• YAKHAK HOEJI
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• v.31 no.2
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• 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.

• 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.

• 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.

• Korean journal of applied entomology
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• v.28 no.3
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• pp.105-112
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• 1989

The nuclear polyhedrosis viruses of Bombyx mori (BmNPV) and Hyphantria cunea (HcNPV) were characterized by electron microscopic observation, SDS-PAGE of polyhedral and virion proteins, and restriction endonuclease analysis of viral DNAs. Polyhedra of BmNPV were octadecahedral in shape with the diameter of $3 \mu\textrm{m}$, whereas those of HcNPV showed irregular appearances having the diameter of $1.5~2\mu\textrm{m}$. Under alkaline protease inactivated condition, polyhedral proteins of two NPVs were resolved into a major polypeptide, 30~31 KD, and several minor polypeptides by SDS-PAGE. Examination of virion proteins by silver staining after SDS-PAGE showed that BmNPV was composed of 47 polypeptides with M.W. range of 9.6~112 KD and HcNPV was composed of 48 polypeptides with M.W. range of 9.4~111 KD. The approximate genome size of two NPVs were determined by restriction endonuclease analysis: BmNPV and HcNPV were 114.6 Kb, respectively.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Korean journal of applied entomology
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• v.34 no.4
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• pp.373-377
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• 1995

Four Bacillus thuringiensis isolates obtained from soil samples in Korea produce parasporal inclusions non-toxic to 10 insect species of three orders, Lepidopera, Diptera and Coleoptera. These four isolates are named NTB-1, NTB-2, NTB-3 and NTB-4, respectively. The morphology of parasporal inclusions of four isolates observed by phase contrast- and scanning electron microscope was all ovoid. Characterization of four non-toxic B. thuringiensis isolates was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis. The results showed that parasporal inclusion proteins and total plasmid DNA profiles of four isolates are different from other known non-toxic B. thuringiensis strains', suggesting that four isolates are novel.

• Pediatric Infection and Vaccine
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• v.3 no.1
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• pp.76-85
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• 1996

Adenoviruses(Ad) are considered to be second only to rotaviruses as the most significant cause of gastroenteritis in young children in Korea and thus it is essential to know the full spectrum of Ad serotypes routinely present in stool specimens from symptomatic patients. Sixty-six Ad isolates and three questionable ones collected over a 2-year peiord were typed by standard microneutralization, restriction endonuclease digestion and PCR of viral DNA to be able to evaluate these assays comprehensively for their ability to identify Ad associated with gastroenteritis. A total of sixty-one isolates(88.4%) were typed: the predominant types were Ad type 41(Ad41)(26.2%), Ad2(19.7%), Ad40(14.8%), Ad5(9.8%), and Ad7(9.8%) which together accounted for almost 80% of the isolates. The remaining virus isolates were typed as Ad1, 31, 34, 3, 25 and a mixture of 40/41. The incidence of Ad31(4.9%) or Ad3(1.6%) was relatively insignificant. DNA restriction analysis(77.5%) proved to be better than serum neutralization but not so when compared to a PCR-based assay for identification of the enteric Ad serotypes(90%) in stool specimens. In this work, the PCR-based assay was evaluated as a tool for the rapid, yet highly sensitive identification of adenoviral DNA sequences in fresh clinical stool specimens.

• Korean journal of applied entomology
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• v.30 no.2
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• pp.144-149
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• 1991

Biochemical characteristics of Spodoptera uigua nuclear polyhedrosis virus (SeNPV) isolated in Jinju were studied. SeNPV contained a number of nucleocapsids within a viral envelope embeded in polyhedra. The polyhedral protein of SeNPV was composed of a single polypeptide with a M. W. of 30kd. Double-immunodiffusion test showed that the polyhedral protein of SeNPV had common antigenic determinants with SINPV and BmNPV. Virion proteins of SeNPV were resolved into 49 polypeptides by silver staining after SDS-PAGE. The approximate genome size of SeNPV by restriction endonuclease analysis was 1l0kb.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.118-125
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• 1999

Porcine Proliferative Enteropathy(PPE) is an infectious enteric disease and a major cause of economic loss in swine industry due to weight loss, poor growth and sudden death in growing and finishing pigs at 6 to 20 weeks of age. PPE has been diagnosed by clinical signs, syndrom and lesions in the intestine in Korea. However, the diagnostic method had several problems in the detection of infected or carrier pigs. Therefore, in this study, we established the polymerase chain reaction(PCR) which was a fast, specific and sensitive method for identification of Lawsonia intracellularis (L intracellularis). We designed and synthesized primer on the 16S rDNA and p78 gene encoding L intracellularis. Specificity of the method was confirmed by comparison of the PCR results using other enteric bacteria and the study has shown that PCR method was sensitive to detect 1ng of genomic DNA as a template. Identity of the PCR products was confirmed by comparison of pattern of restriction endonuclease analysis with restriction enzyme Hae III and Pst I. Also, the PCR method was applicable to the naturally affected pigs with PPE. Based on the results from this study, the PCR method could be used as a fast and specific diagnostic tool for PPE.