• Korean Journal of Microbiology
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• v.35 no.3
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• pp.205-210
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• 1999

16brio cholerne is an important pathogenic organism that causes dimhea in human beings. V ciaoleroe KNIH002 was isolated from patients suffering with dian.heal disease in Korea. From Southern hybridization using the amplified PCR product of 307 bp as a probe. which was obtained from PCR reaction using primer detecting cholera toxin gene, we have found that the c b gene located in 4.5-kb fragmenl double digested with Pstl and BgllI of the chromosome. Therefore, we made mini-libraries of the isolate using PstI and Bgm restriction endonuclease and pBluescript SKU(+) vector. As a result. we cloned 4.5-kb PstI-BglII fragment containing the c a gene encoding a cholera toxin from the constructed mini-libraries of V olzolerae KNlH002 by colony hybridization using the same probes. This recombinant plasmid was named pCTX75. E. coii XL1- Blue harboring pCTX75 showed the cytotoxicity on Chinese Hamster Ovary cells. From the sequencing of he cloned recombinant plasmid, we confinned that it has virulence gene cassette consisting of ace, zot, ctx.4 and cf"~B gene. The ace and zot genes were composed of 291 hp and 1.200 bp with ATG initiation codon and TGA lennination codon, respectively. Nucleotide sequence of the ace gene exhibited 100% identity with that of V cholera E7946 El Tor Ogawa strains. But, nucleolide and amino acid sequence comparison of the zot gene exhibited 99% and 98.8% identity with that of V cholerae 395 Classical Ogawa stram, respectively. Specially. the Ala-100, Ala-272 and Ala-281 sites of Zoi polypeptide presented in V choleme 395 Classical Ogawa strain are replaced by Val in V cholerae KNIH002.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.5
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• pp.432-443
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• 2005

Microarray technology has contributed Significantly to the understanding of bacterial genetics and transcriptional regulation. One neglected aspect of this technology has been optimization of microarray-generated signals and quality of generated information. Full genome microarrays were developed for Clostridium acetobutylicum through spotting of PCR products that were designed with minimal homology with all other genes within the genome. Using statistical analyses it is demonstrated that Signal quality is significantly improved by increasing the hybridization volume. possibly increasing the effective number of transcripts available to bind to a given spot, while changes in labeled probe amounts were found to be less sensitive to improving signal quality. In addition to Q-RT-PCR, array validation was tested by examining the transcriptional program of a mutant (M5) strain lacking the pSOL1 178-gene megaplasmid relative to the wildtype (WT) strain. Under optimal conditions, it is demonstrated that the fraction of false positive genes is 1% when considering differentially expressed genes and 7% when considering all genes with signal above background. To enhance genomic-scale understanding of organismal physiology, using data from these microarrays we estimated that $40{\sim}55%$ of the C. acetobutylicum genome is expressed at any time during batch culture, similar to estimates made for Bacillus subtilis.

• Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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• 2003.09a
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• pp.152-157
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• 2003

Diesel-contaminated soils were ozonated for different times (0 - 900 min) and incubated for 9 wk to monitor petroleum hydrocarbons (PH)-degradative potential of indigenous microorganisms in the soils. Increased ozonation time decreased not only concentration of PH but also number of microorganisms in the soils. Microorganisms in the ozonated soils increased during 9-wk incubation as monitored by culture- and nonculture-based methods. Higher (1-2 orders of magnitude) cell number was observed by quantitative analysis of soil DNA using probes detecting genes encoding 165 rRNA(rrn), naphthalene dioxygenase (nahA), toluene dioxygenase (todC), and alkane hydroxylase (alkB) than microbial abundance estimated by culture-based methods. Such PH-degraders were relatively a few or under detection limit in 900-min ozonated soil. Further PH-removal observed during the incubation period supported the presence of PH-degraders in ozonated soils. Highest reduction (25.4%) of total PH (TPH) was observed in 180-min ozonated soil white negligible reduction was shown in 900-min ozonated soil during the period, resulting in lowest TPH-concentration in 180-min ozonated soil among the ozonated soils. Microbial community composition in 9-wk incubated soils revealed slight difference between 900-min ozonated and unozonated soils as analyzed by whole cell hybridization using group-specific rRNA-targeted oligonucleotides. Results of this study suggest that appropriate ozonation and subsequent biodegradation by indigenous microorganisms may be a cost-effective and successful remediation strategy for PH-contaminated soils.

• 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.

• Tuberculosis and Respiratory Diseases
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• v.58 no.3
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• pp.248-256
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• 2005

Background : Nontuberculous mycobacterial (NTM) infections are increasingly being recognized as a cause of chronic pulmonary disease. This study describes the prevalence of NTM species from clinical specimens and the clinical characteristics of NTM pulmonary disease. Material and Methods : The NTM isolated from March 2003 to December 2003 at the Kosin Medical Center were identified using an oligonucleotide chip containing the internal transcribed space (ITS) sequence. The medical records of the patients with the NTM isolates, who fulfilled the 1997 ATS diagnostic criteria for NTM pulmonary disease, were analyzed, retrospectively. Results : Twenty four species (24.2%) of NTM were isolated from 99 cultured AFB specimens. M. avium complex (MAC) (13 isolates), M. szulgai (3), M. kansasii (2), M. malmoense (2), M. abscessus (1), M. chelonae (1), M. scrofulaceum (1), and unclassified (1). Of the 23 patients with isolated NTM, 11 patients were found to be compatible with a NTM pulmonary infection according to the ATS criteria; MAC was found in 6 cases (54.5%), M. szulgai in 2 cases (18.2%), and M. abscessus, M. szulgai, M. kansasii and M. malmoense in 1 case each (9.1%). Ten patients (91%) were male and the median age at diagnosis was 61 years. In the pre-existing diseases, malignant disease was found in 6 cases including 5 patients with lung cancer, and history of old pulmonary tuberculosis was identified in 4 cases. The radiological patterns showed lung destruction lung in 3 cases, a cavitary mass in 3 cases, a nodular pattern in 2 cases, and reticulonodular, consolidation and a bronchiectasis pattern were in 1 case each. Conclusion : Various types of NTM pulmonary diseases were found in a tertiary hospital at Busan, Korea. The NTM pulmonary diseases were caused by MAC, M. szugai, M. kansasii, M. malmoense, M. abscessus, M. chelonae, and M. scrofulaceum in the order of frequency.

• Journal of Sericultural and Entomological Science
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• v.34 no.2
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• pp.20-25
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• 1992

The location of the polyhedrin gene of Bmbyx mori nuclear polyhedrosis virus(BmNPV) was determined by using a cloned polyhedrin gene from the Autographa californica nuclear polyhedrosis virus(AcNPV) as a hybridization probe. The 7.4 Kb PstⅠ fragment DNA of Bm-NPV was cloned to plasmid pUC19 vector. A fragment containing this gene was mapped and sequenced in its entire polyhedrin reading frame. Nucleotide sequences comparison of the polyhedrin of the BmNPV to that of previously reported by Ⅰatrou(1985) revealed that the sequence varied in 10 base, Comparison of the amino acid sequence of the two structured gene revealed that coding sequence varied 74 valine to isoleucine, 76 aspargine to serine and 155 methionine to valine.

• Biomedical Science Letters
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• v.2 no.2
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• pp.223-229
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• 1996

HLA-B27 gene, one of the HLA-class I molecule, is strongly associated with ankylosing spondylitis. It has been most frequently used as a disease-correlated HLA gene by clinicians. In most laboratories, conventional HLA-B27 typing is still performed by cell cytotoxicity tests or fluorescence serology with specific antibodies. In this study, DNA typing method for HLA-B27 was developed by using group specific Polymerase Chain Reaction (PCR). Four HLA-B27 cell lines (HOM-2, JESTHOM, WT24 and BTB) and fifty six B27 Korean individuals defined by serology were used. The results of control cell and B-27 positive individual samples were correlated well with the data which was performed by serological method. All of B27 positive PCR products gave positive signals on Southern blot hybridization with B27 specific probe. This study shows that the HLA-B27 DNA typing is a relatively simple, fast and practical tool for the determination of the HLA-B27 gene in routine clinical laboratory work.

• Applied Biological Chemistry
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• v.37 no.3
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• pp.148-153
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• 1994

Rice black-streaked dwarf virus (RBSDV), a member of the plant reoviridae fijivirus group, causes a serious damage for rice production in Korea. To characterize the RBSDV genome, virus particles were produced by feeding of planthopper (Laodelphax striatellus F.) carring RBSDV to maize plants for 2 days. In $30{\sim}40$ days after feeding, the viral particles were purified from the infected maize roots by using $10{\sim}40%$ sucrose gradient centrifugation. After treatment of 10% SDS to remove the viral coat proteins, ten viral double-stranded RNAs were resolved in agrose gel electrophoresis. Total dsRNA was then used to synthesize cDNA by reverse transcriptase and a cDNA library was constructed in the ${\lambda}gt11$ vector. The phages that contain RBSDV cDNA fragments were selected by hybridizing with the random-primed probe prepared from RBSDV dsRNAs. After subcloning of several cDNA fragments into the pUC19 plasmid vector, one clone (pRV3) was chosen for sequencing. The pRV3 clone was shown to be located on the RBSDV genome fragment No.3 by RNA gel-blot analysis. Sequence analysis of the clone revealed that the pRV3 contains two partial open reading frames.

• 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.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.1-6
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• 2004

Tetracycline resistant bacterial strains were isolated from 10 batches of Kimchi among 50 batches collected in Taegu restrict. The MIC of tetracycline ranged between 25 and> 100 ㎖/l. Total genomic DNA preparation from all 10 tetracycline resistant lactic acid bacterial isolates were subjected to PCR amplification with class-specific primers for tet(M) and tet(O). In only one isolate, HJ9, tet(M) was detected. By Southern blotting and hybridization with a tet(M)-specific probe, the tet(M) gene of HJ9 isolate could be localized on a plasmid. The partial nucleotide sequence and deduced amino acid sequence of tet(M) of HJ9 showed 90-99% and 94-100% homology to those of Gram positive bacteria, respectively. With sequencing of 16S rRNA, HJ9 isolate from Kimchi was identified as Lactobacillus sakei. From these results, Kimchi can be considered potential vehicle for the spread of antibiotic-resistant lactic acid bacteria along the food chain to the consumer.