• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.2
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• pp.115-121
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• 2002

To develop a new method of conjugation and to determine the distribution of R plasimds, we isolated multi-drug resistant strains from fish pathogenic bacteria in the farms of south and east seacoasts of Korea. Out of the 134 isolates examined, 10 showed resistance to chloramphenicol, tetracycline, streptomycin, ampicillin, colistin, nalidixic acid, oxolinic acid and kanamycin. One out of 10 multi-drug resistance bacteria, Vibfio damsela JE1 (V. damsela JE1), contained transferable R plasmid of chlorarnphenicol- tetracycline resistance genes and other nucleic acids encoding ampicillin and kanamycin resistance. The presence of the R plasmid was confirmed by conjugation using the chromocult medium (CC) as a selective and differential medium for transconiugants with identification based on the growth or colors of the colonies. The frequency of R plasmid transfer with filter mating method was come out much higher than that of broth mating method and appeared to be dependent upon the mating time and temperature. The optimum conditions for filter mating method were found to be 30$^{\circ}C$ and 24hrs as mating temperature and period, respectively, Moreover, donor cells with R plasmid, both isolate and standard bacteria, were shown to have an ability to transfer the plasmid against Escherichia coli K-12 HB101 (E. coli HB101) and Edwardsiella tarda (E. tarda) RE14 at fairly high frequencies, finally, we isolated 3 isolates of Sphingomonas sp., carrying R plasmid from 12 multi-drug resistant bacteria in normal microflora of the flounder (Paralichthys olivaceus) group used for the isolation of V emsela JE1 four months before. The same size and gene transfer chayateristics of R plasimds with those of V damsela JE1 confirmed that normal microflora have the reservoir activity for R plasmid in natural aquatic environment.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.91-98
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• 2005

The hyoscyamine 6${\beta}$-hydroxylase (h6h) gene was introduced into the genome of Scopolia parviflora through the Agrobacterium rhizogenes binary vector system. The enzyme was expressed ally and tissue specific selectively in roots, resulting in five transgenic hairy root lines. The presence of the h6h gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction (PCR), Northern blotting, and Western blotting, respectively. In the transgenic hairy root lines which constitutively expressed the H6H enzyme, hyoscyamine and scopolamine accumulated in high concentration. Among the transgenic hairy root lines that expressed the H6H enzyme, only two were more productive. The levels of tropane alkaloids in transgenic hairy root varied greatly: The best transgenic line (#5) contained 8.12 mg of scopolamine per g dry weight, which produced the compound three times more than wild-type root. These results suggest a possibility of improving the yield of tropane alkaloids in hairy root lines by genetic and metabolic engineering.

• Proceedings of the Korean Society of Tobacco Science Conference
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• 1997.10a
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• pp.134-152
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• 1997

Plant viruses of tobacco including tobacco mosaic virus (TMV) and potato virus Y (PVY) cause severe economic losses in leaf-tobacco production. Cultural practices do not provide sufficient control against the viruses. Use of valuable resistant cultivars is most recommendable for the control of the viruses. However, conventional breeding programs are not always proper for the development of virus-resistant plants mostly owing to the frequent lack of genetic sources and introduction of their unwanted properties. Therefore, we tried to develop virus-resistant tobacco plants by transforming commercial tobacco cultivars, NC 82 and Burley 21, with coat protein (CP) or replicase (Nlb) genes of TMV and PVY necrosis strain (PVY-VN) with or without untranslated region (UTR) and with or without mutation. Each cDNA was cloned and inserted in plant expression vectors with 1 or 2 CaMV 35S promotors, and introduced into tobacco leaf tissues by Agrobacterium tumefaciens LBA 4404. Plants were regenerated in kanamycin-containing MS media. Regenerated plants were tested for resistance to TMV and PVY In these studies, we could obtain a TMV-resistant transgenic line transformed with TMV CP and 6 genetic lines with PVY-VN cDNAs out of 8 CP and replicase genes. In this presentation, resistance rates, verification of gene introduction in resistant plants, stability of resistance through generations, characteristics of viral multiplication and translocation in resistant plants, and resistance responses relative to inoculum potential and to various PVY strains will be shown. Yield and quality of leaf tobacco of a promising resistant tobacco line will be presented.

• Horticultural Science & Technology
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• v.28 no.3
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• pp.449-455
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• 2010

'Moulinrouge' was selected as the best regenerating cultivar among 18 different spray-type chrysanthemum cultivars bred in the Gyeongnam Flowers Breeding Research Institute. When the leaf explants from standard- and spray-type chrysanthemum 'Jinba' and 'Moulinrouge' were incubated on MS basal medium supplemented with $0.5mg{\cdot}L^{-1}$ BA and $1.0mg{\cdot}L^{-1}$ NAA, both 'Jinba' and 'Moulinrouge' induced adventitious shoots that can be regenerated into plantlets. Based on these regeneration conditions, we developed an efficient $Agrobacterium$-mediated chrysanthemum 'Moulinrouge' transformation method by using sequential selection of shoots from low ($10mg{\cdot}L^{-1}$) to high ($30mg{\cdot}L^{-1}$) concentrations of kanamycin after co-cultivation of leaf explants with $Agrobacterium$ for 10 days and induction of shoots. All kanamycin resistant plants investigated with genomic PCR analysis carried the report gene, $AtSICKLE$, in their genome. Although expression levels of the report gene in the transgenic plants investigated with RT-PCR were relatively low because of inefficiency of CaMV 35S promoter in chrysanthemum, transgenic lines expressing $AtSICKLE$ efficiently showed leaf epinasty phenotype. We expect that our results will provide a useful method that can perform a high-throughput investigation of genes isolated and studied well in model plants for molecular breeding of chrysanthemum.

• Journal of Veterinary Clinics
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• v.23 no.2
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• pp.169-174
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• 2006

This work described 3 infection cases caused by Rhodococcus equi in foals between 3 and 5 months of age. The disease histories were not fully taken from local veterinarians. At least 1 sick foal has been treated with cephalothins followed by penicillins during approximately 1 week, but died without effectiveness and other foals rearing with the animal have been suffering from severe pneumonia which show high fever, laboring respiration, cough and/or nasal discharge. There were many abscessations into lungs of 2 foals in postmortem examination and another 1 sample was pus collected from abscess around the shoulder, indicating the osteomyelitis. Those bacteria were grampositive coryneform and were identified as a R. equi by a polymerase chain reaction (PCR) using primers for R. equi-specific vapA gene. The pathogens were usually resistant to penicillin, ampicillin, amoxycillin/clavulanic acid, cefazolin, clindamycin, sulfamethoxazol/trimethoprim, kanamycin, and tetracycline, while were sensitive to ciprofloxacin, norfloxacin, orfloxacin, gentamicin, erythromycin, neomycin, and vancomycin. Some more foals with respiratory symptoms in 1 horse farm were treated by orally administration with erythromycin during 2 weeks. Because the combination of erythromycin and rifampin has recommended as the treatment for R. equi infections in foals, the local equine veterinarian can choose those antibiotics for the treatment of this disease in future. However, another antimicrobial agent may be necessary if R. equi resistant to both agents is isolated.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.124-129
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• 2011

The role of the ferric uptake regulator (Fur) of Helicobacter pylori in the oxidative stress was investigated in this study. A fur knockout mutant of H. pylori was constructed by replacing the fur gene with an aphA (kanamycin resistant marker) gene. Photodynamic therapy using methylene blue (MB) and 660 nm light was chosen to induce oxidative stress. The bactericidal effect of photodynamic therapy (PDT) was compared between wild type H. pylori and fur knockout mutant H. pylori. The degree of oxidative damage of DNA was confirmed using alkaline gel electrophoresis and an assay of 8-hydroxy-2-deoxyguanosine (8-OHdG). In control groups, the number of viable cells was maintained constantly during experiment. After PDT, the mutant H. pylori showed 10,000 times decreased viable cell number compared with wild type H. pylori. Depending on the exposure time of 660 nm light, the 3-fold increase in the concentration of 8-OHdG was observed in mutant H. pylori. The results of this study showed that H. pylori's Fur protein may play a role in oxidative stress induced by PDT.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Korean Journal of Veterinary Research
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• v.46 no.4
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• pp.337-345
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• 2006

The shigellae are common etiological agents of bacillary dysentery in humans and primates. During four years from 2002 to 2005, 22 strains of Shigella spp. were isolated from the diarrheic patients in Seoul region. All of them were identified as S. flexneri by biochemical tests and serotyping. The prevalence of serotypes were variable by year, but the major serotypes were 2a and 3a. In an antimicrobial susceptibility test, all of the isolates were resistant to streptomycin and tetracycline, and susceptible to amikacin, kanamycin, cefoxitin, and gentamicin. All of the isolates showed the multi-resistant patterns over 3 drugs. By analysis of the plasmid profile the isolates were classified into 7 groups (P1~P7). Serotypes 2a and 2b were distributed to P1, P2, P3, and P4. Serotype 3a was differentiated to P5 and serotype 3b, to P6 and serotype 4a, to P7. PCR results showed that all isolates were positive for two virulence genes, ipaH and ial, but none of the strains had stx gene. The set1A and set1B genes were detected from 12 isolates (54.5%) that belonged to serotype 2a and 2b. The sen gene was detected from 19 isolates (86.4%). The 22 isolates showed 12 to 17 DNA fragments in the sizes ranging from 20.5 kb to 1135 kb, resulting in 13 patterns by the PFGE with Not I digestion. The PFGE patterns of the isolates showed the close relation with the serotypes, but no relations with year of isolation and antimicrobial resistance.

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.183-188
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• 1999

The incidence of Yersinia enterocolitica in raw meat was determined over 10 month period. Y. enterocolitica was isolated from 8.5% of beef, 17.0% of pork and 25.6% of chicken. Overall prevalence of Y. enterocolitica in raw meat was 17.5%. Seasonal difference was observed in isolation rate in which pork and chicken samples collected in the second half of the year twice was more than that of the first half of the year. Serotypes of Y. enterocolitica isolates were O:5 (17.3%), O:8 (8.6%), O:3 (6.2%), O:1,2 (1.2%), and others. The antibiotics susceptibility tests showed Y. enterocolitica isolates were resistant to carbenicillin, ampicillin, erythromycin, penicillin and bacitracin. In contrast it showed sensitivity to polymyxin B, kanamycin, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, nalidixic acid, and tetracycline. PCR with specific primers derived from ail gene of Y. enterocolitica was applied to detect and confirm pathogenic Y. enterocolitica. About 10% of the isolated Y. enterocolitica proved to be pathogenic and most of them were found in pork. However, proper cooking and meat process can kill and remove all Y. enterocolitica in meat, because the organism is very sensitive to heat.

• Journal of Life Science
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• v.20 no.10
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• pp.1549-1555
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• 2010

Streptococcus suis is a worldwide pathogen of a variety of porcine infection and has also been described as a pathogen for humans. We studied biochemical characteristics, antimicrobial susceptibility, and identification of polymerase chain reaction (PCR) of S. suis isolated from diseased pigs in Gyeongbuk province from 2004 to 2009. Sixty-one isolates were identified as S. suis by biochemical characteristics and PCR from 40 farms. The biochemical characteristics of S. suis isolates were production of VP-negative, hippurate, esculin, and arginine decarboxylase-positive, and fermentation of carbohydrate was variable lactose, trehalose, inulin, and raffinose, which was typeable 11 phenotype. In an antimicrobial susceptibility test, the majority of isolates were highly susceptible to amoxicillin/clavulanic acid, ampicillin, cephalothin, cefoperazone and florfenicol, while being highly resistant to streptomycin, kanamycin, amikacin, neomycin, erythromycin, clindamycin, and tetracycline. The isolates were divided into 11 phenotypes of biochemistry. By using PCR, the 16S-rRNA gene DNA fragment was detected at 304 bp from all of isolates. These results may provide the basic information needed to establish strategies for the prevention of S. suis infection in pigs.