• Title/Summary/Keyword: DnaK

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Evaluation of Amplified-based Target Preparation Strategies for Toxicogenomics Study : cDNA versus cRNA

  • Nam, Suk-Woo;Lee, Jung-Young
    • Molecular & Cellular Toxicology
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    • v.1 no.2
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    • pp.92-98
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    • 2005
  • DNA microarray analysis of gene expression in toxicogenomics typically requires relatively large amounts of total RNA. This limits the use of DNA microarray when the sample available is small. To confront this limitation, different methods of linear RNA amplification that generate antisense RNA (aRNA) have been optimized for microarray use. The target preparation strategy using amplified RNA in DNA microarray protocol can be divided into direct-incorporation labeling which resulted in cDNA targets (Cy-dye labeled cDNA from aRNA) and indirect-labeling which resulted in cRNA targets (i.e. Cy-dye labeled aRNA), respectively. However, despite the common use of amplified targets (cDNA or cRNA) from aRNAs, no systemic assessment for the use of amplified targets and bias in terms of hybridization performance has been reported. In this investigation, we have compared the hybridization performance of cRNA targets with cDNA targets from aRNA on a 10 K cDNA microarrays. Under optimized hybridization conditions, we found that 43% of outliers from cDNA technique and 86% from the outlier genes were reproducibly detected by both targets hybridization onto cDNA microarray. This suggests that the cRNA labeling method may have a reduced capacity for detecting the differential gene expression when compared to the cDNA target preparation. However, further validation of this discordant result should be pursued to determine which techniques possesses better accuracy in identifying truly differential genes.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

  • Park, Sojin;Choi, Seoyun;Ahn, Byungchan
    • Molecules and Cells
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    • v.39 no.3
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    • pp.204-210
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    • 2016
  • DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

Chiral Separation with DNA-Polyion Complex Membranes

  • Yoshikawa, Masakazu;Maruhashi, Motokazu;Ogata, Naoya
    • Proceedings of the Polymer Society of Korea Conference
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    • 2006.10a
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    • pp.353-353
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    • 2006
  • Deoxyribonucelic acid (DNA) molecules have a huge molecular weight so that DNA was reported to be a promising natural polymer to give durable films. Among many applications of DNA, the authors focused their attention on separation membranes derived from DNA because membranes will play an important role in environmental and energy related processes. DNA-polyion complex membranes were prepared from DNA and corresponding polycations. The DNA-polyion complex membranes showed chiral separation ability toward racemic amino acid mixtures.

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Molecular Cloning and Chaperone Activity of DnaK from Cold-adapted Bacteria, KOPRI22215

  • Sung, Min-Sun;Im, Ha-Na;Lee, Kyung-Hee
    • Bulletin of the Korean Chemical Society
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    • v.32 no.6
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    • pp.1925-1930
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    • 2011
  • Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 $^{\circ}C$: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.

CEO's Innovation DNA and Innovation : Fit of Environment (경영자 혁신DNA와 혁신 : 환경 적합성)

  • Kim, Seung Ho;Huh, Moo Yul
    • Asia-Pacific Journal of Business Venturing and Entrepreneurship
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    • v.10 no.1
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    • pp.95-110
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    • 2015
  • Most innovation related theories including entrepreneurship theory regard the CEO's innovative competencies as the starting point of innovation. The study was investigated the relationship between CEO's innovation DNA and Innovation and the effects of environmental fit in their relation. For the empirical test, the sample was collected from 110 manufacturing companies in Daegu and Gyeongbook region. The results as follows: First, Innovation DNA has generally significant positive effect on innovation. The effect of discovery DNA is stronger than operating DNA to the product innovation, but the operating DNA stronger than the discovery DNA to the process innovation. The fit between CEO's innovative DNA and exogenous environmental turbulence make a strength innovation. The supplementary fit between discovery DNA and technology turbulence and complementary fit between discovery DNA and market turbulence reinforce product innovation. Process innovation were strengthen by the complementary fit between operating DNA and market turbulence.

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DNA Repair Synthesis Induced by Bleomycin in HeLa $S_3$ Cells Pretreated with Base Analogs (鹽基相似體를 前處理한 HeLa $S_3$ 細胞에 있어 Bleomycin에 의한 DNA 回復合成)

  • Um, Kyung-Il;Park, Sang-Dai
    • The Korean Journal of Zoology
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    • v.20 no.1
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    • pp.41-48
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    • 1977
  • Dose response of DNA repair synthesis induced by bleomycin was dose-dependent in lower doses, and maximum rate of it at 5 $\\mu$g/ml represents about 15% of total cells analyzed. At higher doses DNA-repair synthesis was reduced and the rate of it remained unchanged even prolonged treatment. Pretreatment with BUdR or IUdR was found to enhance DNA repair synthesis and also to interfere with semiconservative DNA synthesis at higher doses. Time dependence study showed that DNA repair synthesis occurred as long as for 24 hours after removal of bleomycin. These results seem to suggest that bleomycin is not to be an effective chemical in inducing excision repair and that damages induced in DNA by this drug might include not only strand breaks but other types of DNA damage.

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Photodamage to Double-stranded DNA by Xanthone Analogues Increases Exponentially with Their HOMO Energies

  • Hirakawa, Kazutaka;Yoshida, Mami;Oikawa, Shinji;Kawanishi, Shosuke
    • Journal of Photoscience
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    • v.9 no.2
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    • pp.488-490
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    • 2002
  • DNA photodamage mediated by photosensitizers are believed to play an important role in solar UVA carcinogenesis. We investigated the relationship between the DNA-damaging abilities of photoexcited xanthone analogues (as photosensitizers) and their highest occupied molecular orbital (HOMO) energies. DNA damage was examined using /sup 32/P-labeled DNA fragments obtained from the p53 tumor suppressor gene. These compounds induced DNA photodamage in a similar manner, and the extents of DNA damage were following order: xanthone> thioxanthone > acridone. Photoexcited xanthone caused nucleobase oxidation specifically at 5'-G of GG sequence in double-stranded DNA. An oxidative product of 2'-deoxyguanosine, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was detected, and the amount was decreased by DNA denaturation. These findings suggest that photoexcited xanthone generates 8-OHdG at 5'-G of GG in double-stranded DNA through electron transfer. The calculated HOMO energies of these photosensitizers decreased in the following order: xanthone> thioxanthone > acridone. This study has demonstrated that DNA-damaging abilities of these photosensitizers increased exponentially with an increase in their HOMO energies.

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Comparison of Two Methods to Extract DNA from Formalin-Fixed, Paraffin-Embedded Tissues and their Impact on EGFR Mutation Detection in Non-small Cell Lung Carcinoma

  • Hu, Yu-Chang;Zhang, Qian;Huang, Yan-Hua;Liu, Yu-Fei;Chen, Hong-Lei
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.6
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    • pp.2733-2737
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    • 2014
  • Objective: Molecular pathology tests are often carried for clinicopathological diagnosis and pathologists have established large collections of formalin-fixed, paraffin-embedded tissue (FFPE) banks. However, extraction of DNA from FFPE is a laborious and challenging for researchers in clinical laboratories. The aim of this study was to compare two widely used DNA extraction methods: using a QIAamp DNA FFPE kit from Qiagen and a Cobas Sample Preparation Kit from Roche, and evaluated the effect of the DNA quality on molecular diagnostics. Methods: DNA from FFPE non-small cell lung carcinoma tissues including biopsy and surgical specimens was extracted with both QIAamp DNA FFPE and Cobas Sample Preparation Kits and EGFR mutations of non-small cell lung carcinomas were detected by real-time quantitative PCR using the extracted DNA. Results and Conclusion: Our results showed that DNA extracted by QIAamp and Cobas methods were both suitable to detect downstream EGFR mutation in surgical specimens. Howover, Cobas method could yield more DNA from biopsy specimens, and gain much better EGFR mutation results.

Nuclear rDNA characteristics for DNA taxonomy of the centric diatom Chaetoceros (Bacillariophyceae)

  • Oh, Hye-Young;Cheon, Ju-Yong;Lee, Jin-Hwan;Hur, Sung-Bum;Ki, Jang-Seu
    • ALGAE
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    • v.25 no.2
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    • pp.65-70
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    • 2010
  • The genus Chaetoceros provides highly diversified diatoms in marine systems. Morphological descriptions of the genus are well-documented, yet the DNA taxonomy of Chaetoceros has not been satisfactorily established. Here, the molecular divergences of the 18S-28S rDNA of Chaetoceros were assessed. DNA similarities were relatively low in both 18S (93.1 $\pm$ 3.9%) and 28S rDNA (81.0 $\pm$ 4.6%). Phylogenies of the 18S, 28S rDNAs showed that Chaetoceros was divided according to individual species, clustering the same species into single clades. Statistical analysis with corrected genetic (p-) distance scores showed that nucleotide divergence of Chaetoceros 28S rDNA significantly differed from that of 18S rDNA (Student's t-test, p < 0.05). This finding suggests that the 28S rDNA may be treated as a more suitable marker for species-level taxonomic distinctions of Chaetoceros.

Simple Screening Method for Double-strand DNA Binders Using Hairpin DNA-modified Magnetic Beads

  • Jo, Hun-Ho;Min, Kyoung-In;Song, Kyung-Mi;Ku, Ja-Kang;Han, Min-Su;Ban, Chang-Ill
    • Bulletin of the Korean Chemical Society
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    • v.32 no.1
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    • pp.247-250
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    • 2011
  • We designed an effective screening method for double strand DNA (dsDNA) binders using DNA-modified magnetic particles. Hairpin DNA was immobilized on the surface of magnetic particle for a simple screening of dsDNA binding materials in a solution containing various compounds. Through several magnetic separation and incubation processes, four DNA-binding materials, DAPI, 9AA, AQ2A, and DNR, were successfully screened from among five candidates. Efficiency of screening was demonstrated by HPLC analysis using a C2/18 reverse-phase column. In addition, their relative binding strengths were verified by measuring the melting temperature ($T_m$). If hairpin DNA sequence is modified for other uses, this magnetic bead-based approach can be applied as a high-throughput screening method for various functional materials such as anti-cancer drugs.