• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.235-239
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• 2006

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast to in vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately $650{\mu}g/mL$ of protein was produced after 2h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.

• Korean Journal of Plant Tissue Culture
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• v.21 no.6
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• pp.319-326
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• 1994

For the gene tagging of Armoracia rusticana, maize controlling element Ac and Ds were introduced into A.rusticana via Agrobacterium-mediated transformation method. We established an efficient in via regeneration and transformation system for gene transfer in A. rusticana. The optimum in via regeneration condition has been obtained from leaf, petiole and root organs on modified MS medium supplemented with NAA 0.1 mg/L plus BA 1.0 mg/L for direct shooting and with free growth regulators for root induction for transformation, the leaf, petiole and root explants of A. rusticana were concultivated with Agrobacterium tumefaciens, LBA4404 which carries a binary vector pEND4K containing maize controlling element Ac or Ds, respectively: Selections were performed in the shoot induction medium supplemented with 100 mg/L kanamycin, and 500 mg/L carbenicillin transformation frequency showed about 8 to 10% in case of leaf disks. PCR md Southern blot analyses showed that the Ac and the Ds elements were integrated into the chromosome of donor plants.

• Journal of Genetic Medicine
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• v.1 no.1
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• pp.39-43
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• 1997

The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is inherited maternally, in which the MTTL1*MELAS 3243 mutation has been most commonly found as a heteroplasmy of A to G point mutation in the $tRNA^{Leu(UUR)}$ gene. The MTTL1*MELAS 3271 mutation is known to be the second common mutation, though clinical features of both mutations are not remarkably different. Recently, a variety of minor mutations have been reported in patients with MELAS. In this study, major efforts have been made to investigate the allele frequency of major three mutations including MTTL1*MELAS 3243, 3252, 3271 in 10 Korean families with MELAS probands. The PCR and subsequent direct sequencing of the PCR product in the regions spanning these three mutation sites were employed to identify the mutation in each proband. All family members have been screened for the presence of these three mutations by PCR-RFLP assay using Apa I, Acc I and Bfr I restriction enzymes. The MTTL1*MELAS 3243 mutation was most commonly found (7 out of 10 families tested) followed by the MTTL1*MELAS 3271 which was identified in 1 out of 10 families. In the remaining 2 families none of three mutations were found, indicating the presence of either nuclear mutation or yet unidentified mitochondrial DNA mutation in these families.

• The Plant Pathology Journal
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• v.15 no.3
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• The Plant Pathology Journal
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• v.34 no.4
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• pp.286-296
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• 2018

Maintenance of a beneficial microbial community, especially in the rhizosphere, is indispensable for plant growth and agricultural sustainability. In this sense, plant growth-promoting rhizobacteria (PGPR) have been extensively studied for their role in plant growth promotion and disease resistance. However, the impact of introducing PGPR strains into rhizosphere microbial communities is still underexplored. We previously found that the Proteus vulgaris JBLS202 strain (JBLS202) promoted growth of Kimchi cabbage and altered the relative abundance of total bacteria and Pseudomonas spp. in the treated rhizosphere. To extend these findings, we used pyrosequencing to analyze the changes in bacterial communities in the rhizosphere of Kimchi cabbage after introduction of JBLS202. The alterations were also evaluated by taxon-specific realtime PCR (qPCR). The pyrosequencing data revealed an increase in total bacteria abundance, including specific groups such as Proteobacteria, Acidobacteria, and Actinobacteria, in the treated rhizosphere. Time-course qPCR analysis confirmed the increase in the abundance of Acidobacteria, Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. Furthermore, genes involved in nitrogen cycling were upregulated by JBLS202 treatment indicating changes in ecological function of the rhizosphere soil. Overall, these results indicate that introduction of JBLS202 alters both the composition and function of the rhizosphere bacterial community, which can have direct and indirect effects on plant growth. Therefore, we propose that long-term changes in bacterial composition and community-level function need to be considered for practical use of PGPRs.

• Korean Journal of Veterinary Research
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• v.56 no.3
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• pp.147-153
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• 2016

A total of 782 blood and 465 tissue samples from 1,039 wild animals and 127 dairy goats were collected from January 2011 to December 2013 in 10 provinces of South Korea and tested for the presence of brucellosis. The Rose Bengal test revealed that 8.0% (52/650) of the serum samples were seropositive, while 4.2% (33/782) of the serum samples were positive for Brucella antibodies by competitive enzyme-linked immunosorbent assay. Of the 650 sera examined, only 16 (2.5%) were positive by both serological tests. Direct polymerase chain reaction (PCR) assay using B4/B5 primers for Brucella abortus (BCSP31) revealed the prevalence of Brucella to be 26.5% (129/487) in blood samples and 21% (98/465) in tissue samples while, 16S rRNA PCR detected Brucella DNA in 6.8% (33/487) and 2.6% (12/465) in blood and tissue samples, respectively. Of PCR-positive samples, only 6.2% (30/487) of blood samples and 2.4% (11/465) of tissue samples were found to be positive by both BCSP31 and 16S rRNA PCRs. However, Brucella strains were isolated by blood culture from only two out of 487 blood samples (0.4%). This characterization and identification of pathogenic Brucella isolates is the first to clearly indicate that the organisms were Brucella abortus biovar 1.

• Restorative Dentistry and Endodontics
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• v.28 no.2
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• pp.178-183
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• 2003

The purpose of this study was to investigate the frequency of 7 putative pathogens in endodontic infections. The specimens were collected from infected pulpal tissue of patients who were referred for root canal treatment to the department of conservative dentistry, Chosun University Samples were collected aseptically using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ml of 1 X PBS. DNAs were extracted from the samples by direct DNA extraction method using lysis buffer (0.5% EDTA, 1% Triton X-100). Identification of 7 putative pathogens was performed by PCR based on 16S rDNA. The target species were as follows : Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and Treponema denticola. Our data revealed that the prevalence of P. endodontalis was found in 88.6% (39/54), P. ginivalis 52.3% (23/44), P. nigrescens 18.2% (8/44), P intermedia 15.9% (7/44) B. forsythus 18.2% (8/44), A. actinomycetemcomitans 3.3% (1/44), T. denticola 25% (l1/44) of the samples. The high prevalence of P. endodontalis and P. ginivalis suggests that they may play an important role in the etiology of endodontic infections.

• Tuberculosis and Respiratory Diseases
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• v.50 no.2
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• pp.222-228
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• 2001

Backgrounds : Recent technological developments have introduced a new method to identifying M. tuberculosis complex DNA in clinical samples directly. The direct amplification test (DAT) is approved for identifying M. tuberculosis complex in respiratory specimens that are smear-positive for acid-fast bacilli (AFB). When there is a discrepancy between the AFB smear and DAT, no information on their clinical utility is currently available. In this study, the diagnostic reliability of DAT was investigated in suspected pulmonary tuberculosis patients whose sputum AFB smear was negative. Methods : From June 1, 1998 through May 30, 1999, 909 patients with presumed active pulmonary tuberculosis were enrolled. A sputum AFB stain, culture, DAT and/or biopsy were performed. Using the criteria of clinical tuberculosis or confirmed tuberculosis, the positive predictive value of DAT in diagnosing pulmonary tuberculosis was investigated. Results : The positive predictive value of DAT was 82.1% by the clinically active tuberculosis criteria. However, it decreased to 61.5% when diagnosis was restricted to only to culture positive or biopsy proven cases. The false positive rate of DAT was 18.0%. Conclusion : The DAT is a valuable diagnostic method in suspected patients whose sputum AFB is was negative.

• Pediatric Infection and Vaccine
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• v.22 no.2
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• pp.106-112
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• 2015

Purpose: The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs. Methods: The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs. Results: The detection limit of the PCR assay was approximately $10^4$ colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over $10^6$ CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01). Conclusions: As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA -specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.271-281
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• 1999

The nested polymerase chain reaction (PCR) assay was used to determine the sequences of reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219 of human immunodeficiency virus-1 (HIV-1) pol gene. Template DNA was obtained from uncultured peripheral blood mononuclear cells from 27 Korean HIV-1 infected patients treated with ZDV and Korean red ginseng. The second PCRs were done for 2 separated regions (RT codons $13{\sim}98$ and $152{\sim}259$) with $5\;{\mu}l$ of the first PCR productNucleotide sequences were determined by direct sequencing. In the 27 patients, CD4+ cell count decreased from $230{\pm}117/{\mu}l$ to $152{\pm}162/{\mu}l$ for $46{\pm}26$ months (Mo), and actual duration of ZDV intake was $72{\pm}16$ Mo. In the 16 patients who had been treated with ZDV therapy ${\ge}25$ Mo, the incidences of 70R, 215F/Y, and 41L were 61%, 28% and 22%, respectively and those of 67N, 210W and 219Q were 17%. The incidences of 215F/Y were 6.7% for group ${\le}12$ Mo treatment, 22.7% for group with 13 to 24 Mo, and 27.8% for group ${\ge}25$ Mo. There was no mutation in 9 patients. It might be associated with the interruption of ZDV therapy for more than 6 months in 6 patients. This study shows that the detection of mutation could be useful prognostic marker with other clinical and virological data, and very low mutation rate is dectected compared to overseas reports.