• Proceedings of the Korean Society for Bioinformatics Conference
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• 2004.11a
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• pp.21-28
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• 2004

모든 생명체의 genetic information에는 보존적 염기서열과 다형적 염기서열이 존재한다. 다형적 염기서열과 보존적 염기서열은 하나의 종(species)을 감별하거나, 여러 종류의 종을 동시에 감별할 수 있는 genotyping의 표지자로 각각 이용될 수 있다. 본 논문은 병원성 감염질환 세균, 식중독 유발 세균, 생물의약품 오염 유발 세균 및 환경오염 세균 등 세균의 존재 유무와 속과 종 감별을 위해 대부분 세균 종의 보존적 염기서열과 다형적인 염기서열을 포함하고 있는 23S rDNA 유전자의 표적 염기 서열로부터 고안된 세균 특이적(bacterial-specific), 속 특이적(genus-specific), 종 특이적(species-specific) 올리고 뉴클레오티드프로브와 프라이머를 디자인하는 시스템을 소개한다. 시스템을 통해서 얻어진 프로브와 프라이머들은 PCR을 통한 검증단계를 거쳐서 디자인 결과의 정확성을 확인하였다. 본 시스템의 이용으로 프로브와 프라이머를 디자인하는데 몇 주가 소요되는 시간을 몇 일 내로 줄일 수 있었으며, 체계적인 데이터의 관리로 결과의 정확성을 높일 수 있었다.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.346-353
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• 2005

A 50-kb chromosome DNA region was isolated from Streptomyces kanamyceticus by screening the fosmid genomic library, using the 16S rRNA methylase gene (kmr) as a probe. Sequence analysis of this region revealed 42 putative open reading frames (ORFs), which included biosynthetic genes such as genes responsible for 2-deoxystreptamine (2­DOS) biosynthesis as well as genes for resistance and regulatory function. Also, the kanamycin acetyltransferase gene (kac) was characterized by in vitro enzyme assay, which conferred E. coli BL21 (DE3) with 10, 50, and 80-times higher resistance to kanamycin A, tobramycin, and amikacin, respectively, than the control strain had, thus strongly indicating that the isolated gene cluster is very likely involved in kanamycin biosynthesis. This work provides a solid basis for further elucidation of the kanamycin biosynthesis pathway as well as the productivity improvement and construction of new hybrid antibiotics.

• Fisheries and Aquatic Sciences
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• v.11 no.4
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• pp.205-208
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• 2008

The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.

• Journal of Microbiology
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• v.42 no.2
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• pp.139-142
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• 2004

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.335-341
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• 2007

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.

• The Korean Journal of Mycology
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• v.27 no.3 s.90
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• pp.181-186
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• 1999

Osmotolerant yeast isolated from soy paste could grow on media with 2 M NaCl. This strain was identified as Zygosaccharomyces rouxii by biological characteristics, RFLP of ribosomal DNA and mating with compatible haploid strain. Growing rate of the Z. rouxii YDJ was slower than Saccharomyces cerevisiae. Z. rouxii YDJ accumulated trehalose, which is known as one of the osmolytic protectants, in cells cultured on media with salt. Enzyme activity of trehalose phosphate synthase related to trehalose biosynthesis of the YDJ was lower than those of S. cerevisiae. Trehalase activity related trehalose degradation was also lower in Z. rouxii YDJ than S. cerevisiae. However, as Z. rouxii accumulated trehalose by salt treatment, salt tolerancy of Z. rouxii was assumed to be related to trehalose in additon to glycerol.

• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1133-1140
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• 2018

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene ($phaC_{Pf}$) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (${phaC_{Pf}}^+$, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (${phaC_{Pf}}^+$) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified $PhaC_{Pf}$, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.994-1001
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• 2001

An easier way of understanding the BNR system was proposed from the study on substrate, nutrient removal tendency, microbial community and its metabolic function by applying the municipal settled sewage. During the anaerobic period, the phosphorus release rate per VFACOD we varied depending on the phosphorus content in the sludge. When the phosphorus content in the sludge was $6\%$ VSS, according to influent VFACOD, the phosphorus release rate and PHA production were $0.35 gPO_4P/gVFACOD$ and 1.0 gPHA/gVFACOD, respectively. The $NO_3N$ requirement for the phosphorus uptake as an electron acceptor was about $0.5 gNO_3N/gPO_4P_{uptake}$ based on the proposed equation with PHA, biomass, production, and the concentration of phosphorus release/uptake. Bacterial-community analysis of the sludge, as determined by FISH and 16SrDNA characterization FISH, revealed that the beta-subclass proteobacteria were the most abundant group ($27.9\%$ of the proteobacteria-specific probe EUB338), and it was likely that representative of the beta-subclass played key roles in activated sludge. The next dominant group found was the gamma-protebacteria ($15.4\%$ of probe EUB338). 16S rDNA clone library analysis showed that the members of${\beta}$- and ${\gamma}$-proteobacteria were also the most abundant groups, and $21.5\%$ (PN2 and PN4) and $15.4\%$ (PN1 and PN5) of total clones were the genera of denitrifying bacteria and PAO, respectively. Prediction of the microbial community composition was made with phosphorus content (Pv, $\%$ P/VSS) in wasted sludge and profiles of COD, PHA, $PO_4P,\;and\;NO_3N$ in an anaerobic-anoxic SBR unit. Generally, the predicted microbial composition based upon metabolic function, i.e., as measured by stoichiometry, is fairly similar to that measure by the unculturable dependent method. In this study, a proposal was made on he microbial community composition that was more easily approached to analyze the reactor behavior.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.393-399
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• 2004

Useful genes can be Screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs r were extracted by the SDS-based freezing-thawing method, and then further purified using an $UltraClean^{TM}$ kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoR I, BamH I, and Sac II in Escherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13-15 kb in size. The bphC genes responsible for degradation of aromatic hydrocarbons via mets-cleavage were identified from the metagenomic libraries by colony hybridization using the bphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2, 3-DHBP) dioxygenase gene (bphC ), capable of degradation of 2,3-DHBP, was cloned and its nucleotide Sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and Showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results in-dicated that the bphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.

• The Journal of Korean Society of Virology
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• v.28 no.2
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• pp.175-182
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• 1998

We investigated the rate of hepatitis G virus infection among 50 patients who were not infected with the hepatitis C virus but showed symptoms of hepatitis. Viral RNA was extracted from the patients' sera and cDNA was synthesized and amplified by RT-PCR (reverse transcription-polymerase chain reaction) using random hexamer and 5 primers (470-20-1-77F, 470-20-1-211R, 470-20-1-211R-biotin, GV57-4512MF, GV57-4657MR). The amplified PCR products were confirmed by electrochemiluminescence (ECL), liquid hybridization (LH) and Southern blotting (SB). Among the 50 PCR products, by means of ECL, we found 4 samples to be positive and 5 samples to be indeterminate. The GV45-89M probe (5'-CYCGCTGRTITGGGGTGTACfGGAAGGC-3') was end-labelled with gamma-$^{32}P$ ATP and used for liquid hybridization with the PCR products. By using liquid hybridization, we detected specific bands from 4 positive sera and also from one indeterminate serum as determined by ECL. An 1.5% agarose gel electrophoresis of the 9 PCR products which were HGV positive or indeterminate as determined by ECL showed a 160bp band from 4 positive and one indeterminate serum. The 5 PCR products proved to be positive when SB was applied with the GV45-89M probe as well as when LH was applied. LH and SB were shown to have higher sensitivity and specificity than ECL. Two cases among 5 positive cases had relatively high SGOT, SGPT, ALP values when compared with other 48 cases. In summary, we confirmed hepatitis G virus infection in 5 cases among 50 Korean patients showing symptoms of viral hepatitis.