• Journal of Korean Society of Water and Wastewater
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• v.23 no.2
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• pp.199-205
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• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.669-675
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• 1998

Taq DNA polymerase exhibits a sizable drawback compared to the other thermophilic DNA polymerases in that it demonstrates lower proof-reading activity due to the deficiency of 3'-5'exonuclease activity. A study was undertaken to improve the 3'-5' exonuclease activity in the PCR of Taq DNA polymerase. The three-dimensional structural alignment of the polymerase and 3'-5' exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase has just a background level of 3'-5'exonuclease activity. A comparison indicated that the two polymerase domains are very similar in primary and tertiary conformations, even though Taq DNA polymerase carries a much shorter 3'-5'exonuclease domain than that of E. coli DNA polymerase I. Those two polymerase domains were interchanged between Taq DNA polymerase and E. coli DNA polymerase I. The 3'-5' exonuclease domain from E. coli DNA polymerase I was separated and pasted into the polymerase domain of Taq DNA polymerase I, which resulted in a functional fusion-Stoffel fragment. The 3'-5'exonuclease activity of the fusion-Stoffel fragment increased up to 48% of the value of the Klenow fragment, while that of Taq DNA polymerase remained at 6.0% of the Klenow fragment.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.47-52
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• 1994

The cells of Campylobacter jejuni heat-shocked at 48${\circ}C$ for 30 min synthesized the heat shock proteins of HSP90, HSP66 and HSP60. Those heat shock proteins were found to correspond to the heat shock proteins of HSP87, HSP66 (DnaK), and HSP58 (GroEL) of E. coli, respectively. By Southern blot analysis of the chromosomal DNAs of C. jejuni with groESL and dnaK genes of E. coli as DNA probes, the heat shock genes of C. jejuni which are homologous to the E. coli groESL and dnaK genes were found to exist in the chromosomal DNA. The genomic libraries of C. jejuni were constructed with the cosmid vector pWE15 and the groEL gene of C. jejuni were cloned in E. coli B178 groEL44 temperature senstive mutant. The hybrid plasmid (pLC1) was inserted with the DNA fragment (about 5.7kb in size) containing the groEL gene. E. coli groEL44 mutant cell transformed with the pLC1 could grow at 42${\circ}C$ by synthesizing the HSP60 of C. jejuni and regained the susceptibility to the ${\lambda}$ vir phage by expression of the groEL gene in the cloned cells. These indicated that the groEL products of C. jejuni had chaperon effects by synthesizing the heat shock proteins in the cloned cells of E. coli.

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.164-170
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• 1998

This study was carried out to develop DNA probe for specific detection of Erwinia carotovora subsp. carotovora. Universal rice primer (URP, 20 mer) developed from repetitive sequence of rice was applied for producing PCR DNA fingerprints of Erwinis spp. In E. carotovora subsp. carotovora strains, primer URP2F amplyfied polymorphic bands which are distinguisable from other Erwinia spp. A PCR band of 0.6 kb selected from PCr polymorphic bands of E. carotovora subsp. carotovora strains was cloned and evaluated as a diagnostic DNA probe. Among 28 bacterial strains including 22 Erwinia spp, the probe (pECC2F) only hybridized to total DNAs from e. carotovora subsp. carotovora strains and E. carotovora subsp. wasabiae, but sizes of hybridized bands were different between these subspecies, 10.0 kb and 3.5 kb respectively. In dot blot assays using probe pECC2F, as few as 103 colony forming units (CFU) of E. carotovora subsp. carotovora could be detected in a suspension containing about 1$\times$103 CFU of soil bacteria.

• Journal of Nutrition and Health
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• v.40 no.8
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• pp.708-718
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• 2007

The purpose of this project was to evaluate whether vitamin E supplementation could improve the antioxidant status and lymphocyte DNA damage in Korean postmenopausal women. This was double blinded, placebo-controlled trial. Thirty-five subject were randomized to receive either placebo 400 mg/capsule or natural $\alpha$-tocopherol 400 IU/capsule, 2 times a day for 6 weeks. We measured plasma vitamin C, $\alpha$-tocopherol, $\gamma$-tocopherol, $\alpha$-carotenoid, $\beta$-carotenoid, lycopene concentration and tail length, %DNA in tail, tail moment in lymphocyte DNA damage index. Vitamin E supplementation group had significantly increased plasma vitamin C(p<0.05), $\alpha$-tocopherol(p<0.000), whereas $\gamma$-tocopherol(p<0.000) and tail length(p<0.05) were significantly decreased. However, placebo supplementation group also had significantly increased plasma vitamin C(p<0.05). In conclusion, our study shows that vitamin E supplementation to Korean postmenopausal women may partially improve antioxidant status and lymphocyte DNA damage.

• BMB Reports
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• v.36 no.1
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• pp.12-19
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• 2003

Fragmentation of purine imidazole ring and production of formamidopyrimidines in deoxynucleosides (Fapy lesions) occurs upon DNA oxidation as well as upon spontaneous or alkali-triggered rearrangement of certain alkylated bases. Many chemotherapeutic agents such as cyclophosphamide or thiotepa produce such lesions in DNA. Unsubstituted FapyA and FapyG, formed upon DNA oxidation cause moderate inhibition of DNA synthesis, which is DNA polymerase and sequence dependent. Fapy-7MeG, a methylated counterpart of FapyG-, a efficiently inhibits DNA replication in vitro and in E.coli, however its mutagenic potency is low. This is probably due to preferential incorporation of cytosine opposite Fapy-7MeG and preferential extension of Fapy-7MeG:C pair. In contrast, FapyA and Fapy-7MeA possess miscoding potential. Both lesions in SOS induced E.coli preferentially mispair with cytosine giving rise to A$\rightarrow$G transitions. Fapy lesions substituted with longer chain alkyl groups also show simult aneous lethal and mutagenic properties. Fapy lesions are actively eliminated from DNA by repair glycosylases specific for oxidized purines and pyrimidines both in bacteria and eukaryotic cells. Bacterial enzymes include E.coli formamidopyrimidine-DNA-glycosylase (Fpg protein), endonuclease III (Nth protein) and endonuclease VIII (Nei protein).

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.20-24
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• 2006

Plasmid DNA (pDNA) is a product of interest for many biopharmaceutical companies and research laboratories, because of increase in the number of gene therapy protocols that use nonviral vectors. This work was undertaken to study the effect of antibiotic and dissolved oxygen concentration (DOC) on the production of a ColE 1-type plasmid (pVAX1-LacZ) hosted in Escherichia coli $DH5\alpha$ and cultured in a batch fermentor with 0.751 of Terrific Broth. A decrease in the DOC from $60\%\;to\;5\%$ was shown to increase the specific pDNA concentration approximately 1.5-fold, due to the downregulation of growth. Additionally, this increase in the pDNA concentration led to a 2.2-fold increase in the purity of cell lysates obtained after cell lysis. However, the use of higher DOC led to 2.8-fold higher volumetric productivity as a consequence of a faster growth rate, reducing the fermentation time from 24 to 8 h. Interestingly, the specific pDNA concentration, and pDNA productivity and purity were always higher $(10-15\%)$ in the absence of antibiotic. Overall, the data indicate that nonselective conditions can be used without compromising yield, productivity, and purity of pDNA.

• IMMUNE NETWORK
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• v.11 no.5
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• pp.268-280
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• 2011

Background: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). Methods: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-${\gamma}$ staining. Results: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. Conclusion: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein-specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

• Korean Journal of Environmental Biology
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• v.29 no.3
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• pp.236-240
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• 2011

Mercury known as quicksilver, is the most common cause of heavy metal toxicity. Toxicity caused by excessive mercury exposure is now being recognized as a widespread environmental problem and is continuing to attract a great deal of public concerns. The mercury genotoxicity could be its effect on DNA repair mechanisms, which constitute the defense system designated to protect genome integrity. The objective of this study is to confirm that mercuric chloride inhibits the repair of gamma ray-induced DNA damage. The earthworm of Eisenia fetida was chosen for this study because it is an internationally accepted model species for toxicity testing with a cosmopolitan distribution. Experiments were done to identify the levels of DNA damage and the repair kinetics in the coelomocytes of E. fetida irradiated with 20 Gy gamma rays alone or with gamma rays after 40 mg $kg^{-1}$ $HgCl_2$ treatment by means of the single cell gel electrophoresis assay. The Olive tail moments were measured during 0~96 hours after irradiation. The repair time in the animals treated with the combination of $HgCl_2$ and ionizing radiation was nearly five times longer than that in the animals treated with ionizing radiation alone. Also, E. fetida exposed to mercury showed a statistically lower repair efficiency of gamma ray-induced DNA damage. The results suggest that the mercury could even have deleterious effects on the DNA repair system. Influence of mercury on the DNA repair mechanisms has been confirmed by this study.

• Mycobiology
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• v.30 no.1
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• pp.1-4
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• 2002

Genetic relatedness of medically important Exophiala species such as E. dermatitidis, E. mansonii, and three E. jeanselmei varieties: jeanselmei, lecanii-corni, and heteromorpha was examined using PCR-RFLP(restriction fragment length polymorphism) of ribosomal DNA, M-13, $(GTG)_5$ and nucleotide sequences of ribosomal ITS(internal transcribed space) II regions. Three E. jeanselmei varieties showing distinct band patterns for each DNA markers as well as different nucleotide sequences of ribosomal ITS II regions could be considered as a separate species. E. dermatitidis and E. mansonii demonstrated the identical band patterns of RFLP of ribosomal DNA, M-13, and $(GTG)_5$ markers. However, nucleotides sequences of ribosomal ITS II region were different between these two species.