• Korean Journal of Microbiology
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• v.42 no.1
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• pp.19-25
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• 2006

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.

• Korean Journal of Environmental Biology
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• v.29 no.1
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• pp.68-73
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• 2011

The single cell gel electrophoresis (SCGE) assay is a microelectrophoretic technique for assessments of DNA damage at the level of the individual eukaryotic cell. The SCGE assay, due to its simplicity, sensitivity and need of a few cells, has advantages compared to other genomic damage assays such as sister chromatid exchange, chromosomal aberration and micronucleus test. In this study, investigated were the levels of DNA damage and the repair kinetics in the coelomocytes of Eisenia fetida treated with HgCl2 and ionizing radiation by means of the SCGE assay. For detecting DNA damage and repair in coelomocytes, earthworms (E. fetida) were irradiated with six doses of ${\gamma}$-rays (0, 2.5, 5, 10, 20 and 50 Gy) and in vivo exposed to mercuric chloride at 0, 80 and 160 mg $kg^{-1}$ for 48 hours. Then the Olive tail moments were measured during 0~12 hours after irradiation and 0~72 hours after Hg treatment. The results showed that the more the oxidative stress was induced by mercury and radiation, the longer the repair time was required. Also, the results suggest that the SCGE assay may be used as an important tool for comparison of the sensitivity of different species to oxidative stresses.

• Korean Journal of Microbiology
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• v.41 no.2
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• pp.105-111
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• 2005

Streptomyces produces many kinds of secondary-metabolites including antibiotics. Screening of a new compound and elucidation of a biosynthetic pathway for the secondary metabolites are very important fields of biology, however, there is a main problem that most of the identified compounds are already researched compounds. To solve these problems, a microarray system that is based on the data related to the biosynthetic genes for secondary-metabolites was designed. For the main contents of DNA microarray, the important genes for the bio-synthesis of aminoglycosides, polyenes group, enediyne group, alpha-glucosidase inhibitors, glycopeptide group, and orthosomycin group were chosen. A DNA microarray with 69 genes that were involved in the bio-synthesis for the antibiotics mentioned above was prepared. The usability of the DNA microarray was confirmed with the chromosomal DNA and total RNA extracted from S. coelicolor whose genomic sequence had already been reported.

• Journal of Microbiology
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• v.37 no.2
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• pp.102-110
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• 1999

Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.282-288
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• 2006

Activity-guided fractionations led to the isolation of antitumor compound, ergosterol peroxide ($5{\alpha},\;8{\alpha}-epideoxy-24(R)-methylcholesta-6,\;22-dien-3{\beta}-ol$) from the fruiting body of Pleuratus eryngii that was cultivated artificially. This sterol structure was established by using spectroscopic methods ($^1H\;and\;^{13}C$ nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formular of $C_{28}H_{44}O_3$ displaying characteristic features of epidioxy sterols. The 50% inhibitory concentrations ($IC_{50}$) of ergosterol peroxide against human lung cancer cell line (A549) and human ovarian cell line (SK-OV3) were $7{\mu}M\;and\;14{\mu}M$, respectively. In the DNA fragmentation assay, the compound showed the programmed cell death causing the chromosomal DNA fragmentation. It reveals that ergosterol peroxide arrests G1 phase of the cell division cycle.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.7-12
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• 2010

The full-length cDNA of the porcine peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase (PECI) gene encodes a monofunctional peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase. Cloning and sequencing of the porcine PECI cDNA revealed the presence of an 1185-base pair open reading frame predicted to encode a 394-amino acid protein by the 5'rapid amplification of cDNA ends (5'RACE) and EST sequences. The porcine PECI gene was expressed in seven tissues (heart, liver, spleen, lung, kidney, skeletal muscle, fat) which was revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). The porcine PECI was mapped to SSC71/2 p11-13 using the somatic cell hybrid panel (SCHP) and the radiation hybrid panel (RH) (LOD score 12.84). The data showed that PECI was closely linked to marker S0383. A C/T single nucleotide polymorphism in PECI exon 10 (3'UTR) was detected as a PvuII PCR-RFLP. Association analysis in our experimental pig population showed that different genotypes of PECI gene were significantly associated with the Average Backfat thickness (ABF) (p<0.05) and Buttock backfat thickness (p<0.01).

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• KSBB Journal
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• v.25 no.2
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• pp.167-172
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• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.217-225
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• 1997

PCR amplication using the primers for gag, pol and env genes in BLV (bovine leukemia virus) proviral DNA and syncytium assay were carried out for the Korean native goats experimentally infected with bovine leukemia virus to investigate pathogenesis of BLV in the goats, and to establish a model animal for BLV infection. The oligonucleotide primers used in PCR revealed very high specificity. The minimal amount of FLK-BLV cellular chromosomal DNA to detect the integrated BLV proviral DNA was 10 ng. The peripheral blood lymphocytes from the goat infected with BLV were examined at regular intervals by PCR amplification and syncytium assay. Pol or gag genes were detected in none of three infected goats at the 1st week post-infection (p.i.). At the 4th week p.i., one of three goats showed the amplified gag gene. Thereafter detection rates for the genes were increased, indicating that the BLV proviral genes were integrated in all of the lymphocytes from three goats, at the 16th weeks p.i., when it was evident in syncytium assay that the lymphocytes from all of three goats were infested with infective BLV. Investigating the tissues from the necropsied goats at the 8th month p.i., the amplified BLV proviral genes and infective BLV were detected in all of the peripheral lymphocytes from three infected-goats. Among various tissues examined, the amplified BLV proviral genes were observed in spleen and superficial cervical, mandibular and retropharyngeal lymph nodes, and the infective BLV, in superficial cervical and mandibular lymph nodes. It was assumed that the Korean native goat was quite susceptible to BLV infection, indicating that the goat could be a good model animal for BLV.

• Korean Journal of Medicinal Crop Science
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• v.11 no.4
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• pp.311-315
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• 2003

This study was carried out to compare chromosomal characteristics between Atractylodes japonica and A macrocephala. Cytogenetic analysis was conducted based on karyotype analysis and physical mapping using fluorescence in situ hybridization. As a result of karyotype analysis by feulgen staining, somatic chromosome numbers of A. japonica and A. macrocephala were 2n=24. The length. of the mitotic metaphase chromosomes of A. japonica ranged from $0.70\;to\;1.60{\mu}m$ with a total length. of $12.11{\mu}m$ and the homologous chromosome complement comprised six metacentrics, five submetacentrics and one subtelocentrics. On the other hand, the length of the mitotic metaphase chromosomes of A. macrocephala ranged from $0.90\;to\;2.35{\mu}m$ with a total length of $16.58{\mu}m$ and the homologous chromosome complement comprised seven metacentrics and five submetacentrics. The total length of A. japonica chromosomes was shorter than that of A. macrocephala, but A. japonica had one subtelocentrics (chromosomes 4) different from A. macrocepha1a. chromosomes. The F1SH technique using 17S and 5S rDNA was applied to metaphase chromosomes. The signals for 17S rDNA were detected on the telomeric regions of chromosomes 4 and 5 in both A japonica and A. macrocephala. The 5S rDNA signal was found in the short arm of chromosome 1.