal activity. In addition, our approach can be used effectively for functional analysis of secreted proteins.

• 한국균학회지
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• 제17권4호
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• pp.209-213
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• 1989

사철 느타리 버섯 Uracil 요구성 균주를 Aspergillus nidulans ans 1과 Neurospora crassa pyr 4 유전자를 지닌 백터를 이용하여 형질전환시켰다. 사철 느타리버섯 ura 요구성 균주의 원형질체를 pyr 4 유전자를 지닌 백터 pDJB3과 혼합 후 PEG와 $CaCl_2$를 처리하였다. 형질전환 균주는 버섯 최소배지에서 안정되게 생육하였고 southern hybridization 결과도 백터 DNA가 사철 느타리 염색체에 삽입되었다. 형질전환 균주는 단핵성이므로 다른 단핵 균사와 균사 융합 후 자실체 형태, 포자형성 등을 조사하였다. 형질전환이 안된 균주는 톱니형 자실체를 형성한데 비해 형질전환된 균주는 톱니형, 깔대기형, 우산형, 갓 미발육형의 자실체를 형성하였다.

• 대한미생물학회지
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• 제22권3호
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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.