• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.81-85
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• 1993

Gel electrophoresis has proven to be one of the most useful of DNA separation and purification. The new technique of pulsed field gel electrophoresis (PFGE) is high resolution separation of large size DNA moleculs. Conventional continuous gel electrophresis can not be separation of large DNA fragments(20~50 k base). Field inversion gel electrophoresis(FIGE) is very useful for large DNA molecules. We have found that a pulse ratio ; 2 : 1, time ; 24hrs., volts ; $10^{volts}/_{cm}$, start ; 0.45sec, end ; 1sec, is most effectively resolves DNA fragment in the 6~50k base.

• Journal of Yeungnam Medical Science
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• v.12 no.2
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• pp.366-385
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• 1995

Epidemiological studies are important in both the prevention and treatment of mycobacterial infections. This study was initiated to establish the pulsed-field gel electrophoresis (PFGE) method, which are not yet extensively studied. The most apprpriate restriction endonucleases included DraI, AsnI, and XbaI. The optimal PFGE condition was different according to the enzymes used. Two stage PFGE was performed, in case of DraI first stage was performed with 10 seconds of initial pulse and 15 seconds of final pulse, while the second stage was performed with 60 seconds of initial pulse and 70 seconds of final pulse. The electrophoresis time for DraI-PFGE was 14 hours for each stage. Electrophoresis was performed for 22 hours, in case of XbaI, with 3 seconds of initial pulse and 12 seconds of final pulse. Electrophoresis was performed for 22 hours, in case of AsnI, with 5 seconds of initial pulse and 25 seconds of final pulse. In all cases the voltage of the electrophoresis was maintained constantly at 200 voltage. Standard mycobacterial strains, which included Mycobacterium bovis BCG, M. tuberculosis, and M. fortuitum, could not be differentiated by PFGE analysis. PFGE analysis was performed to differentiate 9 clinically isolated M. fortuitum strains using AsnI. All M. fortuitum strains showed different genotypes except 2 strains. Cluster analysis divided M. fortuitum strains into 2 large groups. PFGE analysis was performed to further differentiate M. fortuitum isolates using XbaI. The undifferentiated 2 M. fortuitum strains showed different PFGE patterns with Xba I. Cluster analysis of the XbaI-PFGE patterns showed more complex grouping than AsnI-PFGE patterns, which showed that XbaI-PFGE analysis was better than AsnI-PFGE in M. fortuitum genotyping. The top dissimilarity values of AsnI-PFGE and XbaI-PFGE were 0.74 and 0.75, respectively. This value was higher than that of arbitrarily primed polymerase chain reaction (AP-PCR) analysis and lower than that of restriction fragment length polymorphism (RFLP) analysis. This suggested that PFGE can be used as a supportive or alternative genotyping method to RFLP analysis.

• Environmental Mutagens and Carcinogens
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• v.17 no.1
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• pp.1-6
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• 1997

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the partial cloning and characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the sequence homologous DNA to RAD4 gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. The level of the transcript did not increase upon UV-irradiation, suggesting that the RAD4 homologous gene in C. cinereus is not UV-inducible.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1650-1655
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• 2006

Bacteria belonging to the genus Paenibacillus are facultatively anaerobic endospore formers and are attracting growing ecological and agricultural interest, yet their genome information is very limited. The present study surveyed the genomic features of P. polymyxa ATCC $842^T$ using pulse-field gel electrophoresis of restriction fragments and sample genome sequencing of 1,747 reads (approximately 17.5% coverage of the genome). Putative functions were assigned to more than 60% of the sequences. Functional classification of the sequences showed a similar pattern to that of B. subtilis. Sequence analysis suggests nitrogen fixation and antibiotic production by P. polymyxa ATCC $842^T$, which may explain its plant growth-promoting effects.

• Journal of Life Science
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• v.10 no.2
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• pp.50-54
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• 2000

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. Here, we report the cloning and characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the sequence homologous DNA to RAD4 gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 3.4 kb BglII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. The isolated gene encodes a protein of 810 amino acids.

• Journal of Life Science
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• v.16 no.4
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• pp.664-671
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• 2006

We obtained 424 Salmonella enterica serovar Typhi isolates from sporadic cases of infection in Busan during 1996 to 2005. We investigated the trend of antimicrobial resistance and molecular typing by pulsed-field gel electrophoresis (PFGE). Of the total 424 isolates, 6 strains (1.4%) were multidrug-resistant (MDR) S. enterica serovar Typhi isolates, 2 strains (0.5%) were resistant to only nalidixic acid, and the remaining 416 strains (98.1%) were fully susceptible to the 18 antimicrobial agent. PFGE of XbaI-digested chromosomal DNA was performed on 50 sporadic S. enterica serovar Typhi isolates with the objective of investigating the extent of genetic diversity of these isolates in our region. We could find that these isolates were much more heterogeneous and at least 32 different PFGE patterns were generated according by dice coefficient, between 0.69 and 1.0. Restriction fragment patterns consisted of 13 to 18 fragments ranged in size from 20 to 630 kb. The results confirmed that PFGE would be an useful tool for investigating surveillance of sporadic or outbreak case and assessing clonality for S. enterica serovar Typhi in Busan area. Our finding will be valuable in developing rational strategies to control this pathogen and setting the basis of an effective PulseNet system in Korea.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.942-945
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• 2008

There have been concerns about possible pathogenicity and antimicrobial resistance in Enterococcus, which constitute more than 50% of probiotics in the worldwide market. In this study, Enterococcus in sixteen products manufactured by ten different companies was tested for the presence of six virulence genes and two vancomycin resistance genes. Results in this study showed the safety of Enterococcus on the Korean market and the importance of screening vanA, vanE, agg, cylA, esp, and gelE. Pulse-field gel electrophoresis showed that the sixteen isolates tested in this study are originated from three strains.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.50-54
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• 2011

This study's objective was to determine the prevalence of Salmonella spp. in pigs and their farm environments in Korea, and to investigate the relationship between the strains based on their phenotypic and genotypic characteristics. A total of 36 Salmonella spp. were isolated in this study: 18 isolates from 492 pigs (3.7%) and 18 isolates from 418 (4.3%) farmhouse environmental samples from 16 different pig farms. Of the Salmonella strains isolated from the numerous environmental samples, the highest prevalence was observed in slurry or manure, followed by partitions, farmer's hands, floors, water/nipples, ventilation sources, and feed, respectively. All the Salmonella isolates originating from different farms were genetically distinct. In three farms, however, identical phage types and pulse-field gel electrophoresis patterns were observed among Salmonella isolates from pig feces and environmental samples. This study suggests that environments contaminated with Salmonella could pose an infection risk to pigs on pig farms.

• Journal of Microbiology
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• v.40 no.1
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• pp.63-69
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• 2002

A total of twenty-five norfloxacin resistant Escherichia coli were isolated from Joongrang-chun stream, a branch of the Han River in Seoul, Korea from May to July in 2000 and their norfloxacin resistance mechanism was characterized for target site mutation, permeability, and efflux pump. Fourteen iso- lates contained the same three mutations, Ser83→Leu and Asp87→Asn in GyrA and Ser90→ lle in ParC. Six isolates had Ser83→Leu and Asp87→Tyr in GyrA and Ser87→lle in ParC while one isolate had Ser83→Leu and Va1103→Ala in GyrA and Ser80→lle in ParC. Two isolates had mutation(s) in GyrA without any mutation in ParC. Two isolates had Ser80→Arg in ParC instead of the commonly found Ser80→lle. Every norfloxacin resistant isolate had an efflux system but the correlation between the efflux activity and MIC was not observed. The amount of OmpF for norfloxacin permeability decreased in resistant isolates compared to the susceptible strains. When amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) were performed, these isolates showed no similarity to each other or clinical isolates.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.775-778
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• 2010

A total of 2,858 meat samples collected during 2003-2008 in Korea were investigated, and methicillin-resistant Staphylococcus aureus (MRSA) isolates were isolated from 1.0% (9/890) of beef, 0.3% (4/1,055) of pork, and 0.3% (3/913) of chicken meat samples, respectively. MRSA isolates showed the two sequence types (STs), ST72 from beef and pork and ST692 from chicken meat. MRSA isolates from beef and pork were Panton-Valentine leukocidin-negative, staphylococcal cassette chromosome mec type IVa strain with ST72, which is the most prevalent type of communityacquired MRSA in Korea. An identical pulse-field gel electrophoresis pattern was detected among 10 of 16 MRSA isolates: 9 strains from beef (n=5) and pork (n=4) in 2008, and one strain from beef in 2005.