• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1336-1344
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• 2011

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

• Korean Journal of Plant Resources
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• v.21 no.1
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• pp.41-46
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• 2008

Molecular relationship and genetic diversity of 21 wild tea collections which grown natural region in Korea were investigated based on PCR-RFLP analysis using DFR genes. Approximately 1.4kb fragment of the DFR gene from wild tea samples were successfully amplified use DFR 4+5 primer pair. On the bases of restriction fragment length polymorphism(RFLP) analysis using Hpa II and Mse I enzymes, three different band patterns shown from Hpa II enzyme and showed genetic diversity between same region wild tea group. Six kind of restriction enzyme profiles obtained from digested with restriction endonuclease Mse I and shown two kind of restriction enzyme profiles collected from same region wild tea at Ungpo. The results of RFLP analysis indicated that wild tea showed genetic diversity among different regions of tea groups, but also between same region wild tea.

• Korean Journal of Veterinary Service
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• v.32 no.3
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• pp.257-264
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• 2009

Subtyping of Brucella abortus isolates is epidemiologically important for monitoring of bovine brucellosis outbreaks. Pulsed-field gel electrophoresis (PFGE) is considered as a gold standard of molecular typing methods to study the DNA polymorphisms of bacteria. In this study, we analyzed using PFGE the DNA fragment profiles of B. abortus isolated in Gyeongbuk province from 1998 to 2006. The genomic DNA was digested with the restriction endonuclease Xba I, Xho I and Smi I followed gel electrophoresis. No distinguishable patterns of the genomic DNA digested with Xba I and Xho I were observed among the field isolates of B. abortus tested in this study. But Smi I restriction enzyme resulted in two PFGE patterns consisting of 13-15 bands that ranged in size from 33 to 668bp by standard marker. The cluster analysis by DNA fingerprinting software showed 93.75% similarity between two PFGE patterns. No different PFGE patterns were recognized among the isolates originated from various years, regions and cow breeds.

• Korean Journal Plant Pathology
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• v.11 no.1
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• pp.66-72
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• 1995

The coat protein (CP) gene of cucumber mosaic virus-As (CMV-As) strain was engineered for expression in the plant by using the cauliflower mosaic virus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone, pCMAS66, was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites in order to re-introduce plant expression vector, pHI121. The resulting subclone pCASCP02 and plant expression vector pBI121 were treated with BamHI-SacI for excising the target gene and removing GUS gene, respectively. After Agrobacterium transformation by freeze-thaw technique, the clone, pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. The results on tobacco plant transformation with the vector system revealed that the system could be successfully introduced and showed high frequency of selection to putative transformations.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.117-121
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• 1998

Bacteriophage SC 921 of Lactobacillus plantarum, isolated from kimchi, showed high lytic effects at 0.2 M.O.I. level. The phage particle contained 4 major proteins (48, 34, 32, 29 kDa). Intact DNA of phage SC 921 is a double stranded linear molecule, and the genomic size is approximately 66.5 kilobase pairs (kbp). Restriction analysis of the genome showed that Sma I gave single site cut and Xba I gave 2 site cuts, while Cla I, Kpn I, and EcoR I formed 4, 5, and 6 cuts, respectively. Hind III digested phage DNA to many fragments. A restriction map of genomic DNA was constructed using the restriction endonuclease Kpn I, Sma I, and Xba I. Bacteriophage SC 921 was compared with B2 phage which had been reported to infect Lactobacillus plantarum ATCC 8014(KCCM l1322). Bacteriophage SC 921 differs from B2 phage at least in thr size of its genome and phage particle proteins.

• Journal of Life Science
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• v.23 no.6
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• pp.770-778
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• 2013

To determine the seasonal variation of bacterial community in the seawater of Gwangyang Bay, three hundred thirty six bacterial strains were isolated on February, May, July and October 2011. Amplified Ribosomal DNA Restriction Analysis (ARDRA) was used to construct the phylotyes of the isolates using the restriction endonuclease, Hae III. Diversity indices of ARDRA patterns were calculated. One hundred and one phylotypes including 40 unique pylotypes were found at the 80% similarity level. Partial 16S rRNA genes of one hundred thirty nine strains representing each phylotypes were sequenced and compared. Bacterial community composed of 4 different phyla which include Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes. Proteobacteria was the prevailing phylum in all seasons, followed by Bacteroidetes in winter, spring and autumn while Actinobacteria in summer. At the family level, Flavobacteriaceae dominated in winter and spring and Pseudoalteromonadaceae did in summer and autumn. Genera Altererythrobacter, Loktanella, Pseudoalteromonas and Vibrio were encountered in all seasons. The most diverse bacterial community was found in autumn followed by the order of spring, winter and summer.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.369-376
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• 1992

This paper dealt with plasmid DNA profile in 98 Salmonella(S) isolated from pigs and cattle sources in Taegu, Gyeongbook and Gyeongnam during the period from 1984 to 1987. Also we were studied for restriction enzyme analysis of the plasmid DNA, and mouse infection, Sereny test and normal setum resistance test in guinea pig for S typhimurium and S enteritidis harbored or cured 60 megadalton(Md) plasmid and 36 Md plasmid, respectively. Of the 13 Salmonella isolated from cattle, 7 Salmonella harbored one or more plasmids and molecular sizes of the large plasmids were 60 Md for S typhimurium and 36 Md for S enteritidis. Of the 85 Salmonella isolated from pigs, 47 Salmonella were confirmed as being one or more plasmids, and all the S typimurium stains harbored 60 Md plasmid. In enzyme digestion with 8 types of restriction endonuclease for 60 Md plasmid DNA of S typhimurium, cleavage patterns were varied to enzymes, and the DNA was segmented into 4 to 15 fragments. In restriction enzyme analysis of 36 Md plasmid DNA obtained from four strains of S. enteritidis, the DNA showed the same cleavage patterns obtained with Eco RI, Hind III and Bam H I, and was segmented into 3 to 5 fragments. In virulence for mice by measuring the 50% lethal dose ($LD_{50}$), the $LD_{50}$ values obtained for 60 Md virulence-associated plasmid harbored strains of S typhimurium and 36 Md virulence-associated plasmid of S enteritidis were up to $10^4$-fold lower than the values obtained for the plasmid-cured strains of the same serotype. Only the plasmid harbored strains were resistant to the bactericidal activity of 90% guinea pig serum, and only they gave positive responses in sereny test. We suggested that their plasmid DNA might be associated with virulence for mice.

• The Journal of Korean Society of Virology
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• v.26 no.2
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• pp.173-179
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• 1996

The gene encoding gIII of bovine herpesvirus type 1 (BHV-1) PQ strain was cloned and expressed in baculovirus. Although the gIII gene is located in Hind III I fragment as the case of the other BHV-1 strains, differences in size and restriction endonuclease site within the fragment were identified. The gIII expression was predominantly detected on the surface on insect cells by indirect immunofluoresecnce assay using monoclonal antibody. The western blotting analysis also revealed the presence of expressed protein of a similar molecular size to the original gIII protein. The immunogenicity of expressed protein were tested in guinea pigs. The immunized guinea pigs with expressed protein developed the neutralizing antibodies against BHV-1.

• The Journal of the Korean Society for Microbiology
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• v.22 no.1
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• pp.95-99
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• 1987

When tetracycline resistancy were cured by ethidium bromide treatment, some of the cured strains lost the tetracycline resistance plasmid while other strains kept the plasmids. Both strains of lost and remained plasmids were digested with restriction endonuclease Hind III and these cleaved plasmids were compared with that of parent strains, two plasmid remained strains showed same cleavage patterns between parent and cured strains, however, one plasmid lost strain showed dissimilarity with parent strain, but in the other one strain, among 4 plasmid lost colonies, 2 showed same but other 2 showed different patterns compared to parent strain. Tetracycline was transfered by conjugation in on set(Staphylococcus aureus donor versus Staphylococcus epidermidis, recipient) with relative high frequency but the other 2 sets showed a low degree of frequency and the other 2 sets exhibited no transfer.

• Journal of environmental and Sanitary engineering
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• v.14 no.4
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• pp.99-109
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• 1999

Enteropathogenic Yersina enterocolitica is an important cause of human and animal disease. Phenotypic and genotypic characteristics currently used to identify Yersinia enterocolitica are not necessarily sufficient to differentiate pathogenic from non-pathogenic strains or to analyze the epidemiology of yersiniae at a molecular level. To improve the characterization of Yersinia enterocolitica, A total of 65 isolates of Yersinia enterocolitica were examined with bioserotyping, antibiotic susceptibilities, PFGE, PCR-ribotyping. Genomic DNA pattern generated by PFGE are highly specific for different strains of an organism and have significant value in epidemiologic investigations. The PFGE analysis of Not I-digested chromosomal DNA of Y. enterocolitica were performed with a CHEF Mapper(Bio-Rad, USA). Not I generated 19 restriction endonuclease digestion profiles(REDP). PCR-ribotyping, performed with primers complementry to conserved regions of 16S and 23S rRNA gene, generated 13 ribotypes. PCR-ribotyping can be considered a good technich for subtyping strains of Y.enterocolitica.